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Archive for the ‘Cell death pathways’ Category


Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

A mutated gene called RAS gives rise to a signalling protein Ral which is involved in tumour growth in the bladder. Many researchers tried and failed to target and stop this wayward gene. Signalling proteins such as Ral usually shift between active and inactive states.

 

So, researchers next tried to stop Ral to get into active state. In inacvtive state Ral exposes a pocket which gets closed when active. After five years, the researchers found a small molecule dubbed BQU57 that can wedge itself into the pocket to prevent Ral from closing and becoming active. Now, BQU57 has been licensed for further development.

 

Researchers have a growing genetic data on bladder cancer, some of which threaten to overturn the supposed causes of bladder cancer. Genetics has also allowed bladder cancer to be reclassified from two categories into five distinct subtypes, each with different characteristics and weak spots. All these advances bode well for drug development and for improved diagnosis and prognosis.

 

Among the groups studying the genetics of bladder cancer are two large international teams: Uromol (named for urology and molecular biology), which is based at Aarhus University Hospital in Denmark, and The Cancer Genome Atlas (TCGA), based at institutions in Texas and Boston. Each team tackled a different type of cancer, based on the traditional classification of whether or not a tumour has grown into the muscle wall of the bladder. Uromol worked on the more common, earlier form, non-muscle-invasive bladder cancer, whereas TCGA is looking at muscle-invasive bladder cancer, which has a lower survival rate.

 

The Uromol team sought to identify people whose non-invasive tumours might return after treatment, becoming invasive or even metastatic. Bladder cancer has a high risk of recurrence, so people whose non-invasive cancer has been treated need to be monitored for many years, undergoing cystoscopy every few months. They looked for predictive genetic footprints in the transcriptome of the cancer, which contains all of a cell’s RNA and can tell researchers which genes are turned on or off.

 

They found three subgroups with distinct basal and luminal features, as proposed by other groups, each with different clinical outcomes in early-stage bladder cancer. These features sort bladder cancer into genetic categories that can help predict whether the cancer will return. The researchers also identified mutations that are linked to tumour progression. Mutations in the so-called APOBEC genes, which code for enzymes that modify RNA or DNA molecules. This effect could lead to cancer and cause it to be aggressive.

 

The second major research group, TCGA, led by the National Cancer Institute and the National Human Genome Research Institute, that involves thousands of researchers across USA. The project has already mapped genomic changes in 33 cancer types, including breast, skin and lung cancers. The TCGA researchers, who study muscle-invasive bladder cancer, have looked at tumours that were already identified as fast-growing and invasive.

 

The work by Uromol, TCGA and other labs has provided a clearer view of the genetic landscape of early- and late-stage bladder cancer. There are five subtypes for the muscle-invasive form: luminal, luminal–papillary, luminal–infiltrated, basal–squamous, and neuronal, each of which is genetically distinct and might require different therapeutic approaches.

 

Bladder cancer has the third-highest mutation rate of any cancer, behind only lung cancer and melanoma. The TCGA team has confirmed Uromol research showing that most bladder-cancer mutations occur in the APOBEC genes. It is not yet clear why APOBEC mutations are so common in bladder cancer, but studies of the mutations have yielded one startling implication. The APOBEC enzyme causes mutations early during the development of bladder cancer, and independent of cigarette smoke or other known exposures.

 

The TCGA researchers found a subset of bladder-cancer patients, those with the greatest number of APOBEC mutations, had an extremely high five-year survival rate of about 75%. Other patients with fewer APOBEC mutations fared less well which is pretty surprising.

 

This detailed knowledge of bladder-cancer genetics may help to pinpoint the specific vulnerabilities of cancer cells in different people. Over the past decade, Broad Institute researchers have identified more than 760 genes that cancer needs to grow and survive. Their genetic map might take another ten years to finish, but it will list every genetic vulnerability that can be exploited. The goal of cancer precision medicine is to take the patient’s tumour and decode the genetics, so the clinician can make a decision based on that information.

 

References:

 

https://www.ncbi.nlm.nih.gov/pubmed/29117162

 

https://www.ncbi.nlm.nih.gov/pubmed/27321955

 

https://www.ncbi.nlm.nih.gov/pubmed/28583312

 

https://www.ncbi.nlm.nih.gov/pubmed/24476821

 

https://www.ncbi.nlm.nih.gov/pubmed/28988769

 

https://www.ncbi.nlm.nih.gov/pubmed/28753430

 

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GE Healthcare has acquired Biosafe Group SA, a supplier of Integrated Cell Bioprocessing Systems for Cell Therapy and Regenerative Medicine Industry

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Researchers of University of Texas at San Antonio, USA, have developed a new, non-invasive method which can kill cancer cells in two hours, an advance that may significantly help people with inoperable or hard-to-reach tumours, as well as young children stricken with the deadly disease.

 

The method involves injecting a chemical compound, nitrobenzaldehyde, into the tumour and allowing it to diffuse into the tissue. A beam of light is then aimed at the tissue, causing the cells to become very acidic inside and, essentially, commit suicide. Within two hours, up to 95 per cent of the targeted cancer cells are estimated to be dead.

 

The method was tested against triple negative breast cancer, one of the most aggressive types of cancer and one of the hardest to treat. The prognosis for triple negative breast cancer is usually very poor. One treatment in the laboratory was able to stop the tumour from growing and doubled the chances of survival in the mice.

 

According to the researchers all forms of cancer attempt to make cells acidic on the outside and attract the attention of blood vessels as an attempt to get rid of the acid. But, instead, the cancer cells latches onto the blood vessel and uses it to make the tumour grow bigger.

 

Chemotherapy treatments target all cells in the body, and certain chemotherapeutics try to keep cancer cells acidic as a way to kill the cancer. This is what causes many cancer patients to lose their hair and become weak. This method however, is more precise and can target just the tumour.

 

This research is presently extended on drug-resistant cancer cells to make this therapy as strong as possible. The researchers also started to develop a nanoparticle that can be injected into the body to target metastasised cancer cells. The nanoparticle is activated with a wavelength of light which can pass harmlessly through skin, flesh and bone and still activate the nanoparticle.

 

This non-invasive method will help cancer patients with tumours in areas that have proven problematic for surgeons, such as the brain stem, aorta or spine. It could also help people who have received the maximum amount of radiation treatment and can no longer cope with the scarring and pain that goes along with it, or children who are at risk of developing mutations from radiation as they grow older.

 

References:

 

http://www.ndtv.com/health/researchers-develop-new-method-to-kill-cancer-cells-in-2-hours-1424509

 

https://www.consumeraffairs.com/news/new-non-invasive-cancer-therapy-shows-promise-062916.html

 

http://www.mirror.co.uk/science/new-cancer-treatment-can-kill-8341452

 

https://www.sciencedaily.com/releases/2016/06/160627214423.htm

 

http://reliawire.com/photodynamic-acidification-therapy/

 

http://www.gizmag.com/making-cancer-cells-acidic/44070/

 

 

http://www.oncologynurseadvisor.com/general-oncology/initial-photodynamic-therapy-tests-promising/article/508292/

 

https://www.sciencedaily.com/releases/2016/06/160627214423.htm

 

http://www.thehindu.com/sci-tech/health/new-method-can-kill-cancer-cells-in-two-hours-shows-study/article8785315.ece

 

http://www.aol.com/article/2016/07/06/new-cancer-treatment-method-causes-cells-to-commit-suicide/21424984/

 

http://zeenews.india.com/news/health/diseases-conditions/new-method-that-can-kill-cancer-cells-in-2-hours-developed_1901377.html

 

http://www.digitaltrends.com/health-fitness/ultraviolet-light-kills-cancer-cells/

 

https://www.thesun.co.uk/news/1385404/light-can-kill-cancer-in-just-two-hours/

 

http://www.techtimes.com/articles/168268/20160704/new-cancer-therapy-method-ultraviolet-light-may-soon-replace-chemotherapy.htm

 

https://www.engadget.com/2016/07/01/scientists-use-light-to-nuke-cancer-cells-in-mice/

 

Nuha Buchanan Kadri, Matthew Gdovin, Nizar Alyassin, Justin Avila, Aryana Cruz, Louis Cruz, Steve Holliday, Zachary Jordan, Cameron Ruiz and Jennifer Watts. Photodynamic acidification therapy to reduce triple negative breast cancer growth in vivo. Journal of Clinical Oncology, Vol 34, No 15_suppl (May 20 Supplement), 2016: e12574.

 

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Novel Discoveries in Molecular Biology and Biomedical Science

Curator: Larry H. Bernstein, MD, FCAP

LPBI

Updated June 1, 2016

The following is a collection of current articles on noncoding DNA, synthetic genome engineering, protein regulation of apoptosis, drug design, and geometrics.

 

No longer ‘junk DNA’ — shedding light on the ‘dark matter’ of the genome

A new tool called “LIGR-Seq” enables scientists to explore in depth what non-coding RNAs actually do in human cells   May 23, 2016

http://www.kurzweilai.net/no-longer-junk-dna-shedding-light-on-the-dark-matter-of-the-genome

http://www.kurzweilai.net/images/LIGR-seq-method.png

he LIGR-seq method for global-scale mapping of RNA-RNA interactions in vivo to reveal unexpected functions for uncharacterized RNAs that act via base-pairing interactions (credit: University of Toronto)

What used to be dismissed by many as “junk DNA” has now become vitally important, as accelerating genomic data points to the importance of non-coding RNAs (ncRNAs) — a genome’s messages that do not specifically code for proteins — in development and disease.

But our progress in understanding these molecules has been slow because of the lack of technologies that allow for systematic mapping of their functions.

Now, professor Benjamin Blencowe’s team at the University of Toronto’s Donnelly Centre has developed a method called “LIGR-seq” that enables scientists to explore in depth what ncRNAs do in human cells.

The study, described in Molecular Cell, was published on May 19, along with two other papers, in Molecular Cell and Cell, respectively, from Yue Wan’s group at the Genome Institute of Singapore and Howard Chang’s group at Stanford University in California, who developed similar methods to study RNAs in different organisms.

So what exactly do ncRNAs do?

http://www.kurzweilai.net/images/ncRNA.png

mRNAs vs. ncRNAs (credit: Thomas Shafee/CC)

Of the 3 billion letters in the human genome, only two per cent make up the protein-coding genes. The genes are copied, or transcribed, into messenger RNA (mRNA) molecules, which provide templates for building proteins that do most of the work in the cell. Much of the remaining 98 per cent of the genome was initially considered by some as lacking in functional importance. However, large swaths of the non-coding genome — between half and three quarters of it — are also copied into RNA.

So then what might the resulting ncRNAs do? That depends on whom you ask. Some researchers believe that most ncRNAs have no function, that they are just a by-product of the genome’s powerful transcription machinery that makes mRNA. However, it is emerging that many ncRNAs do have important roles in gene regulation — some ncRNAs act as carriages for shuttling the mRNAs around the cell, or provide a scaffold for other proteins and RNAs to attach to and do their jobs.

But the majority of available data has trickled in piecemeal or through serendipitous discovery. And with emerging evidence that ncRNAs could drive disease progression, such as cancer metastasis, there was a great need for a technology that would allow a systematic functional analysis of ncRNAs.

Up until now, with existing methods, you had to know what you are looking for because they all require you to have some information about the RNA of interest. The power of our method is that you don’t need to preselect your candidates; you can see what’s occurring globally in cells, and use that information to look at interesting things we have not seen before and how they are affecting biology,” says Eesha Sharma, a PhD candidate in Blencowe’s group who, along with postdoctoral fellow Tim Sterne-Weiler, co-developed the method.

A new ncRNA identification tool

http://www.kurzweilai.net/images/rna-rna-interactions.jpg

The human RNA-RNA interactome, showing interactions detected by LIGR-seq (credit: University of Toronto)

The new ‘‘LIGation of interacting RNA and high-throughput sequencing’’ (LIGR-seq) tool captures interactions between different RNA molecules. When two RNA molecules have matching sequences — strings of letters copied from the DNA blueprint — they will stick together like Velcro. With LIGR-seq, the paired RNA structures are removed from cells and analyzed by state-of-the-art sequencing methods to precisely identify the RNAs that are stuck together.

Most researchers in the life sciences agree that there’s an urgent need to understand what ncRNAs do. This technology will open the door to developing a new understanding of ncRNA function,” says Blencowe, who is also a professor in the Department of Molecular Genetics.

Not having to rely on pre-existing knowledge will boost the discovery of RNA pairs that have never been seen before. Scientists can also, for the first time, look at RNA interactions as they occur in living cells, in all their complexity, unlike in the juices of mashed up cells that they had to rely on before. This is a bit like moving on to explore marine biology from collecting shells on the beach to scuba-diving among the coral reefs, where the scope for discovery is so much bigger.

Actually, ncRNAs come in multiple flavors: there’s rRNA, tRNA, snRNA, snoRNA, piRNA, miRNA, and lncRNA, to name a few, where prefixes reflect the RNA’s place in the cell or some aspect of its function. But the truth is that no one really knows the extent to which these ncRNAs control what goes on in the cell, or how they do this.

Discoveries

Nonetheless, the new technology developed by Blencowe’s group has been able to pick up new interactions involving all classes of RNAs and has already revealed some unexpected findings.

The team discovered new roles for small nucleolar RNAs (snoRNAs), which normally guide chemical modifications of other ncRNAs. It turns out that some snoRNAs can also regulate stability of a set of protein-coding mRNAs. In this way, snoRNAs can also directly influence which proteins are made, as well as their abundance, adding a new level of control in cell biology.

And this is only the tip of the iceberg; the researchers plan to further develop and apply their technology to investigate the ncRNAs in different settings.

“We would like to understand how ncRNAs function during development. We are particularly interested in their role in the formation of neurons. But we will also use our method to discover and map changes in RNA-RNA interactions in the context of human diseases,” says Blencowe.

Abstract of Global Mapping of Human RNA-RNA Interactions

The majority of the human genome is transcribed into non-coding (nc)RNAs that lack known biological functions or else are only partially characterized. Numerous characterized ncRNAs function via base pairing with target RNA sequences to direct their biological activities, which include critical roles in RNA processing, modification, turnover, and translation. To define roles for ncRNAs, we have developed a method enabling the global-scale mapping of RNA-RNA duplexes crosslinked in vivo, “LIGation of interacting RNA followed by high-throughput sequencing” (LIGR-seq). Applying this method in human cells reveals a remarkable landscape of RNA-RNA interactions involving all major classes of ncRNA and mRNA. LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.

references:

 

Venter’s Research Team Creates an Artificial Cell and Reports That 32% of Genes Are Life-Essential but Contain Unknown Functions
http://www.radmailer.com/t/r-l-sttullk-ykogyktt-k/
May 27, 2016

Understanding the unknown functions of these genes may lead to the creation of new diagnostic tests for clinical laboratories and anatomic pathology groups

Once again, J. Craig Venter, PhD, is charting new ground in gene sequencing andgenomic science. This time his research team has built upon the first synthetic cell they created in 2010 to build a more sophisticated synthetic cell. Their findings from this work may give pathologists and medical laboratory scientists new tools to diagnose disease.

Recently the research team at the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) published their latest findings. Among the things they learned is that science still does not understand the functions of about a third of the genes required for their synthetic cells to function.

JCVI-syn3.0 Could Radically Alter Understanding of Human Genome

Based in La Jolla, Calif., and Rockville, Md., JCVI is a not-for-profit research institute aiming to advance genomics. Building upon its first synthetic cell—Mycoplasma mycoides (M. mycoides) JCVI-syn1.0, which JCVI constructed in 2010—the same team of scientists created the first minimal synthetic bacterial cell, which they calledJCVI-syn3.0. This new artificial cell contains 531,560 base pairs and just 473 genes, which means it is the smallest genome of any organism that can be grown in laboratory media, according to a JCVI-SGI statement.

For pathologists and medical laboratory leaders, the creation of a synthetic life form is a milestone toward better understanding genome sequencing and how this new knowledge may help advance both diagnostics and therapeutics.

“What we’ve done is important because it is a step toward completely understanding how a living cell works,” Clyde Hutchison III, PhD, told New Scientist. “If we can really understand how the cell works, then we will be able to design cells efficiently for the production of pharmaceutical and other useful products.” Hutchison is Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill, Distinguished Professor at the J. Craig Venter Institute, a member of the National Academy of Sciences, and a fellow of the American Academy of Arts and Sciences.

Click here to see images

Clyde Hutchison, III, PhD (above), Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill and Distinguished Professor at the J. Craig Venter Institute, stated that his team’s “goal is to have a cell for which the precise biological function of every gene is known.” (Photo credit: JCVI.)

Understanding a Gene’s True Purpose

According to the JCVI researchers, 149 genes have no known purpose. They are, however, necessary for life and health.

“We know about two-thirds of the essential biology, and we’re missing a third,” stated J. Craig Venter, PhD, Founder and CEO of JCVI, in a story published by MedPage Today.

This knowledge is based upon decades of research. JCVI seeks to create a minimal cell operating system to understand biology, while also providing what the JCVI statement called a “chassis for use in industrial applications.”

What Do these Genes Do Anyway?

The JCVI team found that among most genes’ biological functions:

“JCVI-syn3.0 is a working approximation of a minimal cellular genome—a compromise between a small genome size and a workable growth rate for an experimental organism. It retains almost all the genes that are involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions, suggesting the presence of undiscovered functions that are essential for life,” the researchers told the journal Science.

More research is needed, the scientists say, into the 149 genes that appear to lack specific biologic functions.

Unlocking Mystery of the 149 Genes Could Lead to Advances in Genomic Science

“Finding so many genes without a known function is unsettling, but it’s exciting because it’s left us with much still to learn. It’s like the ‘dark matter’ of biology,” said Alistair Elfick, PhD, Chair of Synthetic Biological Engineering, University of Edinburgh, UK, in the New Scientist article.

Studies such as JCVI’s research is key to broadening understanding and framing appropriate questions about scientific, ethical, and economic implications of synthetic biology.

The creation of a synthetic cell will have a profound and positive impact on understanding of biology and how life works, JCVI said.

Such research may inspire new whole genome synthesis tools and semi-automated processes that could dramatically affect clinical laboratory procedures. It also could lead to new techniques and tools for advanced vaccine and pharmaceuticals, JCVI pointed out.

—Donna Marie Pocius

Related Information:

First Minimal Synthetic Bacterial Cell Designed and Constructed by Scientists at Venter Institute and Synthetic Genomics, Inc.

 

CRISPR Versatility Inspires Molecular Biology Innovation

GEN Tech Focus: CRISPR/Gene Editing
No single technique has set the molecular biology field ablaze with excitement and potential like the CRISPR-Cas9 genome editing system has following its introduction only a few short years ago. The following articles represent the flexibility of this technique to potentially treat a host of genetic disorders and possibly even prevent the onset of disease.

 

CRISPR Moves from Butchery to Surgery

Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.

 

New CRISPR System Targets Both DNA and RNA

With a staggering number of papers published in the past several years involving the characterization and use of the CRISPR/Cas9 gene editing system, it is surprising that researchers are still finding new features of the versatile molecular scissor enzyme.

 

High-Fidelity CRISPR-Cas9 Nucleases Virtually Free of Off-Target Noise

If a Cas9 nuclease variant could be engineered that was less grabby, it might loosen its grip on DNA sequences throughout the genome—except those sequences representing on-target sites. That’s the assumption that guided a new investigation by researchers at Massachusetts General Hospital.

 

CRISPR Works Well but Needs Upgrades

The gene-editing technology known as CRISPR-Cas9 is starting to raise expectations in the therapeutic realm. In fact, CRISPR-Cas9 and other CRISPR systems are moving so close to therapeutic uses that the technology’s ethical implications are starting to attract notice.

 

A Guide to CRISPR Gene Activation
http://www.technologynetworks.com/rnai/news.aspx?ID=191776

Published: Tuesday, May 24, 2016
A comparison of synthetic gene-activating Cas9 proteins can help guide research and development of therapeutic approaches.

The CRISPR-Cas9 system has come to be known as the quintessential tool that allows researchers to edit the DNA sequences of many organisms and cell types. However, scientists are also increasingly recognizing that it can be used to activate the expression of genes. To that end, they have built a number of synthetic gene activating Cas9 proteins to study gene functions or to compensate for insufficient gene expression in potential therapeutic approaches.

“The possibility to selectively activate genes using various engineered variants of the CRISPR-Cas9 system left many researchers questioning which of the available synthetic activating Cas9 proteins to use for their purposes. The main challenge was that all had been uniquely designed and tested in different settings; there was no side-by-side comparison of their relative potentials,” said George Church, Ph.D., who is Core Faculty Member at the Wyss Institute for Biologically Inspired Engineering at Harvard University, leader of its Synthetic Biology Platform, and Professor of Genetics at Harvard Medical School. “We wanted to provide that side-by-side comparison to the biomedical research community.”

In a study published on 23 May in Nature Methods, the Wyss Institute team reports how it rigorously compared and ranked the most commonly used artificial Cas9 activators in different cell types from organisms including humans, mice and flies. The findings provide a valuable guide to researchers, allowing them to streamline their endeavors.

The team also included Wyss Core Faculty Member James Collins, Ph.D., who also is the Termeer Professor of Medical Engineering & Science and Professor of Biological Engineering at the Massachusetts Institute of Technology (MIT)’s Department of Biological Engineering and Norbert Perrimon, Ph.D., a Professor of Genetics at Harvard Medical School.

Gene activating Cas9 proteins are fused to variable domains borrowed from proteins with well-known gene activation potentials and engineered so that the DNA editing ability is destroyed. In some cases, the second component of the CRISPR-Cas9 system, the guide RNA that targets the complex to specific DNA sequences, also has been engineered to bind gene-activating factors.

“We first surveyed seven advanced Cas9 activators, comparing them to each other and the original Cas9 activator that served to provide proof-of-concept for the gene activation potential of CRISPR-Cas9. Three of them, provided much higher gene activation than the other candidates while maintaining high specificities toward their target genes,” said Marcelle Tuttle, Research Fellow at the Wyss and a co-lead author of the study.

The team went on to show that the three top candidates were comparable in driving the highest level of gene expression in cells from humans, mice and fruit flies, irrespective of their tissue and developmental origins. The researchers also pinpointed ways to further maximize gene activation employing the three leading candidates.

“In some cases, maximum possible activation of a target gene is necessary to achieve a cellular or therapeutic effect. We managed to cooperatively enhance expression of specific genes when we targeted them with three copies of a top performing activator using three different guide RNAs,” said Alejandro Chavez, Ph.D., a Postdoctoral Fellow and the study’s co-first author.

“The ease of use of CRISPR-Cas9 offers enormous potential for development of genome therapeutics. This study provides valuable new design criteria that will help enable synthetic biologists and bioengineers to develop more effective targeted genome engineering technologies in the future,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, and also Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

 

Engineering T Cells to Functionally Cure HIV-1 Infection

Rachel S Leibman and James L Riley
Molecular Therapy (21 April 2015) |    http://dx.doi.org:/10.1038/mt.2015.70

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients’ lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1–resistant cells, redirecting HIV-1–specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1–specific lymphocytes within a patient’s own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy–mediated functional cure.

 

Automated top-down design technique simplifies creation of DNA origami nanostructures

http://www.kurzweilai.net/automated-top-down-design-technique-simplifies-creation-of-dna-origami-nanostructures

Nanoparticles for drug delivery and cell targeting, nanoscale robots, custom-tailored optical devices, and DNA as a storage medium are among the possible applications

May 27, 2016

The boldfaced line, known as a spanning tree, follows the desired geometric shape of the target DNA origami design method, touching each vertex just once. A spanning tree algorithm is used to map out the proper routing path for the DNA strand. (credit: Public Domain)

MITBaylor College of Medicine, and Arizona State University Biodesign Institute researchers have developed a radical new top-down DNA origami* design method based on a computer algorithm that allows for creating designs for DNA nanostructures by simply inputting a target shape.

DNA origami (using DNA to design and build geometric structures) has already proven wildly successful in creating myriad forms in 2- and 3- dimensions, which conveniently self-assemble when the designed DNA sequences are mixed together. The tricky part is preparing the proper DNA sequence and routing design for scaffolding and staple strands to achieve the desired target structure. Typically, this is painstaking work that must be carried out manually.

The new algorithm, which is reported together with a novel synthesis approach in the journal Science, promises to eliminate all that and expands the range of possible applications of DNA origami in biomolecular science and nanotechnology. Think nanoparticles for drug delivery and cell targeting, nanoscale robots in medicine and industry, custom-tailored optical devices, and most interesting: DNA as a storage medium, offering retention times in the millions of years.**

 

Shape-shifting, top-down software

Unlike traditional DNA origami, in which the structure is built up manually by hand, the team’s radical top-down autonomous design method begins with an outline of the desired form and works backward in stages to define the required DNA sequence that will properly fold to form the finished product.

“The Science paper turns the problem around from one in which an expert designs the DNA needed to synthesize the object, to one in which the object itself is the starting point, with the DNA sequences that are needed automatically defined by the algorithm,” said Mark Bathe, an associate professor of biological engineering at MIT, who led the research. “Our hope is that this automation significantly broadens participation of others in the use of this powerful molecular design paradigm.”

The algorithm, which is known as DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined Structures) after the Greek craftsman and artist who designed labyrinths that resemble origami’s complex scaffold structures, can build any type of 3-D shape, provided it has a closed surface. This can include shapes with one or more holes, such as a torus.

A simplified version of the  top-down procedure used to design scaffolded DNA origami nanostructures. It starts with a polygon corresponding to the target shape. Software translates a wireframe version of this structure into a plan for routing DNA scaffold and staple strands. That enables a 3D DNA-based atomic-level structural model that is then validated using 3D cryo-EM reconstruction. (credit: adapted from Biodesign Institute images)

With the new technique, the target geometric structure is first described in terms of a wire mesh made up of polyhedra, with a network of nodes and edges. A DNA scaffold using strands of custom length and sequence is generated, using a “spanning tree” algorithm — basically a map that will automatically guide the routing of the DNA scaffold strand through the entire origami structure, touching each vertex in the geometric form once. Complementary staple strands are then assigned and the final DNA structural model or nanoparticle self-assembles, and is then validated using 3D cryo-EM reconstruction.

The software allows for fabricating a variety of geometric DNA objects, including 35 polyhedral forms (Platonic, Archimedean, Johnson and Catalan solids), six asymmetric structures, and four polyhedra with nonspherical topology, using inverse design principles — no manual base-pair designs needed.

To test the method, simpler forms known as Platonic solids were first fabricated, followed by increasingly complex structures. These included objects with nonspherical topologies and unusual internal details, which had never been experimentally realized before. Further experiments confirmed that the DNA structures produced were potentially suitable for biological applications since they displayed long-term stability in serum and low-salt conditions.

Biological research uses

The research also paves the way for designing nanoscale systems mimicking the properties of viruses, photosynthetic organisms, and other sophisticated products of natural evolution. One such application is a scaffold for viral peptides and proteins for use as vaccines. The surface of the nanoparticles could be designed with any combination of peptides and proteins, located at any desired location on the structure, in order to mimic the way in which a virus appears to the body’s immune system.

The researchers demonstrated that the DNA nanoparticles are stable for more than six hours in serum, and are now attempting to increase their stability further.

The nanoparticles could also be used to encapsulate the CRISPR-Cas9 gene editing tool. The CRISPR-Cas9 tool has enormous potential in therapeutics, thanks to its ability to edit targeted genes. However, there is a significant need to develop techniques to package the tool and deliver it to specific cells within the body, Bathe says.

This is currently done using viruses, but these are limited in the size of package they can carry, restricting their use. The DNA nanoparticles, in contrast, are capable of carrying much larger gene packages and can easily be equipped with molecules that help target the right cells or tissue.

The most exciting aspect of the work, however, is that it should significantly broaden participation in the application of this technology, Bathe says, much like 3-D printing has done for complex 3-D geometric models at the macroscopic scale.

Hao Yan directs the Biodesign Center for Molecular Design and Biomimetics at Arizona State University and is the Milton D. Glick Distinguished Professor, College of Liberal Arts and Sciences, School of Molecular Sciences at ASU.

DNA origami brings the ancient Japanese method of paper folding down to the molecular scale. The basics are simple: Take a length of single-stranded DNA and guide it into a desired shape, fastening the structure together using shorter “staple strands,” which bind in strategic places along the longer length of DNA. The method relies on the fact that DNA’s four nucleotide letters—A, T, C, & G stick together in a consistent manner — As always pairing with Ts and Cs with Gs.

The DNA molecule in its characteristic double stranded form is fairly stiff, compared with single-stranded DNA, which is flexible. For this reason, single stranded DNA makes for an ideal lace-like scaffold material. Further, its pairing properties are predictable and consistent (unlike RNA).

https://vimeo.com/22349631

** A single gram of DNA can store about 700 terabytes of information — an amount equivalent to 14,000 50-gigabyte Blu-ray disks — and could potentially be operated with a fraction of the energy required for other information storage options.

 

Essential role of miRNAs in orchestrating the biology of the tumor microenvironment

Jamie N. Frediani and Muller Fabbri
Molecular Cancer (2016) 15:42   http://dx.doi.org:/10.1186/s12943-016-0525-3

MicroRNAs (miRNAs) are emerging as central players in shaping the biology of the Tumor Microenvironment (TME). They do so both by modulating their expression levels within the different cells of the TME and by being shuttled among different cell populations within exosomes and other extracellular vesicles. This review focuses on the state-of-the-art knowledge of the role of miRNAs in the complexity of the TME and highlights limitations and challenges in the field. A better understanding of the mechanisms of action of these fascinating micro molecules will lead to the development of new therapeutic weapons and most importantly, to an improvement in the clinical outcome of cancer patients. Keywords: Exosomes, microRNAs, Tumor microenvironment, Cancer

While cancer treatment and survival have improved worldwide, the need for further understanding of the underlying tumor biology remains. In recent years, there has been a significant shift in scientific focus towards the role of the tumor microenvironment (TME) on the development, growth, and metastatic spread of malignancies. The TME is defined as the surrounding cellular environment enmeshed around the tumor cells including endothelial cells, lymphocytes, macrophages, NK cells, other cells of the immune system, fibroblasts, mesenchymal stem cells (MSCs), and the extracellular matrix (ECM). Each of these components interacts with and influences the tumor cells, continually shifting the balance between pro- and anti-tumor phenotype. One of the predominant methods of communication between these cells is through extracellular vesicles and their microRNA (miRNA) cargo. Extracellular vesicles (EVs) are between 30 nm to a few microns in diameter, are surrounded by a phospholipid bilayer membrane, and are released from a variety of cell types into the local environment. There are three well characterized groups of EVs: 1) exosomes, typically 30–100 nm, 2) microvesicles (or ectosomes), typically 100–1000 nm, and 3) large oncosomes, typically 1–10 μm. Each of these categories has a distinctly unique biogenesis and purpose in cellcell communication despite the fact that current laboratory methods do not always allow precise differentiation. EVs are found to be enriched with membrane-bound proteins, lipid raft-associated and cytosolic proteins, lipids, DNA, mRNAs, and miRNAs, all of which can be transferred to the recipient cell upon fusion to allow cell-cell communications [1]. Of these, miRNAs have been of particular interest in cancer research, both as modifiers of transcription and translation as well as direct inhibitors or enhancers of key regulatory proteins. These miRNAs are a large family of small non-coding RNAs (19–24 nucleotides) and are known to be aberrantly expressed, both in terms of content as well as number, in both the tumor cells and the cells of the TME. Synthesis of these mature miRNA is a complex process, starting with the transcription of long, capped, and polyadenylated pri-miRNA by RNA polymerase II. These are cropped into a 60–100 nucleotide hairpinstructure pre-miRNA by the microprocessor, a heterodimer of Drosha (a ribonuclease III enzyme) and DGCR8 (DiGeorge syndrome critical region gene 8). The premiRNA is then exported to the cytoplasm by exportin 5, cleaved by Dicer, and separated into single strands by helicases. The now mature miRNA are incorporated into the RNA-induced silencing complex (RISC), a cytoplasmic effector machine of the miRNA pathway. The primary mechanism of action of the mature miRNA-RISC complex is through their binding to the 3’ untranslated region, or less commonly the 5’ untranslated region, of target mRNA, leading to protein downregulation either via translational repression or mRNA degradation. More recently, it has been shown that miRNAs can also upregulate the expression of target genes [2]. MiRNA genes are mostly intergenic and are transcribed by independent promoters [3] but can also be encoded by introns, sharing the same promoter of their host gene [4]. MiRNAs undergo the same regulatory mechanisms of any other protein coding gene (promoter methylation, histone modifications, etc.…) [5, 6]. Interestingly, each miRNA may have contradictory effects both within varying tumor cell lines and within different cells of the TME. In this review, we provide a state-of-the-art description of the key role that miRNAs have in the communication between tumor cells and the TME and their subsequent effects on the malignant phenotype. Finally, this review has made every effort to clarify, whenever possible, whether the reference is to the −3p or the -5p miRNA. Whenever such clarification has not been provided, this indicates that it was not possible to infer such information from the cited bibliography.

Angiogenesis and miRNAs Cellular plasticity, critical in the development of malignancy, includes the many diverse mechanisms elicited by cancer cells to increase their malignant potential and develop increasing treatment resistance. One such mechanism, angiogenesis, is critical to the development of metastatic disease, affecting both the growth of malignant cells locally and their survival at distant sites. In the last ten years, miRNAs, often packaged in tumor cell-derived exosomes, have emerged as important contributors to the complicated regulation and balance of pro- and anti-angiogenic factors.

Most commonly, miRNAs derived from cancer cells have oncogenic activity, promoting angiogenesis and tumor growth and survival. The most-well characterized of the pro-angiogenic miRNAs, the miR-17-92 cluster encoding six miRNAs (miR-17, −18a, −19a, −19b, −20a, and −92a), is found on chromosome 13, and is highly conserved among vertebrates [7]. The complex and multifaceted functions of the miR-17-92 cluster are summarized in Fig. 1. Amplification, both at the genetic and RNA level, of miR-17-92 was initially found in several lymphoma cell lines and has subsequently been observed in multiple mouse tumor models [7].

Fig. 1   https://static-content.springer.com/image/art%3A10.1186%2Fs12943-016-0525-3/MediaObjects/12943_2016_525_Fig1_HTML.gif

Central role of the miR-17-92 cluster in the biology of the TME. The miR-17-92 cluster encoding miR-17, −18a, −19b, −20a, and -92a is upregulated in multiple tumor types and interacts with various components of the TME to finely “tune” the TME through a complex combination of pro- and anti-tumoral effects

Most commonly, miRNAs derived from cancer cells have oncogenic activity, promoting angiogenesis and tumor growth and survival. The most-well characterized of the pro-angiogenic miRNAs, the miR-17-92 cluster encoding six miRNAs (miR-17, −18a, −19a, −19b, −20a, and −92a), is found on chromosome 13, and is highly conserved among vertebrates [7]. The complex and multifaceted functions of the miR-17-92 cluster are summarized in Fig. 1. Amplification, both at the genetic and RNA level, of miR-17-92 was initially found in several lymphoma cell lines and has subsequently been observed in multiple mouse tumor models [7]. Up-regulation of this particular locus has further been confirmed in miRnome analysis across multiple different tumor types, including lung, breast, stomach, prostate, colon, and pancreatic cancer [8]. The miR-17-92 cluster is directly activated by Myc and modulates a variety of downstream transcription factors important in cell cycle regulation and apoptosis including activation of E2F family and Cyclin-dependent kinase inhibitor (CDKN1A) and downregulation of BCL2L11/BIM and p21 [7]. In addition to promoting cell cycle progression and inhibiting apoptosis, the miR-17-92 cluster also downregulates thrombospondin-1 (Tsp1) and connective tissue growth factor (CTGF), important antiangiogenic proteins [7]. Similarly, microvesicles from colorectal cancer cells contain miR-1246 and TGF-β which are transferred to endothelial cells to silence promyelocytic leukemia protein (PML) and activate Smad 1/5/8 signaling promoting proliferation and migration [9]. Likewise, lung cancer cell line derived microvesicles contain miR-494, in response to hypoxia, which targets PTEN in the endothelial cells promoting angiogenesis through the Akt/eNOS pathway [10]. Lastly, exosomal miR-135b from multiple myeloma cells suppresses the HIF-1/FIH-1 pathway in endothelial cells, increasing angiogenesis [11]. A summary of the studies showing the functions of exosomal miRNAs in shaping the biology of the TME is provided in Table 1.

 

Table 1

Actions of exosomal miRNAs exchanged between cells of the TME

 

Angiogenesis:

 miRNA

Cell of origin

Accepting cell

Pathway/target

Effect on TME

Ref.

 miR-135b

Multiple myeloma

Endothelial cells

HIF-1/FIH-1

↑angiogenesis

[11]

 miR-494

Lung cancer

Endothelial cells

PTEN/AKT/eNOS

↑angiogenesis

[10]

 miR-503

Endothelial cells

Breast cancer

Cyclin D2 and D3

↓Tumor growth and invasion

[22]

 miR-1246

Colorectal cancer

Endothelial Cells

PML/Smad 1/5/8

↑ Growth & migration

[9]

Stromal compartment:

 miR-105

Breast cancer

Endothelial cells

ZO-1

↓Tight junctions

↑Metastatic progression

[68]

 miR-202-3p

CLL

Stromal cells

c-fos/ATM

↑Tumor growth

[53]

Immune system:

 miR-29a

NSCLC

TAM

TLR8/NF-κB

↑Growth & metastasis

[75]

 miR-21

NSCLC

TAM

TLR8/NF-κB

↑Growth & metastasis

[75]

NBL

TAM

TLR8/NF-κB

↑miR-155

[76]

 miR-155

TAM

NBL

TERF1

↑ Drug resistance

[76]

 miR-23a

Hypoxic tumor derived

NK cells

CD107a

↓ NK cell response

[95]

 miR-210

 miR-214

Tumor cells (various)

Regulatory T cells

PTEN

↑Immunosuppression

[96]

 miR-223

TAM

Breast cancer

Mef2c/β-catenin

↑ Invasion

[82]

Abbreviations: TAMs Tumor Associated Macrophages, CLL chronic lymphocytic leukemia, NSCLCnon-small cell lung cancer, NBL Neuroblastoma

The most common target of anti-angiogenic therapy is VEGF, and not unsurprisingly, multiple miRNAs (including miR-9, miR-20b, miR-130, miR-150, and miR-497) promote angiogenesis through the induction of the VEGF pathway. The most studied of these is the up-regulation of miR-9 which has been linked to a poor prognosis in multiple tumor types, including breast cancer, non-small cell lung cancer, and melanoma [12]. The two oncogenes MYC and MYCN activate miR-9 and cause E-cadherin downregulation resulting in the upregulated transcription of VEGF [13]. In addition, miR-9 has been shown to upregulate the JAK-STAT pathway, supporting endothelial cell migration and tumor angiogenesis [13]. Both amplification of miR-20b and miR-130 as well as miR-497 suppression regulate VEGF through hypoxia inducible factor 1α (HIF-1α) supporting increased angiogenesis [14, 15, 16, 17]. …..

The pivotal discovery in 2012 by Mitra et al. laid the ground-work for our current knowledge on the interactions between tumor-derived miRNAs and fibroblasts. In combination, the down-regulation of miR-214 and miR-31 and the up-regulation of miR-155 trigger the reprogramming of quiescent fibroblasts to CAFs [32]. As expected, the reverse regulation of these miRNAs reduced the migration and invasion of co-cultured ovarian cancer cells [32]. While the pathway of miR-155’s involvement in CAF biology is still being elucidated, the pathways of miR-214 and miR-31 have been established. In endometrial cancer, miR-31 was found to target the homeobox gene SATB2, leading to enhanced tumor cell migration and invasion [33]. MiR-214 similarly has an inverse correlation with its chemokine target, C-C motif Ligand 5 (CCL5) [32]. CCL5 secretion has been associated with enhanced motility, invasion, and metastatic potential through NF-κB-mediated MMP9 activation and through generation and differentiation of myeloid-derived suppressor cells (MDSCs) [34, 35, 36]. Furthermore, miR-210 and miR-133b overexpression and miR-149 suppression have been subsequently found to independently trigger the conversion to CAFs, possibly through paracrine stimulation, and to additionally promote EMT in prostate and gastric cancer, respectively [37, 38,39]. MiR-210 additionally enlists monocytes and encourages angiogenesis [37].   …

Another function of CAFs is the destruction of the ECM and its remodeling with a tumor-supportive composition and structure which includes modulation of specific integrins and metalloproteinases as some of the most studied miRNA targets. The 23 matrix metalloproteinases (MMPs) are critical in the ECM degradation, disruption of the growth signal balance, resistance to apoptosis, establishment of a favorable metastatic niche, and promotion of angiogenesis [54]. As expected, miRNAs have been found to regulate the actions of MMPs, together working to promote cancer cell growth, invasiveness, and metastasis. In HCC, MMP2 and 9 expression is up-regulated by miR-21 via PTEN pathway downregulation. Similarly, in cholangiocarcinoma it was observed that reduced levels of miR-138 induced up-regulation of RhoC, leading to increased levels of the same two MMPs [55, 56]. ….

As has been shown throughout this review, miRNAs have an important and varied effect on human carcinogenesis by shaping the biology of the TME towards a more permissive pro-tumoral phenotype. The complex events leading to such an outcome are currently quite universally defined as the “educational” process of cancer cells on the surrounding TME. While the initial focus was on the direction from the cancer cell to the surrounding TME, increasingly interest is centered on the implications of a more dynamic bidirectional exchange of genetic information. MiRNAs represent only part of the cargo of the extracellular vesicles, but an increasing scientific literature points towards their pivotal role in creating the micro-environmental conditions for cancer cell growth and dissemination. The nearby future will have to address several questions still unanswered. First, it is absolutely necessary to clarify which miRNAs and to what extent they are involved in this process. The contradictory results of some studies can be explained by the differences in tumor-types and by different concentrations of miRNAs used for functional studies. Understanding whether different concentrations of the same miRNA elicit different target effects and therefore changes the biology of the TME, will represent a significant consideration in the development of this field. It is certainly very attractive (especially in an attempt to develop new and desperately needed better cancer biomarkers) to think that concentrations of miRNAs within the TME are reflected systemically in the circulating levels of that same miRNA, however this has not yet been irrefutably demonstrated. Moreover, the study of the paracrine interactions among different cell populations of the TME and their reciprocal effects has been limited to two, maximum three cell populations. This is still way too far from describing the complexity of the TME and only the development of new tridimensional models of the TME will be able to cast a more conclusive light on such complexity. Finally, the pharmacokinetics of miRNA-containing vesicles is in its infancy at best, and needs to be further developed if the goal is development of new therapies based on the use of exosomic miRNAs. Therefore, the future of miRNA research, particularly in its role in the TME, holds still a lot of questions that need answering. However, for these exact same reasons, this is an incredibly exciting time for research in this field. We can envision a not too far future in which these concerns will be satisfactorily addressed and our understanding of the role of miRNAs within the TME will allow us to use them as new therapeutic weapons to successfully improve the clinical outcome of cancer patients.

 

 

 

Triggering the protein that programs cancer cells to kill themselves
http://www.kurzweilai.net/triggering-the-protein-that-programs-cancer-cells-to-kill-themselves

May 24, 2016

https://youtu.be/DR80Huxp4y8
WEHI | Apoptosis

Researchers at the Walter and Eliza Hall Institute in Australia have discovered a new way to trigger cell death that could lead to drugs to treat cancer and autoimmune disease.

Programmed cell death (a.k.a. apoptosis) is a natural process that removes unwanted cells from the body. Failure of apoptosis can allow cancer cells to grow unchecked or immune cells to inappropriately attack the body.

The protein known as Bak is central to apoptosis. In healthy cells, Bak sits in an inert state but when a cell receives a signal to die, Bak transforms into a killer protein that destroys the cell.

Triggering the cancer-apoptosis trigger

Institute researchers Sweta Iyer, PhD, Ruth Kluck, PhD, and colleagues unexpectedly discovered that an antibody they had produced to study Bak actually bound to the Bak protein and triggered its activation. They hope to use this discovery to develop drugs that promote cell death.

The researchers used information about Bak’s three-dimensional structure to find out precisely how the antibody activated Bak. “It is well known that Bak can be activated by a class of proteins called ‘BH3-only proteins’ that bind to a groove on Bak. We were surprised to find that despite our antibody binding to a completely different site on Bak, it could still trigger activation,” Kluck said.  “The advantage of our antibody is that it can’t be ‘mopped up’ and neutralized by pro-survival proteins in the cell, potentially reducing the chance of drug resistance occurring.”

Drugs that target this new activation site could be useful in combination with other therapies that promote cell death by mimicking the BH3-only proteins. The researchers are now working with collaborators to develop their antibody into a drug that can access Bak inside cells.

Their findings have just been published in the open-access journal Nature Communications. The research was supported by the National Health and Medical Research Council, the Australian Research Council, the Victorian State Government Operational Infrastructure Support Scheme, and the Victorian Life Science Computation Initiative.

Abstract of Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.

references:

 

Catching metastatic cancer cells before they grow into tumors: a new implant shows promise

https://62e528761d0685343e1c-f3d1b99a743ffa4142d9d7f1978d9686.ssl.cf2.rackcdn.com/files/122764/width926/image-20160516-15899-18cgw3m.jpg

Cure” is a word that’s dominated the rhetoric in the war on cancer for decades. But it’s a word that medical professionals tend to avoid. While the American Cancer Society reports that cancer treatment has improved markedly over the decades and the five-year survival rate is impressively high for many cancers, oncologists still refrain from declaring their cancer-free patients cured. Why?

Patients are declared cancer-free (also called complete remission) when there are no more signs of detectable disease.

However, minuscule clusters of cancer cells below the detection level can remain in a patient’s body after treatment. Moreover, such small clusters of straggler cells may undergo metastasis, where they escape from the initial tumor into the bloodstream and ultimately settle in a distant site, often a vital organ such as the lungs, liver or brain.

Cancer cells can move throughout the body, like these metastatic melanoma cells. NIH Image Gallery/FlickrCC BY

When a colony of these metastatic cells reaches a detectable size, the patient is diagnosed with recurrent metastatic cancer. About one in three breast cancer patients diagnosed with early-stage cancer later develop metastatic disease, usually within five years of initial remission.

By the time metastatic cancer becomes evident, it is much more difficult to treat than when it was originally diagnosed.

What if these metastatic cells could be detected earlier, before they established a “foothold” in a vital organ? Better yet, could these metastatic cancer cells be intercepted, preventing them them from lodging in a vital organ in the first place?

To catch a cancer cell

With these goals in mind, our biomaterials lab joined forces with surgical oncologist Jacqueline Jeruss to create an implantable medical device that acts as a metastatic cancer cell trap.

The implant is a tiny porous polymer disc (basically a miniature sponge, no larger than a pencil eraser) that can be inserted just under a patient’s skin. Implantation triggers the immune system’s “foreign body response,” and the implant starts to soak up immune cells that travel to it. If the implant can catch mobile immune cells, then why not mobile metastatic cancer cells?

The disc can detect cancer cells in mice. Lab mouse via www.shutterstock.com.

We gave implants to mice specially bred to model metastatic breast cancer. When the mice had palpable tumors but no evidence of metastatic disease, the implant was removed and analyzed.

Cancer cells were indeed present in the implant, while the other organs (potential destinations for metastatic cells) still appeared clean. This means that the implant can be used to spot previously undetectable metastatic cancer before it takes hold in an organ.

For patients with cancer in remission, an implant that can detect tumor cells as they move through the body would be a diagnostic breakthrough. But having to remove it to see if it has captured any cancer cells is not the most convenient or pleasant detection method for human patients.

Detecting cancer cells with noninvasive imaging

There could be a way around this, though: a special imaging method under development at Northwestern University called Inverse Spectroscopic Optical Coherence Tomography (ISOCT). ISOCT detects molecular-level differences in the way cells in the body scatter light. And when we scan our implant with ISOCT, the light scatter pattern looks different when it’s full of normal cells than when cancer cells are present. In fact, the difference is apparent when even as few as 15 out of the hundreds of thousands of cells in the implant are cancer cells.

There’s a catch – ISOCT cannot penetrate deep into tissue. That means it is not a suitable imaging technology for finding metastatic cells buried deep in internal organs. However, when the cancer cell detection implant is located just under the skin, it may be possible to detect cancer cells trapped in it using ISOCT. This could offer an early warning sign that metastatic cells are on the move.

This early warning could prompt doctors to monitor their patients more closely or perform additional tests. Conversely, if no cells are detected in the implant, a patient still in remission could be spared from unneeded tests.

The ISOCT results show that noninvasive imaging of the implant is feasible. But it’s a method still under development, and thus it’s not widely available. To make scanning easier and more accessible, we’re working to adapt more ubiquitous imaging technologies like ultrasound to detect tiny quantities of tumor cells in the implant.

Detect and capture. Joseph Xu, Michigan EngineeringCC BY-NC-ND

Not just detecting, but quarantining cancer

Besides providing a way to detect tiny numbers of cancer cells before they can form new tumors in other parts of the body, our implant offers an even more intriguing possibility: diverting metastatic cells away from vital organs, and sequestering them where they cannot cause any damage.

In our mouse studies, we found that metastatic cells got caught in the implant before they were apparent in vital organs. When metastatic cells eventually made their way into the organs, the mice with implants still had significantly fewer tumor cells in their organs than implant-free controls. Thus, the implant appears to provide a therapeutic benefit, most likely by taking the metastatic cells it catches out of the circulation, preventing them from lodging anywhere vital.

Interestingly, we have not seen cancer cells leave the implant once trapped, or form a secondary tumor in the implant. Ongoing work aims to learn why this is. Whether the cells can stay safely immobilized in the implant or if it would need to be removed periodically will be important questions to answer before the implant could be used in human patients.

What the future may hold

For now, our work aims to make the implant more effective at drawing and detecting cancer cells. Since we tested the implant with metastatic breast cancer cells, we also want to see if it will work on other types of cancer. Additionally, we’re studying the cells the implant traps, and learning how the implant interacts with the body as a whole. This basic research should give us insight into the process of metastasis and how to treat it.

In the future (and it might still be far off), we envision a world where recovering cancer patients can receive a detector implant to stand guard for disease recurrence and prevent it from happening. Perhaps the patient could even scan their implant at home with a smartphone and get treatment early, when the disease burden is low and the available therapies may be more effective. Better yet, perhaps the implant could continually divert all the cancer cells away from vital organs on its own, like Iron Man’s electromagnet that deflects shrapnel from his heart.

This solution is still not a “cure.” But it would transform a formidable disease that one out of three cancer survivors would otherwise ultimately die from into a condition with which they could easily live.

 

New PSA Test Examines Protein Structures to Detect Prostate Cancers

5/16/2016  by Cleveland Clinic

A promising new test is detecting prostate cancer more precisely than current tests, by identifying molecular changes in the prostate specific antigen (PSA) protein, according to Cleveland Clinic research presented today at the American Urological Association annual meeting.

The study – part of an ongoing multicenter prospective clinical trial – found that the IsoPSATM test can also differentiate between high-risk and low-risk disease, as well as benign conditions.

Although widely used, the current PSA test relies on detection strategies that have poor specificity for cancer – just 25 percent of men who have a prostate biopsy due to an elevated PSA level actually have prostate cancer, according to the National Cancer Institute – and an inability to determine the aggressiveness of the disease.

The IsoPSA test, however, identifies prostate cancer in a new way. Developed by Cleveland Clinic, in collaboration with Cleveland Diagnostics, Inc., IsoPSA identifies the molecular structural changes in protein biomarkers. It is able to detect cancer by identifying these structural changes, as opposed to current tests that simply measure the protein’s concentration in a patient’s blood.

“While the PSA test has undoubtedly been one of the most successful biomarkers in history, its limitations are well known. Even currently available prostate cancer diagnostic tests rely on biomarkers that can be affected by physiological factors unrelated to cancer,” said Eric Klein, M.D., chair of Cleveland Clinic’s Glickman Urological & Kidney Institute. “These study results show that using structural changes in PSA protein to detect cancer is more effective and can help prevent unneeded biopsies in low-risk patients.”

The clinical trial involves six healthcare institutions and 132 patients, to date. It examined the ability of IsoPSA to distinguish patients with and without biopsy-confirmed evidence of cancer. It also evaluated the test’s precision in differentiating patients with high-grade (Gleason = 7) cancer from those with low-grade (Gleason = 6) disease and benign findings after standard ultrasound-guided biopsy of the prostate.

Substituting the IsoPSA structure-based composite index for the standard PSA resulted in improvement in diagnostic accuracy. Compared with serum PSA testing, IsoPSA performed better in both sensitivity and specificity.

“We took an ‘out of the box’ approach that has shown success in detecting prostate cancer but also has the potential to address other clinically important questions such as clinical surveillance of patients after treatment,” said Mark Stovsky, M.D., staff member, Cleveland Clinic Glickman Urological & Kidney Institute’s Department of Urology. Stovsky has a leadership position (Chief Medical Officer) and investment interest in Cleveland Diagnostics, Inc. “In general, the clinical utility of prostate cancer early detection and screening tests is often limited by the fact that biomarker concentrations may be affected by physiological processes unrelated to cancer, such as inflammation, as well as the relative lack of specificity of these biomarkers to the cancer phenotype. In contrast, clinical research data suggests that the IsoPSA assay can interrogate the entire PSA isoform distribution as a single stand-alone diagnostic tool which can reliably identify structural changes in the PSA protein that correlate with the presence or absence and aggressiveness of prostate cancer.”

 

Point of Care, Highly Accurate Cervical Cancer Screening

5/20/2016 by Avi Rosenzweig, VP of Business Development, Biop Medical
http://www.mdtmag.com/article/2016/05/point-care-highly-accurate-cervical-cancer-screening

Fifty-five million times a year, American women go to their gynecologist for a Pap Smear. After waiting a few weeks for the results, more than 3.5 million of them are called back to the physician for a follow up visualization of the cervix. Beyond the stress related to possibly having cancer, the women are then subjected to a colposcopic exam, and all too often, a painful biopsy. Then more stressful waiting for a final diagnosis from the pathologist.

Cervical cancer develops slowly, allowing for successful treatment, when identified on time. Regions with high screening compliancy have low mortality rates from this cancer. In the US, for instance, where screening rates are close to 90%, only 4,200 women die from cervical cancer, annually, or 2.6 women per 100,000. However, the screening process in the developed world is long, complicated and not optimized.

In developing regions however, cervical cancer is a leading cause of women death. Over 85% of the total deaths from this cancer are in developing countries. Regions suffering from low screening rates include not only Africa, India and China, but many Eastern European countries as well. According to an OECD report from 2014, the cervical cancer screening rates in Romania and Hungary are as low as 14.6% and 36.7% respectively. The mortality rates in these countries are high, 16 in 100,000 women in Romania and 7.7 in 100,000 in Hungary.

The current screening process for cervical cancer detection is long, beginning with a Pap or HPV test. Cytology results take weeks to receive. A positive result requires follow-up testing by colposcopy and often biopsy. In countries where there is little access to medical care, or where screening compliancy is low, the chances of successful detection via this multi-step process are small. Developing regions and non-compliant countries require a point of care diagnostic method, which eliminates the need for return visits.

Additional limitations to cervical cancer screening are the low sensitivity and specificity rates of Pap tests and the high false positive rates of HPV test, leading to unnecessary colposcopies. Both cytology and colposcopy testing are highly dependent on operator proficiency for accurate diagnosis.

Biop has developed a new technology for the optimization of this process, into one, three minute, painless optical scan. The vaginal probe uses advanced optical, imaging and non-imaging technologies to identify and classify epithelium based cancers and pre-cancerous lesions. The probe is inserted into the vaginal canal, and scans the entire cervix. The resulting images and optical signatures created from the light, and captured by the sensors, are analyzed by the proprietary algorithm. The result is two pictures, on the physician’s screen; a high resolution photograph of the patient’s cervix, immediately next to a hot/cold map indicating a precise classification and location of any diseased lesions.

 

Deep learning applied to drug discovery and repurposing

May 27, 2016  http://www.kurzweilai.net/deep-learning-applied-to-drug-discovery-and-repurposing

Deep neural networks for drug discovery (credit: Insilico Medicine, Inc.)

Scientists from Insilico Medicine, Inc. have trained deep neural networks (DNNs) to predict the potential therapeutic uses of 678 drugs, using gene-expression data obtained from high-throughput experiments on human cell lines from Broad Institute’s LINCS databases and NIH MeSH databases.

The supervised deep-learning drug-discovery engine used the properties of small molecules, transcriptional data, and literature to predict efficacy, toxicity, tissue-specificity, and heterogeneity of response.

“We used LINCS data from Broad Institute to determine the effects on cell lines before and after incubation with compounds, co-author and research scientist Polina Mamoshina explained to KurzweilIAI.

“We used gene expression data of total mRNA from cell lines extracted and measured before incubation with compound X and after incubation with compound X to identify the response on a molecular level. The goal is to understand how gene expression (the transcriptome) will change after drug uptake. It is a differential value, so we need a reference (molecular state before incubation) to compare.”

The research is described in a paper in the upcoming issue of the journal Molecular Pharmaceutics.

Helping pharmas accelerate R&D

Alex Zhavoronkov, PhD, Insilico Medicine CEO, who coordinated the study, said the initial goal of their research was to help pharmaceutical companies significantly accelerate their R&D and increase the number of approved drugs. “In the process we came up with more than 800 strong hypotheses in oncology, cardiovascular, metabolic, and CNS spaces and started basic validation,” he said.

The team measured the “differential signaling pathway activation score for a large number of pathways to reduce the dimensionality of the data while retaining biological relevance.” They then used those scores to train the deep neural networks.*

“This study is a proof of concept that DNNs can be used to annotate drugs using transcriptional response signatures, but we took this concept to the next level,” said Alex Aliper, president of research, Insilico Medicine, Inc., lead author of the study.

Via Pharma.AI, a newly formed subsidiary of Insilico Medicine, “we developed a pipeline for in silico drug discovery — which has the potential to substantially accelerate the preclinical stage for almost any therapeutic — and came up with a broad list of predictions, with multiple in silico validation steps that, if validated in vitro and in vivo, can almost double the number of drugs in clinical practice.”

Despite the commercial orientation of the companies, the authors agreed not to file for intellectual property on these methods and to publish the proof of concept.

Deep-learning age biomarkers

According to Mamoshina, earlier this month, Insilico Medicine scientists published the first deep-learned biomarker of human age — aiming to predict the health status of the patient — in a paper titled “Deep biomarkers of human aging: Application of deep neural networks to biomarker development” by Putin et al, in Aging; and an overview of recent advances in deep learning in a paper titled “Applications of Deep Learning in Biomedicine” by Mamoshina et al., also in Molecular Pharmaceutics.

Insilico Medicine is located in the Emerging Technology Centers at Johns Hopkins University in Baltimore, Maryland, in collaboration with Datalytic Solutions and Mind Research Network.

* In this study, scientists used the perturbation samples of 678 drugs across A549, MCF-7 and PC-3 cell lines from the Library of Integrated Network-Based Cellular Signatures (LINCS) project developed by the National Institutes of Health (NIH) and linked those to 12 therapeutic use categories derived from MeSH (Medical Subject Headings) developed and maintained by the National Library of Medicine (NLM) of the NIH.

To train the DNN, scientists utilized both gene level transcriptomic data and transcriptomic data processed using a pathway activation scoring algorithm, for a pooled dataset of samples perturbed with different concentrations of the drug for 6 and 24 hours. Cross-validation experiments showed that DNNs achieve 54.6% accuracy in correctly predicting one out of 12 therapeutic classes for each drug.

One peculiar finding of this experiment was that a large number of drugs misclassified by the DNNs had dual use, suggesting possible application of DNN confusion matrices in drug repurposing.
FutureTechnologies Media Group | Video presentation Insilico medicine

Abstract of Deep learning applications for predicting pharmacological properties of drugs and drug repurposing using transcriptomic data

Deep learning is rapidly advancing many areas of science and technology with multiple success stories in image, text, voice and video recognition, robotics and autonomous driving. In this paper we demonstrate how deep neural networks (DNN) trained on large transcriptional response data sets can classify various drugs to therapeutic categories solely based on their transcriptional profiles. We used the perturbation samples of 678 drugs across A549, MCF-7 and PC-3 cell lines from the LINCS project and linked those to 12 therapeutic use categories derived from MeSH. To train the DNN, we utilized both gene level transcriptomic data and transcriptomic data processed using a pathway activation scoring algorithm, for a pooled dataset of samples perturbed with different concentrations of the drug for 6 and 24 hours. When applied to normalized gene expression data for “landmark genes,” DNN showed cross-validation mean F1 scores of 0.397, 0.285 and 0.234 on 3-, 5- and 12-category classification problems, respectively. At the pathway level DNN performed best with cross-validation mean F1 scores of 0.701, 0.596 and 0.546 on the same tasks. In both gene and pathway level classification, DNN convincingly outperformed support vector machine (SVM) model on every multiclass classification problem. For the first time we demonstrate a deep learning neural net trained on transcriptomic data to recognize pharmacological properties of multiple drugs across different biological systems and conditions. We also propose using deep neural net confusion matrices for drug repositioning. This work is a proof of principle for applying deep learning to drug discovery and development.

references:

 

Transistor-based biosensor detects molecules linked to cancer, Alzheimer’s, and Parkinson’s

May 23, 2016  http://www.kurzweilai.net/transistor-based-biosensor-detects-molecules-linked-to-cancer-alzheimers-and-parkinsons

An inexpensive portable biosensor developed by researchers at Brazil’s National Nanotechnology Laboratory (credit: LNNano)  http://www.kurzweilai.net/images/Biosensor-LNNano.jpg

A novel nanoscale organic transistor-based biosensor that can detect molecules associated with neurodegenerative diseases and some types of cancer has been developed by researchers at the National Nanotechnology Laboratory (LNNano) in Brazil.

The transistor, mounted on a glass slide, contains the reduced form of the peptide glutathione (GSH), which reacts in a specific way when it comes into contact with the enzyme glutathione S-transferase (GST), linked to Parkinson’s, Alzheimer’s and breast cancer, among other diseases.

http://www.kurzweilai.net/images/CuPc-transistor.png

Sensitive water-gated copper phthalocyanine (CuPc) thin-film transistor (credit: Rafael Furlan de Oliveira et al./Organic Electronics)

“The device can detect such molecules even when they’re present at very low levels in the examined material, thanks to its nanometric sensitivity,” explained Carlos Cesar Bof Bufon, Head of LNNano’s Functional Devices & Systems Lab (DSF).

Bufon said the system can be adapted to detect other substances by replacing the analytes (detection compounds). The team is working on paper-based biosensors to further lower the cost, improve portability, and facilitate fabrication and disposal.

The research is published in the journal Organic Electronics.

Abstract of Water-gated phthalocyanine transistors: Operation and transduction of the peptide–enzyme interaction

The use of aqueous solutions as the gate medium is an attractive strategy to obtain high charge carrier density (1012 cm−2) and low operational voltages (<1 V) in organic transistors. Additionally, it provides a simple and favorable architecture to couple both ionic and electronic domains in a single device, which is crucial for the development of novel technologies in bioelectronics. Here, we demonstrate the operation of transistors containing copper phthalocyanine (CuPc) thin-films gated with water and discuss the charge dynamics at the CuPc/water interface. Without the need for complex multilayer patterning, or the use of surface treatments, water-gated CuPc transistors exhibited low threshold (100 ± 20 mV) and working voltages (<1 V) compared to conventional CuPc transistors, along with similar charge carrier mobilities (1.2 ± 0.2) x 10−3 cm2 V−1 s−1. Several device characteristics such as moderate switching speeds and hysteresis, associated with high capacitances at low frequencies upon bias application (3.4–12 μF cm−2), indicate the occurrence of interfacial ion doping. Finally, water-gated CuPc OTFTs were employed in the transduction of the biospecific interaction between tripeptide reduced glutathione (GSH) and glutathione S-transferase (GST) enzyme, taking advantage of the device sensitivity and multiparametricity.

references:

 

First Large-Scale Proteogenomic Study of Breast Cancer    

Tues, May 31, 2016     http://www.technologynetworks.com/rnai/news.aspx?ID=191934

The study offers understanding of potential therapeutic targets.

Building on data from The Cancer Genome Atlas (TCGA) project, a multi-institutional team of scientists have completed the first large-scale “proteogenomic” study of breast cancer, linking DNA mutations to protein signaling and helping pinpoint the genes that drive cancer. Conducted by members of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), including Baylor College of Medicine, Broad Institute of MIT and Harvard, Fred Hutchinson Cancer Research Center, New York University Langone Medical Center, and Washington University School of Medicine, the study takes aim at proteins, the workhorses of the cell, and their modifications to better understand cancer.

Appearing in the Advance Online Publication of Nature, the study illustrates the power of integrating genomic and proteomic data to yield a more complete picture of cancer biology than either analysis could do alone. The effort produced a broad overview of the landscape of the proteome (all the proteins found in a cell) and the phosphoproteome (the sites at which proteins are tagged by phosphorylation, a chemical modification that drives communication in the cell) across a set of 77 breast cancer tumors that had been genomically characterized in the TCGA project. Although the TCGA produced an extensive catalog of somatic mutations found in cancer, the effects of many of those mutations on cellular functions or patients’ outcomes are unknown.

In addition, not all mutated genes are true “drivers” of cancer — some are merely “passenger” mutations that have little functional consequence. And some mutations are found within very large DNA regions that are deleted or present in extra copies, so winnowing the list of candidate genes by studying the activity of their protein products can help identify therapeutic targets. “We don’t fully understand how complex cancer genomes translate into the driving biology that causes relapse and mortality,” said Matthew Ellis, director of the Lester and Sue Smith Breast Center at Baylor College of Medicine and a senior author of the paper.

“These findings show that proteogenomic integration could one day prove to be a powerful clinical tool, allowing us to traverse the large knowledge gap between cancer genomics and clinical action.” In this study, the researchers at the Broad Institute analyzed breast tumors using accurate mass, high-resolution mass spectrometry, a technology that extends the coverage of the proteome far beyond the coverage that can be achieved by traditional antibody-based methods. This allowed them to scale their efforts and quantify more than 12,000 proteins and 33,000 phosphosites, an extremely deep level of coverage.

 

Breakthrough Approach to Breast Cancer Treatment

May 24, 2016    http://www.technologynetworks.com/rnai/news.aspx?ID=191771

Scripps scientists have designed a drug candidate that decreases growth of breast cancer cells.

In a development that could lead to a new generation of drugs to precisely treat a range of diseases, scientists from the Florida campus of The Scripps Research Institute (TSRI) have for the first time designed a drug candidate that decreases the growth of tumor cells in animal models in one of the hardest to treat cancers—triple negative breast cancer.

“This is the first example of taking a genetic sequence and designing a drug candidate that works effectively in an animal model against triple negative breast cancer,” said TSRI Professor Matthew Disney. “The study represents a clear breakthrough in precision medicine, as this molecule only kills the cancer cells that express the cancer-causing gene—not healthy cells. These studies may transform the way the lead drugs are identified—by using the genetic makeup of a disease.”

The study, published by the journal Proceedings of the National Academy of Sciences, demonstrates that the Disney lab’s compound, known as Targaprimir-96, triggers breast cancer cells to kill themselves via programmed cell death by precisely targeting a specific RNA that ignites the cancer.

Short-Cut to Drug Candidates

While the goal of precision medicine is to identify drugs that selectively affect disease-causing biomolecules, the process has typically involved time-consuming and expensive high-throughput screens to test millions of potential drug candidates to identify those few that affect the target of interest. Disney’s approach eliminates these screens.

The new study uses the lab’s computational approach called Inforna, which focuses on developing designer compounds that bind to RNA folds, particularly microRNAs.

MicroRNAs are short molecules that work within all animal and plant cells, typically functioning as a “dimmer switch” for one or more genes, binding to the transcripts of those genes and preventing protein production. Some microRNAs have been associated with diseases. For example, microRNA-96, which was the target of the new study, promotes cancer by discouraging programmed cell death, which can rid the body of cells that grow out of control.

In the new study, the drug candidate was tested in animal models over a 21-day course of treatment. Results showed decreased production of microRNA-96 and increased programmed cell death, significantly reducing tumor growth. Since targaprimir-96 was highly selective in its targeting, healthy cells were unaffected.

In contrast, Disney noted, a typical cancer therapeutic targets and kills cells indiscriminately, often leading to side effects that can make these drugs difficult for patients to tolerate.

Benjamin Zealley and Aubrey D.N.J. de Grey
Commentary on Some Recent Theses Relevant to Combating Aging: June 2015

REJUVENATION RESEARCH 2015; 18(3), 282 – 287   http://dx.doi.org:/10.1089/rej.2015.1728

Cancer Autoantibody Biomarker Discovery and Validation Using Nucleic Acid Programmable Protein Array
Jie Wang, PhD, Arizona State University

Currently in the United States, many patients with cancer do not benefit from population-based screening due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and are diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies are a promising class of molecules that have the potential to serve as early detection biomarkers for cancers. This PhD thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancers, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformations on a protein array. This method adopted a novel protein tag technology, called a HaloTag, to immobilize proteins covalently on the surface of a glass slide. The covalent attachment allowed these proteins to endure harsh treatment without becoming dissociated from the slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to increase significantly the signal-to-noise ratio of protein array–based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases, 145 controls, 70 non-basal breast cancer) by enzyme-linked immunosorbent assay (ELISA), a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. A similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed tomography–positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA levels in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights into the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins.

Comment: There are two widely supported models for cancer development and progression—the clonal evolution (CE) model and the cancer stem cell (CSC) model. Briefly, the former claims that most or all cells in a tumor contribute to its maintenance; as newer and more aggressive clones develop by random mutation, they become responsible for driving growth. The range of different mutational profiles generated is assumed to be large enough to account for disease recurrence after therapy (due to rare resistant clones) and metastasis (clones arising with the ability to travel to distant sites). The CSC model instead asserts that a small number of mutated stem cells are the origin of the primary cell mass, drive metastasis through the intermittent release of undifferentiated, highly mobile progeny, and account for recurrence due to a generally quiescent metabolic profile conferring potent resistance to chemotherapy. In either case, the immunological visibility of an early tumor may be highly sporadic. Clones arising early in CE differ little in proteomic terms from healthy host cells; those that do trigger a response are unlikely to have acquired robust resistance to immune attack, so are destroyed quickly in favor of their stealthier brethren. Likewise, CSCs share some of the immune privilege of normal stem cells and, due to their inherent ability to produce differentiated progeny with distinct proteomic signatures, are partially protected from attacks on their descendants. Consequently, such well-hidden cells may remain in the body for years to decades. The autoantibody panel developed in this study for basal-like breast cancer exhibits exceptional specificity despite a comparatively small training set. Given its ease of application, this suggests great promise for a more exhaustively trained classifier as a populationlevel screening tool.

 

Condition-Specific Differential Sub-Network Analysis for Biological Systems
Deepali Jhamb, PhD, Indiana University

Biological systems behave differently under different conditions. Advances in sequencing technology over the last decade have led to the generation of enormous amounts of condition-specific data. However, these measurements often fail to identify low-abundance genes and proteins that can be biologically crucial. In this work, a novel textmining system was first developed to extract condition-specific proteins from the biomedical literature. The literaturederived data was then combined with proteomics data to construct condition-specific protein interaction networks. Furthermore, an innovative condition-specific differential analysis approach was designed to identify key differences, in the form of sub-networks, between any two given biological systems. The framework developed here was implemented to understand the differences between limb regenerationcompetent Ambystoma mexicanum and regeneration-deficient Xenopus laevis. This study provides an exhaustive systems-level analysis to compare regeneration competent and deficient sub-networks to show how different molecular entities inter-connect with each other and are rewired during the formation of an accumulation blastema in regenerating axolotl limbs. This study also demonstrates the importance of literature-derived knowledge, specific to limb regeneration, to augment the systems biology analysis. Our findings show that although the proteins might be common between the two given biological conditions, they can have a high dissimilarity based on their biological and topological properties in the sub-network. The knowledge gained from the distinguishing features of limb regeneration in amphibians can be used in future to induce regeneration chemically in mammalian systems. The approach developed in this dissertation is scalable and adaptable to understanding differential sub-networks between any two biological systems. This methodology will not only facilitate the understanding of biological processes and molecular functions that govern a given system, but will also provide novel intuitions about the pathophysiology of diseases/conditions.

Comment: We have long advocated a principle of directly comparing young and old bodies as a means to identify the classes of physical damage that accumulate in the body during aging. This approach circumvents our ignorance of the full etiology of each particular disease manifestation, a phenomenally difficult question given the ethical issues of experimenting on human subjects, the lengthy ‘‘incubation time’’ of aging-related diseases, and the complex interconnections between their risk factors—innate and environmental. Repairing such damage has the potential to prevent pathology before symptoms appear, an approach now becoming increasingly mainstream.11 However, a naı¨ve comparison faces a number of difficulties, even given a sufficiently large sample set to compensate for inter-individual variation. Most importantly, the causal significance of a given species cannot be reliably determined from its simple prevalence.12 The catalytic nature of cell biology means that those entities whose abundance changes the most profoundly in absolute terms are quite unlikely to be the drivers of that change and may even spontaneously revert to baseline levels in the absence of on-going stimulation. Meanwhile, functionality is often heavily influenced independently of abundance by post-translational modifications that may escape direct detection. Sub-network analysis uses computational means to identify groups of genes and/or proteins that vary in a synchronized way with some parameter, indicating functional connectivity. The application of methods such as those developed here to the comparison of a wide range of younger and older conditions will facilitate the identification of processes—not merely individual factors—that are impaired with age, and thus will help greatly in identifying the optimal points for intervention.

 

Development of a Light Actuated Drug Delivery-on-Demand System
Chase Linsley, PhD, University of California, Los Angeles

The need for temporal–spatial control over the release of biologically active molecules has motivated efforts to engineer novel drug delivery-on-demand strategies actuated via light irradiation. Many systems, however, have been limited to in vitro proof-of-concept due to biocompatibility issues with the photo-responsive moieties or the light wavelength, intensity, and duration. To overcome these limitations, the objective of this dissertation was to design a light-actuated drug delivery-on-demand strategy that uses biocompatible chromophores and safe wavelengths of light, thereby advancing the clinical prospects of light-actuated drug delivery-on-demand systems. This was achieved by: (1) Characterizing the photothermal response of biocompatible visible light and near-infrared-responsive chromophores and demonstrating the feasibility and functionality of the light actuated on-demand drug delivery system in vitro; and (2) designing a modular drug delivery-on-demand system that could control the release of biologically active molecules over an extended period of time. Three biocompatible chromophores—Cardiogreen, Methylene Blue, and riboflavin—were identified and demonstrated significant photothermal response upon exposure to near-infrared and visible light, and the amount of temperature change was dependent upon light intensity, wavelength, as well as chromophore concentration. As a proof-of-concept, pulsatile release of a model protein from a thermally responsive delivery vehicle fabricated from poly(N-isopropylacrylamide) was achieved over 4 days by loading the delivery vehicle with Cardiogreen and irradiating with near-infrared light. To extend the useful lifetime of the light-actuated drug delivery-on-demand system, a modular, reservoir-valve system was designed. Using poly(ethylene glycol) as a reservoir for model small molecule drugs combined with a poly(N-isopropylacrylamide) valve spiked with chromophore-loaded liposomes, pulsatile release was achieved over 7 days upon light irradiation. Ultimately, this drug delivery strategy has potential for clinical applications that require explicit control over the presentation of biologically active molecules. Further research into the design and fabrication of novel biocompatible thermally responsive delivery vehicles will aid in the advancement of the light-actuated drug delivery-on-demand strategy described here. Comment: Our combined comments on this thesis and the next one appear after the next abstract.

 

Light-Inducible Gene Regulation in Mammalian Cells
Lauren Toth, PhD, Duke University

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue-engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible. The recent emergence of the field of optogenetics—the ability to control gene expression using light—has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific 3-bp DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or enhanced green fluorescent protein (eGFP) transgene enabled expression of the transgene in response to blue light. Gene activation was both reversible and tunable on the basis of duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be patterned spatially by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore, we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a lightactivated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the amino-terminal fragment of CIB1 to the amino, carboxyl, or amino and carboxyl terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic ‘‘on’’ switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase–mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system, could be used to express MyoD or the synthetic transcription factor VP64- MyoD in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated up-regulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T1/2 cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased micro-vessel density compared to mice that were not illuminated. Analysis of human vascular endothelial growth factor (VEGF) and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

Comment: Although it is easy to characterize technological progress as following in the wake of scientific discoveries, the reverse is almost equally true; advances in technique open the door to types of experiment previously intractable or impossible. Such is currently the case for the field of optically controlled biotechnology, which has exploded into prominence, particularly over the last half-decade. Light of an appropriate wavelength can penetrate mammalian tissues to a depth of up to a couple of centimeters, rendering much of the living body accessible to optical study and control—still more if the detector/source is integrated into an endoscopic or fiber optic probe. Techniques borrowed from the semiconductor industry allow patterns of illumination to be controlled down to the nanometer scale, ideal for addressing individual cells. The highly controlled time course of such experiments, as compared to traditional means of gene activation, such as the addition of a chemical agent to the medium, eliminates confounding variables, and simplifies data analysis. Furthermore, this level of immediate control opens the door to closed-loop systems where the activity of entities under optical control can be continuously tuned in relation to some parameter(s). In the first of these two illuminating theses, a vehicle is developed that permits light-driven release of a small molecule. Such a system could be employed to target a systemically administered antibiotic or anti-neoplastic agent to a site of infection or cancer while sparing other bodily tissues from toxicity. Because most modern drugs cannot be produced in the body, even given arbitrarily good control of cellular biochemistry, this technique will have lasting value in numerous clinical contexts. In the second thesis, the level of precision achieved is even more profound; the CRISPR/Cas9 system has received much recent attention13 in its own right for its capacity to target arbitrary genetic sequences without an arduous protein-engineering step. The LACE system described stands to permit genetic manipulation with almost arbitrarily good spatial, temporal, and genomic site-specific control, using only means available to a typical university laboratory.

 

Targeting T Cells for the Immune-Modulation of Human Diseases
Regina Lin, PhD, Duke University

Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers, and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity, and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell–mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and pre-clinically characterizing their mechanisms of action and in vivo therapeutic efficacy.

CD8+ cytotoxic T cells have potent anti-tumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo– expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA (miRNA)-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR- 23a was up-regulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired anti-tumor potential of patient CD8+ T cells. We determined that tumor-derived transforming growth factor-b (TGF-b) directly suppresses CD8+ T cell immune function by elevating miR-23a and down-regulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-bmediated tumor immune-evasion pathway

Having established that miR-23a-inhibition can enhance the quality and functional resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers—such as TGF-b—within the tumor microenvironment. Because TGF-b poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wild-type and miR-23a–deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumorspecific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wild-type EGFRvIIICAR T cells were vulnerable to functional suppression by TGF-b, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF-b. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23adeficient EGFRvIII-CAR T cells for GBM immunotherapy.

Last, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immunesuppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro. Subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory interferon-c (IFN-c) and interleukin-17 (IL-17) production by fully differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.

Comment: Immunotherapy is among the most promising approaches to cancer treatment, having the specificity and scope to selectively target transformed cells wherever they may reside within the body and the potential to install a permanent defense against disease recurrence. By the time a typical cancer is clinically diagnosed, however, it has already found means to survive a prolonged period of potential immune attack. The mechanisms by which tumors evade immune surveillance are beginning to be elucidated,15,16 and include both direct suppression of effector cells and progressive editing of the host’s immune repertoire to disfavor future attack. It is inherently difficult to interfere with these defenses directly, due to the selection pressures in genetically heterogeneous neoplastic tissue. Much effort is thus being focused on methods for rendering therapeutically delivered immune cells resistant to their effects. The cytokine TGF-b is paradoxically known to function as both a tumor suppressor in healthy tissue and as a tumorderived species associated with multiple cancer-promoting activities, including enhanced immune evasion. This work identifies the pathway by which TGF-b compromises cytotoxic T cell function in the tumor microenvironment, and demonstrates an effective method for blocking this signal. In many clinical cases, however, editing of the patient’s immune repertoire has already removed or rendered anergic those immune cells able to recognize their cancer. Thus, the finding that blocking TGF-b signaling also appears to enhance the effectiveness of CAR-modified T cells— engineered with an antibody fragment targeting them with high affinity to a particular tumor-associated epitope—is a welcome addition to these already promising results.

 

Novel Fibonacci and non-Fibonacci structure in the sunflower: results of a citizen science experiment

Jonathan Swinton, Erinma Ochu, The MSI Turing’s Sunflower Consortium

Published 18 May 2016. DOI http://dx.doi.org:/10.1098/rsos.160091

This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower (Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.

  1. Introduction

Fibonacci structure can be found in hundreds of different species of plants [1]. This has led to a variety of competing conceptual and mathematical models that have been developed to explain this phenomenon. It is not the purpose of this paper to survey these: reviews can be found in [14], with more recent work including [510]. Instead, we focus on providing empirical data useful for differentiating them.

These models are in some ways now very mathematically satisfying in that they can explain high Fibonacci numbers based on a small number of plausible assumptions, though they are not so satisfying to experimental scientists [11]. Despite an increasingly detailed molecular and biophysical understanding of plant organ positioning [1214], the very parsimony and generality of the mathematical explanations make the generation and testing of experimental hypotheses difficult. There remains debate about the appropriate choice of mathematical models, and whether they need to be central to our understanding of the molecular developmental biology of the plant. While sunflowers provide easily the largest Fibonacci numbers in phyllotaxis, and thus, one might expect, some of the stronger constraints on any theory, there is a surprising lack of systematic data to support the debate. There have been only two large empirical studies of spirals in the capitulum, or head, of the sunflower: Weisse [15] and Schoute [16], which together counted 459 heads; Schoute found numbers from the main Fibonacci sequence 82% of the time and Weise 95%. The original motivation of this study was to add a third replication to these two historical studies of a widely discussed phenomenon. Much more recently, a study of a smaller sample of 21 seedheads was carried out by Couder [17], who specifically searched for non-Fibonacci examples, whereas Ryan et al. [18] studied the arrangement of seeds more closely in a small sample of Helianthus annuus and a sample of 33 of the related perennial H. tuberosus.

Neither the occurrence of Fibonacci structure nor the developmental biology leading to it are at all unique to sunflowers. As common in other species, the previous sunflower studies found not only Fibonacci numbers, but also the occasional occurrence of the double Fibonacci numbers, Lucas numbers and F4 numbers defined below [1]. It is worth pointing out the warning of Cooke [19] that numbers from these sequences make up all but three of the first 17 integers. This means that it is particularly valuable to look at specimens with large parastichy numbers, such as the sunflowers, where the prevalence of Fibonacci structure is at its most striking.

Neither Schoute nor Weisse reported their precise technique for assigning parastichy numbers to their samples, and it is noteworthy that neither author reported any observation of non-Fibonacci structure. One of the objectives of this study was to rigorously define Fibonacci structure in advance and to ensure that the assignment method, though inevitably subjective, was carefully documented.

This paper concentrates on the patterning of seeds towards the outer rim of sunflower seedheads. The number of ray florets (the ‘petals’, typically bright yellow) or the green bracts behind them tends to have a looser distribution around a Fibonacci number. In the only mass survey of these, Majumder & Chakravarti [20] counted ray florets on 1002 sunflower heads and found a distribution centred on 21.

This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower (Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.

Incorporation of irregularity into the mathematical models of phyllotaxis is relatively recent: [17] gave an example of a disordered pattern arising directly from the deterministic model while more recently the authors have begun to consider the effects of stochasticity [10,21]. Differentiating between these models will require data that go beyond capturing the relative prevalence of different types of Fibonacci structure, so this study was also designed to yield the first large-scale sample of disorder in the head of the sunflower.

The Fibonacci sequence is the sequence of integers 1,2,3,5,8,13,21,34,55,89,144… in which each member after the second is the sum of the two preceding. The Lucas sequence is the sequence of integers 1,3,4,7,11,18,29,47,76,123… obeying the same rule but with a different starting condition; the F4 sequence is similarly 1,4,5,9,14,23,37,60,97,…. The double Fibonacci sequence 2,4,6,10,16,26,42,68,110,… is double the Fibonacci sequence. We say that a parastichy number which is any of these numbers has Fibonacci structure. The sequencesF5=1,5,6,11,17,28,45,73,… and F8=1,8,9,17,26,43,69,112… also arise from the same rule, but as they had not been previously observed in sunflowers we did not include these in the pre-planned definition of Fibonacci structure for parsimony. One example of adjacent pairs from each of these sequences was, in fact, observed but both examples are classified as non-Fibonacci below. A parastichy number which is any of 12,20,33,54,88,143 is also not classed as having Fibonacci structure but is distinguished as a Fibonacci number minus one in some of the analyses, and similarly 14,22,35,56,90,145 as Fibonacci plus one.

When looking at a seedhead such as in figure 1 the eye naturally picks out at least one family of parastichies or spirals: in this case, there is a clockwise family highlighted in blue in the image on the right-hand side.

http://d3hu9binmobce5.cloudfront.net/content/royopensci/3/5/160091/F1.medium.gif

Distribution and type of parastichy pairs

Figure 5 plots the individual pairs observed. On the reference line, the ratio of the numbers is equal to the golden ratio so departures from the line mark departures from Fibonacci structure, which are less evident in the more reliable photoreviewed dataset. It can be seen from table 3 that Fibonacci pairings dominate the dataset.

 

http://d3hu9binmobce5.cloudfront.net/content/royopensci/3/5/160091/F5.medium.gif

Table 3.

Observed pairings of Fibonacci types of clockwise and anticlockwise parastichy numbers. Other means any parastichy number which neither has Fibonacci structure nor is Fibonacci ±1. Of all the Fibonacci ±1/Fibonacci pairs, only sample 191, a (21,20) pair, was not close to an adjacent Fibonacci pair.

One typical example of a Fibonacci pair is shown in figure 6, with a double Fibonacci case infigure 1 and a Lucas one in figure 7. There was no photoreviewed example of an F4 pairing. The sole photoreviewed assignment of a parastichy number to the F4 sequence was the anticlockwise parastichy number 37 in sample 570, which was relatively disordered. The clockwise parastichy number was 55, lending support to the idea this may have been a perturbation of a (34,55) pattern. We also found adjacent members of higher-order Fibonacci series. Figures 8 and 9 each show well-ordered examples with parastichy counts found adjacent in the F5 and F8 series, respectively: neither of these have been previously reported in the sunflower.

Figure 6.

 

http://d3hu9binmobce5.cloudfront.net/content/royopensci/3/5/160091/F6.medium.gif

Sunflower 095. An (89,55) example with 89 clockwise parastichies and 55 anticlockwise ones, extending right to the rim of the head. Because these are clear and unambiguous, the other parastichy families which are visible towards the centre are not counted here.

Figure 7.   Sunflower 171. A Lucas series (76,47) example.

Sunflower 667. Anticlockwise parastichies only, showing competing parastichy families which are distinct but in some places overlapping.

Our core results are twofold. First, and unsurprisingly, Fibonacci numbers, and Fibonacci structure more generally, are commonly found in the patterns in the seedheads of sunflowers. Given the extent to which Fibonacci patterns have attracted pseudo-scientific attention [33], this substantial replication of limited previous studies needs no apology. We have also published, for the first time, examples of seedheads related to the F5 and F8 sequences but by themselves they do not add much to the evidence base. Our second core result, though, is a systematic survey of cases where Fibonacci structure, defined strictly or loosely, did not appear. Although not common, such cases do exist and should shed light on the underlying developmental mechanisms. This paper does not attempt to shed that light, but we highlight the observations that any convincing model should explain. First, the prevalence of Lucas numbers is higher than those of double Fibonacci numbers in all three large datasets in the literature, including ours, and there are sporadic appearances of F4, F5 and F8 sequences. Second, counts near to but not exactly equal to Fibonacci structure are also observable: we saw a parastichy count of 54 more often than the most common Lucas count of 47. Sometimes, ambiguity arises in the counting process as to whether an exact Fibonacci-structured number might be obtained instead, but there are sufficiently many unambiguous cases to be confident this is a genuine phenomenon. Third, among these approximately Fibonacci counts, those which are a Fibonacci number minus one are significantly more likely to be seen than a Fibonacci number plus one. Fourth, it is not uncommon for the parastichy families in a seedhead to have strong departures from rotational symmetry: this can have the effect of yielding parastichy numbers which have large departures from Fibonacci structure or which are completely uncountable. This is related to the appearance of competing parastichy families. Fifth, it is common for the parastichy count in one direction to be more orderly and less ambiguous than that in the other. Sixth, seedheads sometimes possess completely disordered regions which make the assignment of parastichy numbers impossible. Some of these observations are unsurprising, some can be challenged by different counting protocols, and some are likely to be easily explained by the mathematical properties of deformed lattices, but taken together they pose a challenge for further research.

It is in the nature of this crowd-sourced experiment with multiple data sources that it is much easier to show variability than it is to find correlates of that variability. We tried a number of cofactor analyses that found no significant effect of geography, growing conditions or seed type but if they do influence Fibonacci structure, they are likely to be much easier to detect in a single-experimenter setting.

We have been forced by our results to extend classifications of seedhead patterns beyond structured Fibonacci to approximate Fibonacci ones. Clearly, the more loose the definition of approximate Fibonacci, the easier it is to explain away departures from model predictions. Couder [17] found one case of a (54,87) pair that he interpreted as a triple Lucas pair 3×(18,29). While mathematically true, in the light of our data, it might be more compellingly be thought of as close to a (55,89) ideal than an exact triple Lucas one. Taken together, this need to accommodate non-exact patterns, the dominance of one less over one more than Fibonacci numbers, and the observation of overlapping parastichy families suggest that models that explicitly represent noisy developmental processes may be both necessary and testable for a full understanding of this fascinating phenomenon. In conclusion, this paper provides a testbed against which a new generation of mathematical models can and should be built.

 

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Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

 

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

https://pharmaceuticalintelligence.com/2016/04/24/new-class-of-immune-system-stimulants-cyclic-di-nucleotides-cdn-shrink-tumors-and-bolster-vaccines-re-arm-the-immune-systems-natural-killer-cells-which-attack-cancer-cells-and-virus-inf/

A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

 

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

 

Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu1,2, R B Parmigiani1,2 and P A Marks1

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list).   Full table

http://www.nature.com/common/images/table_thumb.gif

In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD+ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005Chavez-Blanco et al., 2005Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials.  Full table

http://www.nature.com/common/images/table_thumb.gif

Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005Marks and Dokmanovic, 2005;Rosato and Grant, 2005Bolden et al., 2006Minucci and Pelicci, 2006Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1.  Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

http://www.nature.com/onc/journal/v26/n37/images/1210620f1.jpg

HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003Yoshida et al., 2003Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC50, at 0.3 m, compared to HDAC3 with an IC50 of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

  • HDACi induces cell cycle arrest
  • HDACi activates the extrinsic apoptotic pathways
  • HDACi activates the intrinsic apoptotic pathways
  • HDACi induces mitotic cell death
  • HDACi induces autophagic cell death and senescence
  • ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
  • Antitumor effects of HDAC6 inhibition
  • Activation of protein phosphatase 1
  • Disruption of the function of chaperonin HSP90
  • Disruption of the aggresome pathway
  • HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.

 

Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004Bhalla, 2005Marks and Dokmanovic, 2005Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)

RESEARCH   

Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:

 

The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.

 

The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology.  Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.

 

The work that has been described covers only cyclic dinucleotides and HDACi’s.  Some of the activities described have relevance to microorganisms as well as cancer.  As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.

 

The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

 

The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004).  The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.

 

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi.

 

 

 

 

 

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Effect of mitochondrial stress on epigenetic modifiers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Early Mitochondrial Stress Alters Epigenetics, Secures Lifelong Health Benefits

GEN 5/3/2016  http://www.genengnews.com/gen-news-highlights/early-mitochondrial-stress-alters-epigenetics-secures-lifelong-health-benefits/81252685/

A little adversity builds character, or so the saying goes. True or not, the saying does seem an apt description of a developmental phenomenon that shapes gene expression. While it knows nothing of character, the gene expression apparatus appears to respond well to short-term mitochondrial stress that occurs early in development. In fact, transient stress seems to result in lasting benefits. These benefits, which include improved metabolic function and increased longevity, have been observed in both worms and mice, and may even occur—or be made to occur—in humans.

Gene expression is known to be subject to reprogramming by epigenetic modifiers, but such modifiers generally affect metabolism or lifespan, not both. A new set of epigenetic modifiers, however, has been found to trigger changes that do just that—both improve metabolism and extend lifespan.

Scientists based at the University of California, Berkeley, and the École Polytechnique Fédérale de Lausanne (EPFL) have discovered enzymes that are ramped up after mild stress during early development and continue to affect the expression of genes throughout the animal’s life. When the scientists looked at strains of inbred mice that have radically different lifespans, those with the longest lifespans had significantly higher expression of these enzymes than did the short-lived mice.

“Two of the enzymes we discovered are highly, highly correlated with lifespan; it is the biggest genetic correlation that has ever been found for lifespan in mice, and they’re both naturally occurring variants,” said Andrew Dillin, a UC Berkeley professor of molecular and cell biology. “Based on what we see in worms, boosting these enzymes could reprogram your metabolism to create better health, with a possible side effect of altering lifespan.”

Details of the work, which appeared online April 29 in the journal Cell, are presented in a pair of papers. One paper (“Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity”) resulted from an effort led by Dillin and the EPFL’s Johan Auwerx. The other paper (“Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt”) resulted from an effort led by Dillin and his UC Berkeley colleague Barbara Meyer.

According to these papers, mitochondrial stress activates enzymes in the brain that affect DNA folding, exposing a segment of DNA that contains the 1500 genes involved in the work of the mitochondria. A second set of enzymes then tags these genes, affecting their activation for much or all of the lifetime of the animal and causing permanent changes in how the mitochondria generates energy.

The first set of enzymes—methylases, in particular LIN-65—add methyl groups to the DNA, which can silence promoters and thus suppress gene expression. By also opening up the mitochondrial genes, these methylases set the stage for the second set of enzymes—demethylases, in this case jmjd-1.2 and jmjd-3.1—to ramp up transcription of the mitochondrial genes. When the researchers artificially increased production of the demethylases in worms, all the worms lived longer, a result identical to what is observed after mitochondrial stress.

“By changing the epigenetic state, these enzymes are able to switch genes on and off,” Dillin noted. This happens only in the brain of the worm, however, in areas that sense hunger or satiety. “These genes are expressed in neurons that are sensing the nutritional status of the animal, and these signals emanate out to the periphery to change peripheral metabolism,” he continued.

When the scientists profiled enzymes in short- and long-lived mice, they found upregulation of these genes in the brains of long-lived mice, but not in other tissues or in the brains of short-lived mice. “These genes are expressed in the hypothalamus, exactly where, when you eat, the signals are generated that tell you that you are full. And when you are hungry, signals in that region tell you to go and eat,” Dillin explained said. “These genes are all involved in peripheral feedback.”

Among the mitochondrial genes activated by these enzymes are those involved in the body’s response to proteins that unfold, which is a sign of stress. Increased activity of the proteins that refold other proteins is another hallmark of longer life.

These observations suggest that the reversal of aging by epigenetic enzymes could also take place in humans.

“It seems that, while extreme metabolic stress can lead to problems later in life, mild stress early in development says to the body, ‘Whoa, things are a little bit off-kilter here, let’s try to repair this and make it better.’ These epigenetic switches keep this up for the rest of the animal’s life,” Dillin stated.

 

Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity

Carsten Merkwirth6, Virginija Jovaisaite6, Jenni Durieux,…., Reuben J. Shaw, Johan Auwerx, Andrew Dillin

Highlights
  • H3K27 demethylases jmjd-1.2 and jmjd-3.1 are required for ETC-mediated longevity
  • jmjd-1.2 and jmjd-3.1 extend lifespan and are sufficient for UPRmt activation
  • UPRmt is required for increased lifespan due to jmjd-1.2 or jmjd-3.1 overexpression
  • JMJD expression is correlated with UPRmt and murine lifespan in inbred BXD lines

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPRmt), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPRmt signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPRmt induction, while gain of function is sufficient to extend lifespan in a UPRmt-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPRmt signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.

Figure thumbnail fx1

 

Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt
Ye Tian, Gilberto Garcia, Qian Bian, Kristan K. Steffen, Larry Joe, Suzanne Wolff, Barbara J. Meyer, Andrew Dillincorrespondence
http://dx.doi.org/10.1016/j.cell.2016.04.011             Publication stage: In Press Corrected Proof
Highlights
  • LIN-65 accumulates in the nucleus in response to mitochondrial stress
  • Mitochondrial stress-induced chromatin changes depend on MET-2 and LIN-65
  • LIN-65 and DVE-1 exhibit interdependence in nuclear accumulation
  • met-2 and atfs-1 act in parallel to affect mitochondrial stress-induced longevity

Organisms respond to mitochondrial stress through the upregulation of an array of protective genes, often perpetuating an early response to metabolic dysfunction across a lifetime. We find that mitochondrial stress causes widespread changes in chromatin structure through histone H3K9 di-methylation marks traditionally associated with gene silencing. Mitochondrial stress response activation requires the di-methylation of histone H3K9 through the activity of the histone methyltransferase met-2 and the nuclear co-factor lin-65. While globally the chromatin becomes silenced by these marks, remaining portions of the chromatin open up, at which point the binding of canonical stress responsive factors such as DVE-1 occurs. Thus, a metabolic stress response is established and propagated into adulthood of animals through specific epigenetic modifications that allow for selective gene expression and lifespan extension

 Siddharta Mukherjee’s Writing Career Just Got Dealt a Sucker Punch
Author: Theral Timpson

Siddharha Mukherjee won the 2011 Pulitzer Prize in non-fiction for his book, The Emperor of All Maladies.  The book has received widespread acclaim among lay audience, physicians, and scientists alike.  Last year the book was turned into a special PBS series.  But, according to a slew of scientists, we should all be skeptical of his next book scheduled to hit book shelves this month, The Gene, An Intimate History.

Publishing an article on epigenetics in the New Yorker this week–perhaps a selection from his new book–Mukherjee has waltzed into one of the most active scientific debates in all of biology: that of gene regulation, or epigenetics.

Jerry Coyne, the evolutionary biologist known for keeping journalists honest, has published a two part critique of Mukherjee’s New Yorker piece.  The first part–wildly tweeted yesterday–is a list of quotes from Coyne’s colleagues and those who have written in to the New Yorker, including two Nobel prize winners, Wally Gilbert and Sidney Altman, offering some very unfriendly sentences.

Wally Gilbert: “The New Yorker article is so wildly wrong that it defies rational analysis.”

Sidney Altman:  “I am not aware that there is such a thing as an epigenetic code.  It is unfortunate to inflict this article, without proper scientific review, on the audience of the New Yorker.”

The second part is a thorough scientific rebuttal of the Mukherjee piece.  It all serves as a great drama about one of the most contested ideas in biology and also as a cautionary tale to journalists, even experienced writers such as Mukherjee, about the dangers of wading into scientific arguments.  Readers may remember that a few years ago, science writer, David Dobbs, similarly skated into the same topic with his piece, Die, Selfish Gene, Die, and which raised a similar shitstorm, much of it from Coyne.

Mukherjee’s mistake is in giving credence to only one side of a very fierce debate–that the environment causes changes in the genome which can be passed on; another kind of evolution–as though it were settled science.   Either Mukherjee, a physicisan coming off from a successful book and PBS miniseries on cancer, is setting himself up as a scientist, or he has been a truly naive science reporter.   If he got this chapter so wrong, what does it mean about an entire book on the gene?

Coyne quotes one of his colleagues who raised some questions about the New Yorker’s science reporting, one particular question we’ve been asking here at Mendelspod.  How do we know what we know?  Does science now have an edge on any other discipline for being able to create knowledge?

Coyne’s colleague is troubled by science coverage in the New Yorker, and goes so far as to write that the New Yorker has been waging a “war on behalf of cultural critics and literary intellectuals against scientists and technologists.”

From my experience, it’s not quite that tidy.  First of all, the New Yorker is the best writing I read each week.  Period.  Second, I haven’t found their science writing to have the slant claimed in the quote above.  For example, most other mainstream outlets–including the New York Times with the Amy Harmon pieces–have given the anti-GMO crowd an equal say in the mistaken search for a “balance” on whether GMOs are harmful.  (Remember John Stewart’s criticism of Fox News?  That they give a false equivalent between two sides even when there is no equivalent on the other side?)

But the New Yorker has not fallen into this trap on GMOs and most of their pieces on the topic–mainly by Michael Specter–have been decidedly pro science and therefore decided pro GMO.

So what led Mukherjee to play scientist as well as journalist?  There’s no question about whether I enjoy his prose.  His writing beautifully whisks me away so that I don’t feel that I’m really working to understand.  There is a poetic complexity that constantly brings different threads effortlessly together, weaving them into the same light.  At one point he uses the metaphor of a web for the genome, with the epigenome being the stuff that sticks to the web.  He borrows the metaphor from the Hindu notion of “being”, or jaal.

“Genes form the threads of the web; the detritus that adheres to it transforms every web into a singular being.”

There have been a few writers on Twitter defending Mukherjee’s piece.  Tech Review’s Antonio Regalado called Coyne and his colleagues “tedious literalists” who have an “issue with epigenetic poetry.”

At his best, Mukherjee can take us down the sweet alleys of his metaphors and family stories with a new curiosity for the scientific truth.  He can hold a mirror up to scientists, or put the spotlight on their work.   At their worst, Coyne and his scientific colleagues can reek of a fear of language and therefore metaphor.  The always outspoken scientist and author, Richard Dawkins, who made his name by personifying the gene, was quick to personify epigentics in a tweet:   “It’s high time the 15 minutes of underserved fame for “epigenetics” came to an overdue end.”  Dawkins is that rare scientist who has consistently been as comfortable with rhetoric and language as he is with data.

Hats off to Coyne who reminds us that a metaphor–however lovely–does not some science make. If Mukherjee wants to play scientist, let him create and gather data. If it’s the role of science journalist he wants, let him collect all the science he can before he begins to pour it into his poetry.

 

Same but Different  

How epigenetics can blur the line between nature and nurture.

Annals of Science MAY 2, 2016 ISSUE     BY

The author’s mother (right) and her twin are a study in difference and identity. CREDIT: PHOTOGRAPH BY DAYANITA SINGH FOR THE NEW YORKER

October 6, 1942, my mother was born twice in Delhi. Bulu, her identical twin, came first, placid and beautiful. My mother, Tulu, emerged several minutes later, squirming and squalling. The midwife must have known enough about infants to recognize that the beautiful are often the damned: the quiet twin, on the edge of listlessness, was severely undernourished and had to be swaddled in blankets and revived.

The first few days of my aunt’s life were the most tenuous. She could not suckle at the breast, the story runs, and there were no infant bottles to be found in Delhi in the forties, so she was fed through a cotton wick dipped in milk, and then from a cowrie shell shaped like a spoon. When the breast milk began to run dry, at seven months, my mother was quickly weaned so that her sister could have the last remnants.
Tulu and Bulu grew up looking strikingly similar: they had the same freckled skin, almond-shaped face, and high cheekbones, unusual among Bengalis, and a slight downward tilt of the outer edge of the eye, something that Italian painters used to make Madonnas exude a mysterious empathy. They shared an inner language, as so often happens with twins; they had jokes that only the other twin understood. They even smelled the same: when I was four or five and Bulu came to visit us, my mother, in a bait-and-switch trick that amused her endlessly, would send her sister to put me to bed; eventually, searching in the half-light for identity and difference—for the precise map of freckles on her face—I would realize that I had been fooled.

But the differences were striking, too. My mother was boisterous. She had a mercurial temper that rose fast and died suddenly, like a gust of wind in a tunnel. Bulu was physically timid yet intellectually more adventurous. Her mind was more agile, her tongue sharper, her wit more lancing. Tulu was gregarious. She made friends easily. She was impervious to insults. Bulu was reserved, quieter, and more brittle. Tulu liked theatre and dancing. Bulu was a poet, a writer, a dreamer.

….. more

Why are identical twins alike? In the late nineteen-seventies, a team of scientists in Minnesota set out to determine how much these similarities arose from genes, rather than environments—from “nature,” rather than “nurture.” Scouring thousands of adoption records and news clips, the researchers gleaned a rare cohort of fifty-six identical twins who had been separated at birth. Reared in different families and different cities, often in vastly dissimilar circumstances, these twins shared only their genomes. Yet on tests designed to measure personality, attitudes, temperaments, and anxieties, they converged astonishingly. Social and political attitudes were powerfully correlated: liberals clustered with liberals, and orthodoxy was twinned with orthodoxy. The same went for religiosity (or its absence), even for the ability to be transported by an aesthetic experience. Two brothers, separated by geographic and economic continents, might be brought to tears by the same Chopin nocturne, as if responding to some subtle, common chord struck by their genomes.

One pair of twins both suffered crippling migraines, owned dogs that they had named Toy, married women named Linda, and had sons named James Allan (although one spelled the middle name with a single “l”). Another pair—one brought up Jewish, in Trinidad, and the other Catholic, in Nazi Germany, where he joined the Hitler Youth—wore blue shirts with epaulets and four pockets, and shared peculiar obsessive behaviors, such as flushing the toilet before using it. Both had invented fake sneezes to diffuse tense moments. Two sisters—separated long before the development of language—had invented the same word to describe the way they scrunched up their noses: “squidging.” Another pair confessed that they had been haunted by nightmares of being suffocated by various metallic objects—doorknobs, fishhooks, and the like.

The Minnesota twin study raised questions about the depth and pervasiveness of qualities specified by genes: Where in the genome, exactly, might one find the locus of recurrent nightmares or of fake sneezes? Yet it provoked an equally puzzling converse question: Why are identical twins different? Because, you might answer, fate impinges differently on their bodies. One twin falls down the crumbling stairs of her Calcutta house and breaks her ankle; the other scalds her thigh on a tipped cup of coffee in a European station. Each acquires the wounds, calluses, and memories of chance and fate. But how are these changes recorded, so that they persist over the years? We know that the genome can manufacture identity; the trickier question is how it gives rise to difference.

….. more

But what turns those genes on and off, and keeps them turned on or off? Why doesn’t a liver cell wake up one morning and find itself transformed into a neuron? Allis unpacked the problem further: suppose he could find an organism with two distinct sets of genes—an active set and an inactive set—between which it regularly toggled. If he could identify the molecular switches that maintain one state, or toggle between the two states, he might be able to identify the mechanism responsible for cellular memory. “What I really needed, then, was a cell with these properties,” he recalled when we spoke at his office a few weeks ago. “Two sets of genes, turned ‘on’ or ‘off’ by some signal.”

more…

“Histones had been known as part of the inner scaffold for DNA for decades,” Allis went on. “But most biologists thought of these proteins merely as packaging, or stuffing, for genes.” When Allis gave scientific seminars in the early nineties, he recalled, skeptics asked him why he was so obsessed with the packing material, the stuff in between the DNA.  …. A skein of silk tangled into a ball has very different properties from that same skein extended; might the coiling or uncoiling of DNA change the activity of genes?

In 1996, Allis and his research group deepened this theory with a seminal discovery. “We became interested in the process of histone modification,” he said. “What is the signal that changes the structure of the histone so that DNA can be packed into such radically different states? We finally found a protein that makes a specific chemical change in the histone, possibly forcing the DNA coil to open. And when we studied the properties of this protein it became quite clear that it was also changing the activity of genes.” The coils of DNA seemed to open and close in response to histone modifications—inhaling, exhaling, inhaling, like life.

Allis walked me to his lab, a fluorescent-lit space overlooking the East River, divided by wide, polished-stone benches. A mechanical stirrer, whirring in a corner, clinked on the edge of a glass beaker. “Two features of histone modifications are notable,” Allis said. “First, changing histones can change the activity of a gene without affecting the sequence of the DNA.” It is, in short, formally epi-genetic, just as Waddington had imagined. “And, second, the histone modifications are passed from a parent cell to its daughter cells when cells divide. A cell can thus record ‘memory,’ and not just for itself but for all its daughter cells.”

…..

 

 

The New Yorker screws up big time with science: researchers criticize the Mukherjee piece on epigenetics

Jerry Coyne
https://whyevolutionistrue.wordpress.com/2016/05/05/the-new-yorker-screws-up-big-time-with-science-researchers-criticize-the-mukherjee-piece-on-epigenetics/

Abstract: This is a two part-post about a science piece on gene regulation that just appeared in the New Yorker. Today I give quotes from scientists criticizing that piece; tomorrow I’ll present a semi-formal critique of the piece by two experts in the field.

esterday I gave readers an assignment: read the new New Yorkerpiece by Siddhartha Mukherjee about epigenetics. The piece, called “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”) was brought to my attention by two readers, both of whom praised it.  Mukherjee, a physician, is well known for writing the Pulitzer-Prize-winning book (2011) The Emperor of All Maladies: A Biography of Cancer. (I haven’t read it yet, but it’s on my list.)  Mukherjee has a new book that will be published in May: The Gene: An Intimate History. As I haven’t seen it, the New Yorker piece may be an excerpt from this book.

Everyone I know who has read The Emperor of All Maladies gives it high praise. I wish I could say the same for Mukherjee’s New Yorker piece. When I read it at the behest of the two readers, I found his analysis of gene regulation incomplete and superficial. Although I’m not an expert in that area, I knew that there was a lot of evidence that regulatory proteins called “transcription factors”, and not “epigenetic markers” (see discussion of this term tomorrow) or modified histones—the factors emphasized by Mukherjee—played hugely important roles in gene regulation. The speculations at the end of the piece about “Lamarckian evolution” via environmentally induced epigenetic changes in the genome were also unfounded, for we have no evidence for that kind of adaptive evolution. Mukherjee does, however, mention that lack of evidence, though I wish he’d done so more strongly given that environmental modification of DNA bases is constantly touted as an important and neglected factor in evolution.

Unbeknownst to me, there was a bit of a kerfuffle going on in the community of scientists who study gene regulation, with many of them finding serious mistakes and omissions in Mukherjee’s piece.  There appears to have been some back-and-forth emailing among them, and several wrote letters to the New Yorker, urging them to correct the misconceptions, omissions, and scientific errors in “Same but different.” As I understand it, both Mukherjee and the New Yorker simply batted these criticisms away, and, as far as I know, will not publish any corrections.  So today and tomorrow I’ll present the criticisms here, just so they’ll be on the record.

Because Mukherjee writes very well, and because even educated laypeople won’t know the story of gene regulation revealed over the last few decades,  they may not see the big lacunae in his piece. It is, then,  important to set matters straight, for at least we should know what science has told us about how genes are turned on and off. The criticism of Mukherjee’s piece, coming from scientists who really are experts in gene regulation, shows a lack of care on the part of Mukherjee and theNew Yorker: both a superficial and misleading treatment of the state of the science, and a failure of the magazine to properly vet this piece (I have no idea whether they had it “refereed” not just by editors but by scientists not mentioned in the piece).

Let me add one thing about science and the New Yorker. I believe I’ve said this before, but the way the New Yorker treats science is symptomatic of the “two cultures” problem. This is summarized in an email sent me a while back by a colleague, which I quote with permission:

The New Yorker is fine with science that either serves a literary purpose (doctors’ portraits of interesting patients) or a political purpose (environmental writing with its implicit critique of modern technology and capitalism). But the subtext of most of its coverage (there are exceptions) is that scientists are just a self-interested tribe with their own narrative and no claim to finding the truth, and that science must concede the supremacy of literary culture when it comes to anything human, and never try to submit human affairs to quantification or consilience with biology. Because the magazine is undoubtedly sophisticated in its writing and editing they don’t flaunt their postmodernism or their literary-intellectual proprietariness, but once you notice it you can make sense of a lot of their material.

. . . Obviously there are exceptions – Atul Gawande is consistently superb – but as soon as you notice it, their guild war on behalf of cultural critics and literary intellectuals against scientists, technologists, and analytic scholars becomes apparent.

…. more

Researchers criticize the Mukherjee piece on epigenetics: Part 2

Trigger warning: Long science post!

Yesterday I provided a bunch of scientists’ reactions—and these were big names in the field of gene regulation—to Siddhartha Mukherjee’s ill-informed piece in The New Yorker, “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”). Today, in part 2, I provide a sentence-by-sentence analysis and reaction by two renowned researchers in that area. We’ll start with a set of definitions (provided by the authors) that we need to understand the debate, and then proceed to the critique.

Let me add one thing to avoid confusion: everything below the line, including the definition (except for my one comment at the end) was written by Ptashne and Greally.

by Mark Ptashne and John Greally

Introduction

Ptashne is The Ludwig Professor of Molecular Biology at the Memorial Sloan Kettering Cancer Center in New York. He wrote A Genetic Switch, now in its third edition, which describes the principles of gene regulation and the workings of a ‘switch’; and, with Alex Gann, Genes and Signals, which extends these principles and ideas to higher organisms and to other cellular processes as well.  John Greally is the Director of the Center for Epigenomics at the Albert Einstein College of Medicine in New York.

 

The New Yorker  (May 2, 2016) published an article entitled “Same But Different” written by Siddhartha Mukherjee.  As readers will have gathered from the letters posted yesterday, there is a concern that the article is misleading, especially for a non-scientific audience. The issue concerns our current understanding of “gene regulation” and how that understanding has been arrived at.

First some definitions/concepts:

Gene regulation refers to the “turning on and off of genes”.  The primary event in turning a gene “on” is to transcribe (copy) it into messenger RNA (mRNA). That mRNA is then decoded, usually, into a specific protein.  Genes are transcribed by the enzyme called RNA polymerase.

Development:  the process in which a fertilized egg (e.g., a human egg) divides many times and eventually forms an organism.  During this process, many of the roughly 23,000 genes of a human are turned “on” or “off” in different combinations, at different times and places in the developing organism. The process produces many different cell types in different organs (e.g. liver and brain), but all retain the original set of genes.

Transcription factors: proteins that bind to specific DNA sequences near specific genes and turn transcription of those genes on and off. A transcriptional ‘activator’, for example, bears two surfaces: one binds a specific sequence in DNA, and the other binds to, and thereby recruits to the gene, protein complexes that include RNA polymerase. It is widely acknowledged that the identity of a cell in the body depends on the array of transcription factors present in the cell, and the cell’s history.  RNA molecules can also recognize specific genomic sequences, and they too sometimes work as regulators.  Neither transcription factors nor these kinds of RNA molecules – the fundamental regulators of gene expression and development – are mentioned in the New Yorker article.

Signals:  these come in many forms (small molecules like estrogen, larger molecules (often proteins such as cytokines) that determine the ability of transcription factors to work.  For example, estrogen binds directly to a transcription factor (the estrogen receptor) and, by changing its shape, permits it to bind DNA and activate transcription.

Memory”:  a dividing cell can (often does) produce daughters that are identical, and that express identical genes as does the mother cell.  This occurs because the transcription factors present in the mother cell are passively transmitted to the daughters as the cell divides, and they go to work in their new contexts as before.  To make two different daughters, the cell must distribute its transcription factors asymmetrically.

Positive Feedback: An activator can maintain its own expression by  positive feedback.  This requires, simply, that a copy of the DNA sequence to which the activator binds is  present  near its own gene. Expression of the activator  then becomes self-perpetuating.  The activator (of which there now are many copies in the cell) activates  other target genes as it maintains its own expression. This kind of ‘memory circuit’, first described  in  bacteria, is found in higher organisms as well.  Positive feedback can explain how a fully differentiated cell (that is, a cell that has reached its developmental endpoint) maintains its identity.

Nucleosomes:  DNA in higher organisms (eukaryotes) is wrapped, like beads on a string, around certain proteins (called histones), to form nucleosomes.  The histones are subject to enzymatic modifications: e.g., acetyl, methyl, phosphate, etc. groups can be added to these structures. In bacteria there are no nucleosomes, and the DNA is more or less ‘naked’.

“Epigenetic modifications: please don’t worry about the word ”epigenetic”; it is misused in any case. What Mukherjee refers to by this term are the histone modifications mentioned above, and a modification to DNA itself: the addition of methyl groups. Keep in mind that the organisms that have taught us the most about development – flies (Drosophila) and worms (C. elegans)—do not have the enzymes required for DNA methylation. That does not mean that DNA methylation cannot do interesting things in humans, for example, but it is obviously not at the heart of gene regulation.

Specificity Development requires the highly specific sequential turning on and off of sets of genes.  Transcription factors and RNA supply this specificity, but   enzymes that impart modifications to histones  cannot: every nucleosome (and hence every gene) appears the same to the enzyme.  Thus such enzymes cannot pick out particular nucleosomes associated with particular genes to modify.  Histone modifications might be imagined to convey ‘memory’ as cells divide – but there are no convincing indications that this happens, nor are there molecular models that might explain why they would have the imputed effects.

Analysis and critique of Mukherjee’s article

The picture we have just sketched has taken the combined efforts of many scientists over 50 years to develop.  So what, then, is the problem with the New Yorker article?

There are two: first, the picture we have just sketched, emphasizing the primary role of transcription factors and RNA, is absent.  Second, that picture is replaced by highly dubious speculations, some of which don’t make sense, and none of which has been shown to work as imagined in the article.

(Quotes from the Mukherjee article are indented and in plain text; they are followed by comments, flush left and in bold, by Ptashne and Greally.)

In 1978, having obtained a Ph.D. in biology at Indiana University, Allis began to tackle a problem that had long troubled geneticists and cell biologists: if all the cells in the body have the same genome, how does one become a nerve cell, say, and another a blood cell, which looks and functions very differently?

The problems referred to were recognized long before 1978.  In fact, these were exactly the problems that the great French scientists François Jacob and Jacques Monod took on in the 1950s-60s.  In a series of brilliant experiments, Jacob and Monod showed that in bacteria, certain genes encode products that regulate (turn on and off) specific other genes.  Those regulatory molecules turned out to be proteins, some of which respond to signals from the environment.  Much of the story of modern biology has been figuring out how these proteins – in bacteria and in higher organisms  – bind to and regulate specific genes.  Of note is that in higher organisms, the regulatory proteins look and act like those in bacteria, despite the fact that eukaryotic DNA is wrapped in nucleosomes  whereas bacterial DNA is not.   We have also learned that certain RNA molecules can play a regulatory role, a phenomenon made possible by the fact that RNA molecules, like regulatory proteins, can recognize specific genomic sequences.

In the nineteen-forties, Conrad Waddington, an English embryologist, had proposed an ingenious answer: cells acquired their identities just as humans do—by letting nurture (environmental signals) modify nature (genes). For that to happen, Waddington concluded, an additional layer of information must exist within a cell—a layer that hovered, ghostlike, above the genome. This layer would carry the “memory” of the cell, recording its past and establishing its future, marking its identity and its destiny but permitting that identity to be changed, if needed. He termed the phenomenon “epigenetics”—“above genetics.”

This description greatly misrepresents the original concept.  Waddington argued that development proceeds not by the loss (or gain) of genes, which would be a “genetic” process, but rather that some genes would be selectively expressed in specific and complex cellular patterns as development proceeds.  He referred to this intersection of embryology (then called “epigenesis”) and genetics as “epigenetic”.We now understand that regulatory proteins work in combinations to turn on and off genes, including their own genes, and that sometimes the regulatory proteins respond to signals sent by other cells.  It should be emphasized that Waddington never proposed any “ghost-like” layer of additional information hovering above the gene.  This is a later misinterpretation of a literal translation of the term epigenetics, with “epi-“ meaning “above/upon” the genetic information encoded in DNA sequence.  Unfortunately, this new and pervasive definition encompasses all of transcriptional regulation and is of no practical value.

…..more

By 2000, Allis and his colleagues around the world had identified a gamut of proteins that could modify histones, and so modulate the activity of genes. Other systems, too, that could scratch different kinds of code on the genome were identified (some of these discoveries predating the identification of histone modifications). One involved the addition of a chemical side chain, called a methyl group, to DNA. The methyl groups hang off the DNA string like Christmas ornaments, and specific proteins add and remove the ornaments, in effect “decorating” the genome. The most heavily methylated parts of the genome tend to be dampened in their activity.

It is true that enzymes that modify histones have been found—lots of them.  A striking problem is that, after all this time, it is not at all clear what the vast majority of these modifications do.  When these enzymatic activities are eliminated by mutation of their active sites (a task substantially easier to accomplish in yeast than in higher organisms) they mostly have little or no effect on transcription.  It is not even clear that histones are the biologically relevant substrates of most of these enzymes.  

 In the ensuing decade, Allis wrote enormous, magisterial papers in which a rich cast of histone-modifying proteins appear and reappear through various roles, mapping out a hatchwork of complexity. . . These protein systems, overlaying information on the genome, interacted with one another, reinforcing or attenuating their signals. Together, they generated the bewildering intricacy necessary for a cell to build a constellation of other cells out of the same genes, and for the cells to add “memories” to their genomes and transmit these memories to their progeny. “There’s an epigenetic code, just like there’s a genetic code,” Allis said. “There are codes to make parts of the genome more active, and codes to make them inactive.”

By ‘epigenetic code’ the author seems to mean specific arrays of nucleosome modifications, imparted over time and cell divisions, marking genes for expression.  This idea has been tested in many experiments and has been found not to hold.

….. and more

 

Larry H. Bernstein, MD, FCAP

I hope that this piece brings greater clarity to the discussion.  I have heard the use of the term “epigenetics” for over a decade.  The term was never so clear.  I think that the New Yorker article was a reasonable article for the intended audience.  It was not intended to clarify debates about a mechanism for epigenetic based changes in evolutionary science.  I think it actually punctures the “classic model” of the cell depending only on double stranded DNA and transcription, which deflates our concept of the living cell.  The concept of epigenetics was never really formulated as far as I have seen, and I have done serious work in enzymology and proteins at a time that we did not have the technology that exists today.  I have considered with the critics that protein folding, protein misfolding, protein interactions with proximity of polar and nonpolar groups, and the regulatory role of microRNAs that are not involved in translation, and the evolving concept of what is “dark (noncoding) DNA” lend credence to the complexity of this discussion.  Even more interesting is the fact that enzymes (and isoforms of enzymes) have a huge role in cellular metabolic differences and in the function of metabolic pathways.  What is less understood is the extremely fast reactions involved in these cellular reactions.  These reactions are in my view critical drivers.  This is brought out by Erwin Schroedinger in the book What is Life? which infers that there can be no mathematical expression of life processes.

 

 

 

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Biology, Physiology and Pathophysiology of Heat Shock Proteins

Curation: Larry H. Bernstein, MD, FCAP

 

 

Heat Shock Proteins (HSP)

  1. Exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes

Hsp70 chaperones: Cellular functions and molecular mechanism

M. P. MayerB. Bukau
Cell and Molec Life Sci  Mar 2005; 62:670  http://dx.doi.org:/10.1007/s00018-004-4464-6

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [17]. Hsp70s have thus housekeeping functions in the cell in which they are built-in components of folding and signal transduction pathways, and quality control functions in which they proofread the structure of proteins and repair misfolded conformers. All of these activities appear to be based on the property of Hsp70 to interact with hydrophobic peptide segments of proteins in an ATP-controlled fashion. The broad spectrum of cellular functions of Hsp70 proteins is achieved through

  • the amplification and diversification of hsp70genes in evolution, which has generated specialized Hsp70 chaperones,
  • co-chaperones which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and
  • cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines.

Protein folding processes assisted by Hsp70

The role of Hsp70s in the folding of non-native proteins can be divided into three related activities: prevention of aggregation, promotion of folding to the native state, and solubilization and refolding of aggregated proteins. In the cellular milieu, Hsp70s exert these activities in the quality control of misfolded proteins and the co- and posttranslational folding of newly synthesized proteins. Mechanistically related but less understood is the role of Hsp70s in the disassembly of protein complexes such as clathrin coats, viral capsids and the nucleoprotein complex, which initiates the replication of bacteriophage λ DNA. A more complex folding situation exists for the Hsp70-dependent control of regulatory proteins since several steps in the folding and activation process of these substrates are assisted by multiple chaperones.

Hsp70 proteins together with their co-chaperones of the J-domain protein (JDP) family prevent the aggregation of non-native proteins through association with hydrophobic patches of substrate molecules, which shields them from intermolecular interactions (‘holder’ activity). Some JDPs such as Escherichia coli DnaJ and Saccharomyces cerevisiae Ydj1 can prevent aggregation by themselves through ATP-independent transient and rapid association with the substrates. Only members of the Hsp70 family with general chaperone functions have such general holder activity.

Hsp70 chaperone systems assist non-native folding intermediates to fold to the native state (‘folder’ activity). The mechanism by which Hsp70-chaperones assist the folding of non-native substrates is still unclear. Hsp70-dependent protein folding in vitro occurs typically on the time scale of minutes or longer. Substrates cycle between chaperone-bound and free states until the ensemble of molecules has reached the native state. There are at least two alternative modes of action. In the first mechanism Hsp70s play a rather passive role. Through repetitive substrate binding and release cycles they keep the free concentration of the substrate sufficiently low to prevent aggregation, while allowing free molecules to fold to the native state (‘kinetic partitioning’). In the second mechanism, the binding and release cycles induce local unfolding in the substrate, e.g. the untangling of a misfolded β-sheet, which helps to overcome kinetic barriers for folding to the native state (‘local unfolding’) [8–11]. The energy of ATP may be used to induce such conformational changes or alternatively to drive the ATPase cycle in the right direction.

Hsp70 in cellular physiology and pathophysiology

Two Hsp70 functions are especially interesting, de novo folding of nascent polypeptides and interaction with signal transduction proteins, and therefore some aspects of these functions shall be discussed below in more detail. Hsp70 chaperones were estimated to assist the de novo folding of 10–20% of all bacterial proteins whereby the dependence on Hsp70 for efficient folding correlated with the size of the protein [12]. Since the average protein size in eukaryotic cells is increased (52 kDa in humans) as compared to bacteria (35 kDa in E. coli) [25], it is to be expected that an even larger percentage of eukaryotic proteins will be in need of Hsp70 during de novo folding. This reliance on Hsp70 chaperones increases even more under stress conditions. Interestingly, mutated proteins [for example mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxid dismutase (SOD) 1] seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein [2629]. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations [30], a function demonstrated earlier for Hsp90 [3132]. Such mutations are only uncovered when the overall need for Hsp70 action exceeds the chaperone capacity of the Hsp70 proteins, for example during stress conditions [30], at certain stages in development or during aging, when the magnitude of stress-induced increase in Hsp70 levels declines [3334]. Alternatively, the mutant protein can be targeted by Hsp70 and its co-chaperones to degradation as shown e.g. for CFTRΔF508 and some of the SOD1 mutant proteins [35,36]. Deleterious mutant proteins may then only accumulate when Hsp70 proteins are overwhelmed by other, stress-denatured proteins. Both mechanisms may contribute to pathological processes such as oncogenesis (mutant p53) and neurodegenerative diseases, including amyotrophic, lateral sclerosis (SOD1 mutations), Parkinsonism (α-synuclein mutations), Huntington’s chorea (huntingtin with polyglutamin expansions) and spinocerebellar ataxias (proteins with polyglutamin expansions).

De novo folding is not necessarily accelerated by Hsp70 chaperones. In some cases folding is delayed for different reasons. First, folding of certain proteins can only proceed productively after synthesis of the polypeptide is completed as shown, e.g. for the reovirus lollipop-shaped protein sigma 1 [37]. Second, proteins destined for posttranslational insertion into organellar membranes are prevented from aggregation and transported to the translocation pore [38]. Third, in the case of the caspase-activated DNase (CAD), the active protein is dangerous for the cell and therefore can only complete folding in the presence of its specific inhibitor (ICAD). Hsp70 binds CAD cotranslationally and mediates folding only to an intermediate state. Folding is completed after addition of ICAD, which is assembled into a complex with CAD in an Hsp70-dependent manner [39]. Similar folding pathways may exist also for other potentially dangerous proteins.

As mentioned above Hsp70 interacts with key regulators of many signal transduction pathways controlling cell homeostasis, proliferation, differentiation and cell death. The interaction of Hsp70 with these regulatory proteins continues in activation cycles that also involve Hsp90 and a number of co-chaperones. The regulatory proteins, called clients, are thereby kept in an inactive state from which they are rapidly activated by the appropriate signals. Hsp70 and Hsp90 thus repress regulators in the absence of the upstream signal and guarantee full activation after the signal transduction pathway is switched on [6]. Hsp70 can be titrated away from these clients by other misfolded proteins that may arise from internal or external stresses. Consequently, through Hsp70 disturbances of the cellular system induced by environmental, developmental or pathological processes act on these signal transduction pathways.

In this way stress response and apoptosis are linked to each other. Hsp70 inhibits apoptosis acting on the caspase-dependent pathway at several steps both upstream and downstream of caspase activation and on the caspase-independent pathway. Overproduction of Hsp70 leads to increased resistance against apoptosis-inducing agents such as tumor necrosis factor-α(TNFα), staurosporin and doxorubicin, while downregulation of Hsp70 levels by antisense technology leads to increased sensitivity towards these agents [1840]. This observation relates to many pathological processes, such as oncogenesis, neurodegeneration and senescence. In many tumor cells increased Hsp70 levels are observed and correlate with increased malignancy and resistance to therapy. Downregulation of the Hsp70 levels in cancer cells induce differentiation and cell death [41]. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s corea and spinocerebellar ataxias are characterized by excessive apoptosis. In several different model systems overexpression of Hsp70 or one of its co-chaperones could overcome the neurodegenerative symptoms induced by expression of a disease-related gene (huntingtin, α-synuclein or ataxin) [20,42]. Senescence in cell culture as well as aging in vivo is correlated with a continuous decline in the ability to mount a stress response [3443]. Age-related symptoms and diseases reflect this decreased ability to cope with cellular stresses. Interestingly, centenarians seem to be an exception to the rule, as they show a significant induction of Hsp70 production after heat shock challenge [44].

ATPase domain and ATPase cycle

Substrate binding

The coupling mechanism: nucleotide-controlled opening and closing of the substrate binding cavity

The targeting activity of co-chaperones

J-domain proteins

Bag proteins

Hip, Hop and CHIP

Perspectives

The Hsp70 protein family and their co-chaperones constitute a complex network of folding machines which is utilized by cells in many ways. Despite considerable progress in the elucidation of the mechanistic basis of these folding machines, important aspects remain to be solved. With respect to the Hsp70 proteins it is still unclear whether their activity to assist protein folding relies on the ability to induce conformational changes in the bound substrates, how the coupling mechanism allows ATP to control substrate binding and to what extent sequence variations within the family translate into variations of the mechanism. With respect to the action of co-chaperones we lack a molecular understanding of the coupling function of JDPs and of how co-chaperones target their Hsp70 partner proteins to substrates. Furthermore, it can be expected that more cellular processes will be discovered that depend on the chaperone activity of Hsp70 chaperones.

 

  1. The biochemistry and ultrastructure of molecular chaperones

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Laurence H. Pearl and Chrisostomos Prodromou
Ann Rev of Biochem July 2006;75:271-294
http://dx.doi.org:/10.1146/annurev.biochem.75.103004.142738

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90’s diverse “clientele.” Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

 

  1. Properties of heat shock proteins (HSPs) and heat shock factor (HSF)

Heat shock factors: integrators of cell stress, development and lifespan

Malin Åkerfelt,*‡ Richard I. Morimoto,§ and Lea Sistonen*‡
Nat Rev Mol Cell Biol. 2010 Aug; 11(8): 545–555.  doi:  10.1038/nrm2938

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

In the early 1960s, Ritossa made the seminal discovery of temperature-induced puffs in polytene chromosomes of Drosophila melanogaster larvae salivary glands1. A decade later, it was shown that the puffing pattern corresponded to a robust activation of genes encoding the heat shock proteins (HSPs), which function as molecular chaperones2. The heat shock response is a highly conserved mechanism in all organisms from yeast to humans that is induced by extreme proteotoxic insults such as heat, oxidative stress, heavy metals, toxins and bacterial infections. The conservation among different eukaryotes suggests that the heat shock response is essential for survival in a stressful environment.

The heat shock response is mediated at the transcriptional level by cis-acting sequences called heat shock elements (HSEs; BOX 1) that are present in multiple copies upstream of the HSP genes3. The first evidence for a specific transcriptional regulator, the heat shock factor (HSF) that can bind to the HSEs and induce HSP gene expression, was obtained through DNA–protein interaction studies on nuclei isolated from D. melanogaster cells4,5. Subsequent studies showed that, in contrast to a single HSF in invertebrates, multiple HSFs are expressed in plants and vertebrates68. The mammalian HSF family consists of four members: HSF1,HSF2, HSF3 and HSF4. Distinct HSFs possess unique and overlapping functions (FIG. 1), exhibit tissue-specific patterns of expression and have multiple post-translational modifications (PTMs) and interacting protein partners7,9,10. Functional crosstalk between HSF family members and PTMs facilitates the fine-tuning of HSF-mediated gene regulation. The identification of many targets has further extended the impact of HSFs beyond the heat shock response. Here, we present the recent discoveries of novel target genes and physiological functions of HSFs, which have changed the view that HSFs act solely in the heat shock response. Based on the current knowledge of small-molecule activators and inhibitors of HSFs, we also highlight the potential for pharmacologic modulation of HSF-mediated gene regulation.

Box 1

The heat shock element

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u1.jpg

Heat shock factors (HSFs) act through a regulatory upstream promoter element, called the heat shock element (HSE). In the DNA-bound form of a HSF, each DNA-binding domain (DBD) recognizes the HSE in the major groove of the double helix6. The HSE was originally identified using S1 mapping of transcripts of the Drosophila melanogaster heat shock protein (HSP) genes3 (see the figure; part a). Residues –47 to –66 are necessary for heat inducibility. HSEs in HSP gene promoters are highly conserved and consist of inverted repeats of the pentameric sequence nGAAn132. The type of HSEs that can be found in the proximal promoter regions of HSP genes is composed of at least three contiguous inverted repeats: nTTCnnGAAnnTTCn132134. The promoters of HSF target genes can also contain more than one HSE, thereby allowing the simultaneous binding of multiple HSFs. The binding of an HSF to an HSE occurs in a cooperative manner, whereby binding of an HSF trimer facilitates binding of the next one135. More recently, Trinklein and colleagues used chromatin immunoprecipitation to enrich sequences bound by HSF1 in heat-shocked human cells to define the HSE consensus sequence. They confirmed the original finding of Xiao and Lis, who identified guanines as the most conserved nucleotides in HSEs87,133 (see the figure; part b). Moreover, in a pair of inverted repeats, a TTC triplet 5′ of a GAA triplet is separated by a pyrimidine–purine dinucleotide, whereas the two nucleotides separating a GAA triplet 5′ from a TTC triplet is unconstrained87. The discovery of novel HSF target genes that are not involved in the heat shock response has rendered it possible that there may be HSEs in many genes other than the HSP genes. Although there are variations in these HSEs, the spacing and position of the guanines are invariable7. Therefore, both the nucleotides and the exact spacing of the repeated units are considered as key determinants for recognition by HSFs and transcriptional activation. Part b of the figure is modified, with permission, from REF. 87 © (2004) The American Society for Cell Biology.

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f1.gif

The mammalian HSF machinery

HSFs as stress integrators

A hallmark of stressed cells and organisms is the increased synthesis of HSPs, which function as molecular chaperones to prevent protein misfolding and aggregation to maintain protein homeostasis, also called proteostasis11. The transcriptional activation of HSP genes is mediated by HSFs (FIG. 2a), of which HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance1215. HSF1 is constitutively expressed in most tissues and cell types16, where it is kept inactive in the absence of stress stimuli. Thus, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple PTMs, protein–protein interactions and subcellular localization. HSF1 also has an intrinsic stress-sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a homotrimer in vitro in response to thermal or oxidative stress1719.

Figure 2    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f2.gif

Members of the mammalian HSF family

Functional domains

HSFs, like other transcription factors, are composed of functional domains. These have been most thoroughly characterized for HSF1 and are schematically presented in FIG. 2b. The DNA-binding domain (DBD) is the best preserved domain in evolution and belongs to the family of winged helix-turn-helix DBDs2022. The DBD forms a compact globular structure, except for a flexible wing or loop that is located between β-strands 3 and 4 (REF. 6). This loop generates a protein– protein interface between adjacent subunits of the HSF trimer that enhances high-affinity binding to DNA by cooperativity between different HSFs23. The DBD can also mediate interactions with other factors to modulate the transactivating capacity of HSFs24. Consequently, the DBD is considered as the signature domain of HSFs for target-gene recognition.

The trimerization of HSFs is mediated by arrays of hydrophobic heptad repeats (HR-A and HR-B) that form a coiled coil, which is characteristic for many Leu zippers6,25 (FIG. 2b). The trimeric assembly is unusual, as Leu zippers typically facilitate the formation of homodimers or heterodimers. Suppression of spontaneous HSF trimerization is mediated by yet another hydrophobic repeat, HR-C2628. Human HSF4 lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity29. Positioned at the extreme carboxyl terminus of HSFs is the transactivation domain, which is shared among all HSFs6except for yeast Hsf, which has transactivation domains in both the amino and C termini, and HSF4A, which completely lacks a transactivation domain2931. In HSF1, the transactivation domain is composed of two modules — AD1 and AD2, which are rich in hydrophobic and acidic residues (FIG. 3a) — that together ensures a rapid and prolonged response to stress32,33. The transactivation domain was originally proposed to provide stress inducibility to HSF1 (REFS 34,35), but it soon became evident that an intact regulatory domain, located between the HR-A and HR-B and the transactivation domain, is essential for the responsiveness to stress stimuli32,33,36,37. Because several amino acids that are known targets for different PTMs reside in the regulatory domain33,3842, the structure and function of this domain are under intensive investigation.

Figure 3    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f3.gif

HSF1 undergoes multiple PTMs on activation

Regulation of the HSF1 activation–attenuation cycle

The conversion of the inactive monomeric HSF1 to high-affinity DNA-binding trimers is the initial step in the multistep activation process and is a common feature of all eukaryotic HSFs43,44 (FIG. 3b). There is compelling evidence for HSF1 interacting with multiple HSPs at different phases of its activation cycle. For example, monomeric HSF1 interacts weakly with HSP90 and, on stress, HSF1 dissociates from the complex, allowing HSF1 trimerization45,46 (FIG. 3b). Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex of HSP90, co-chaperone p23 (also known as PTGES3) and immunophilin FK506-binding protein 5 (FKBP52; also known as FKBP4)4651. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation on heat shock52. Activated HSF1 trimers also interact with HSP70 and the co-chaperone HSP40 (also known as DNAJB1), but instead of suppressing the DNA-binding activity of HSF1, this interaction inhibits its transactivation capacity5254. Although the inhibitory mechanism is still unknown, the negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides.

A ribonucleoprotein complex containing eukaryotic elongation factor 1A (eEF1A) and a non-coding RNA, heat shock RNA-1 (HSR-1), has been reported to possess a thermosensing capacity. According to the proposed model, HSR-1 undergoes a conformational change in response to heat stress and together with eEF1A facilitates trimerization of HSF1 (REF. 55). How this activation mode relates to the other regulatory mechanisms associated with HSFs remains to be elucidated.

Throughout the activation–attenuation cycle, HSF1 undergoes extensive PTMs, including acetylation, phosphorylation and sumoylation (FIG. 3). HSF1 is also a phosphoprotein under non-stress conditions, and the results from mass spectrometry (MS) analyses combined with phosphopeptide mapping experiments indicate that at least 12 Ser residues are phosphorylated41,5659. Among these sites, stress-inducible phosphorylation of Ser230 and Ser326 in the regulatory domain contributes to the transactivation function of HSF1 (REFS 38,41). Phosphorylation-mediated sumoylation on a single Lys residue in the regulatory domain occurs rapidly and transiently on exposure to heat shock; Ser303 needs to be phosphorylated before a small ubiquitin-related modifier (SUMO) can be conjugated to Lys298 (REF. 39). The extended consensus sequence ΨKxExxSP has been named the phosphorylation-dependent sumoylation motif (PDSM; FIG. 3)40. The PDSM was initially discovered in HSF1 and subsequently found in many other proteins, especially transcriptional regulators such as HSF4, GATA1, myocyte-specific enhancer factor 2A (MEF2A) and SP3, which are substrates for both SUMO conjugation and Pro-directed kinases40,6062.

Recently, Mohideen and colleagues showed that a conserved basic patch on the surface of the SUMO-conjugating enzyme ubiquitin carrier protein 9 (UBC9; also known as UBE2I) discriminates between the phosphorylated and non-phosphorylated PDSM of HSF1 (REF. 63). Future studies will be directed at elucidating the molecular mechanisms for dynamic phosphorylation and UBC9-dependent SUMO conjugation in response to stress stimuli and establishing the roles of kinases, phosphatases and desumoylating enzymes in the heat shock response. The kinetics of phosphorylation-dependent sumoylation of HSF1 correlates inversely with the severity of heat stress, and, as the transactivation capacity of HSF1 is impaired by sumoylation and this PTM is removed when maximal HSF1 activity is required40, sumoylation could modulate HSF1 activity under moderate stress conditions. The mechanisms by which SUMO modification represses the transactivating capacity of HSF1, and the functional relationship of this PTM with other modifications that HSF1 is subjected to, will be investigated with endogenous substrate proteins.

Phosphorylation and sumoylation of HSF1 occur rapidly on heat shock, whereas the kinetics of acetylation are delayed and coincide with the attenuation phase of the HSF1 activation cycle. Stress-inducible acetylation of HSF1 is regulated by the balance of acetylation by p300–CBP (CREB-binding protein) and deacetylation by the NAD+-dependent sirtuin, SIRT1. Increased expression and activity of SIRT1 enhances and prolongs the DNA-binding activity of HSF1 at the human HSP70.1promoter, whereas downregulation of SIRT1 enhances the acetylation of HSF1 and the attenuation of DNA-binding without affecting the formation of HSF1 trimers42. This finding led to the discovery of a novel regulatory mechanism of HSF1 activity, whereby SIRT1 maintains HSF1 in a state that is competent for DNA binding by counteracting acetylation (FIG. 3). In the light of current knowledge, the attenuation phase of the HSF1 cycle is regulated by a dual mechanism: a dependency on the levels of HSPs that feed back directly by weak interactions with HSF1, and a parallel step that involves the SIRT1-dependent control of the DNA-binding activity of HSF1. Because SIRT1 has been implicated in caloric restriction and ageing, the age-dependent loss of SIRT1 and impaired HSF1 activity correlate with an impairment of the heat shock response and proteostasis in senescent cells, connecting the heat shock response to nutrition and ageing (see below).

HSF dynamics on the HSP70 promoter

For decades, the binding of HSF to the HSP70.1 gene has served as a model system for inducible transcription in eukaryotes. In D. melanogaster, HSF is constitutively nuclear and low levels of HSF are associated with the HSP70promoter before heat shock6466. The uninduced HSP70 promoter is primed for transcription by a transcriptionally engaged paused RNA polymerase II (RNAP II)67,68. RNAP II pausing is greatly enhanced by nucleosome formation in vitro, implying that chromatin remodelling is crucial for the release of paused RNAP II69. It has been proposed that distinct hydrophobic residues in the transactivation domain of human HSF1 can stimulate RNAP II release and directly interact withBRG1, the ATPase subunit of the chromatin remodelling complex SWI/SNF70,71. Upon heat shock, RNAP II is released from its paused state, leading to the synthesis of a full-length transcript. Rapid disruption of nucleosomes occurs across the entire HSP70 gene, at a rate that is faster than RNAP II-mediated transcription72. The nucleosome displacement occurs simultaneously with HSF recruitment to the promoter in D. melanogaster. Downregulation of HSF abrogates the loss of nucleosomes, indicating that HSF provides a signal for chromatin rearrangement, which is required for HSP70 nucleosome displacement. Within seconds of heat shock, the amount of HSF at the promoter increases drastically and HSF translocates from the nucleoplasm to several native loci, including HSP genes. Interestingly, the levels of HSF occupying the HSP70 promoter reach saturation soon after just one minute65,73.

HSF recruits the co-activating mediator complex to the heat shock loci, which acts as a bridge to transmit activating signals from transcription factors to the basal transcription machinery. The mediator complex is recruited by a direct interaction with HSF: the transactivation domain of D. melanogaster HSF binds to TRAP80(also known as MED17), a subunit of the mediator complex74. HSF probably has other macromolecular contacts with the preinitiation complex as it binds to TATA-binding protein (TBP) and the general transcription factor TFIIB in vitro75,76. In contrast to the rapid recruitment and elongation of RNAP II on heat shock, activated HSF exchanges very slowly at the HSP70 promoter. HSF stays stably bound to DNA in vivo and no turnover or disassembly of transcription activator is required for successive rounds of HSP70 transcription65,68.

Functional interplay between HSFs

Although HSF1 is the principal regulator of the heat shock response, HSF2 also binds to the promoters of HSP genes. In light of our current knowledge, HSF2 strictly depends on HSF1 for its stress-related functions as it is recruited to HSP gene promoters only in the presence of HSF1 and this cooperation requires an intact HSF1 DBD77. Nevertheless, HSF2 modulates, both positively and negatively, the HSF1-mediated inducible expression of HSP genes, indicating that HSF2 can actively participate in the transcriptional regulation of the heat shock response. Coincident with the stress-induced transcription of HSP genes, HSF1 and HSF2 colocalize and accumulate rapidly on stress into nuclear stress bodies (NSBs; BOX 2), where they bind to a subclass of satellite III repeats, predominantly in the human chromosome 9q12 (REFS 7880). Consequently, large and stable non-coding satellite III transcripts are synthesized in an HSF1-dependent manner in NSBs81,82. The function of these transcripts and their relationship with other HSF1 targets, and the heat shock response in general, remain to be elucidated.

 

Box 2

Nuclear stress bodies  

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u2.jpg

The cell nucleus is highly compartmentalized and dynamic. Many nuclear factors are diffusely distributed throughout the nucleoplasm, but they can also accumulate in distinct subnuclear compartments, such as nucleoli, speckles, Cajal bodies and promyelocytic leukaemia (PML) bodies136. Nuclear stress bodies (NSBs) are different from any other known nuclear bodies137,138. Although NSBs were initially thought to contain aggregates of denatured proteins and be markers of heat-shocked cells, their formation can be elicited by various stresses, such as heavy metals and proteasome inhibitors137. NSBs are large structures, 0.3–3 μm in diameter, and are usually located close to the nucleoli or nuclear envelope137,138. NSBs consist of two populations: small, brightly stained bodies and large, clustered and ring-like structures137.

NSBs appear transiently and are the main site of heat shock factor 1 (HSF1) and HSF2 accumulation in stressed human cells80. HSF1 and HSF2 form a physically interacting complex and colocalize into small and barely detectable NSBs after only five minutes of heat shock, but the intensity and size of NSBs increase after hours of continuous heat shock. HSF1 and HSF2 colocalize in HeLa cells that have been exposed to heat shock for one hour at 42°C (see the figure; confocal microscopy image with HSF1–green fluorescent protein in green and endogenous HSF2 in red). NSBs form on specific chromosomal loci, mainly on q12 of human chromosome 9, where HSFs bind to a subclass of satellite III repeats78,79,83. Stress-inducible and HSF1-dependent transcription of satellite III repeats has been shown to produce non-coding RNA molecules, called satellite III transcripts81,82. The 9q12 locus consists of pericentromeric heterochromatin, and the satellite III repeats provide scaffolds for docking components, such as splicing factors and other RNA-processing proteins139143.

HSF2 also modulates the heat shock response through the formation of heterotrimers with HSF1 in the NSBs when bound to the satellite III repeats83 (FIG. 4). Studies on the functional significance of heterotrimerization indicate that HSF1 depletion prevents localization of HSF2 to NSBs and abolishes the stress-induced synthesis of satellite III transcripts. By contrast, increased expression of HSF2 leads to its own activation and the localization of both HSF1 and HSF2 to NSBs, where transcription is spontaneously induced in the absence of stress stimuli. These results suggest that HSF2 can incorporate HSF1 into a transcriptionally competent heterotrimer83. It is possible that the amounts of HSF2 available for heterotrimerization with HSF1 influence stress-inducible transcription, and that HSF1–HSF2 heterotrimers regulate transcription in a temporal manner. During the acute phase of heat shock, HSF1 is activated and HSF1–HSF2 heterotrimers are formed, whereas upon prolonged exposures to heat stress the levels of HSF2 are diminished, thereby limiting heterotrimerization83. Intriguingly, in specific developmental processes such as corticogenesis and spermatogenesis, the expression of HSF2 increases spatiotemporarily, leading to its spontaneous activation. Therefore, it has been proposed that HSF-mediated transactivation can be modulated by the levels of HSF2 to provide a switch that integrates the responses to stress and developmental stimuli83 (FIG. 4). Functional relationships between different HSFs are emerging, and the synergy of DNA-binding activities among HSF family members offers an efficient way to control gene expression in a cell- and stimulus-specific manner to orchestrate the differential upstream signalling and target-gene networks.

Figure 4   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f4.gif

 

Interactions between different HSFs provide distinct functional modes in transcriptional regulation

A new member of the mammalian HSF family, mouse HSF3, was recently identified10. Avian HSF3 was shown to be activated at higher temperatures and with different kinetics than HSF1 (REF. 84), whereas in mice, heat shock induces the nuclear translocation of HSF3 and activation of stress-responsive genes other than HSP genes10. Future experiments will determine whether HSF3 is capable of interacting with other HSFs, potentially through heterocomplex formation. HSF4 has not been implicated in the heat shock response, but it competes with HSF1 for common target genes in mouse lens epithelial cells85, which will be discussed below. It is important to elucidate whether the formation of homotrimers or hetero trimers between different family members is a common theme in HSF-mediated transcriptional regulation.

 

HSFs as developmental regulators

Evidence is accumulating that HSFs are highly versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physioogical functions, especially during development. The initial observations using deletion experiments of the D. melanogaster Hsf gene revealed defective oogenesis and larvae development86. These effects were not caused by obvious changes in HSP gene expression patterns, which is consistent with the subsequent studies showing that basal expression of HSP genes during mouse embryogenesis is not affected by the lack of HSF1 (REF. 13). These results are further supported by genome-wide gene expression studies revealing that numerous genes, not classified as HSP genes or molecular chaperones, are under HSF1-dependent control87,88.

Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility13. Fertilized oocytes do not develop past the zygotic stage when HSF1-deficient female mice are mated with wild-type male mice, indicating that HSF1 is a maternal factor that is essential for early post-fertilization development89. Recently, it was shown that HSF1 is abundantly expressed in maturing oocytes, where it regulates specifically Hsp90α transcription90. The HSF1-deficient oocytes are devoid of HSP90α and exhibit a blockage of meiotic maturation, including delayed G2–M transition or germinal vesicle breakdown and defective asymmetrical division90. Moreover, intra-ovarian HSF1-depleted oocytes contain dysfunctional mitochondria and are sensitive to oxidative stress, leading to reduced survival91. The complex phenotype of Hsf1-knockout mice also demonstrates the involvement of HSF1 in placenta formation, placode development and the immune system15,85,92,93, further strengthening the evidence for a protective function of HSF1 in development and survival.

Both HSF1 and HSF2 are key regulators in the developing brain and in maintaining proteostasis in the central nervous system. Disruption of Hsf1 results in enlarged ventricles, accompanied by astrogliosis, neurodegeneration, progressive myelin loss and accumulation of ubiquitylated proteins in specific regions of the postnatal brain under non-stressed conditions94,95. The expression of HSP25 (also known as HSPB1) and α-crystallin B chain (CRYAB), which are known to protect cells against stress-induced protein damage and cell death, is dramatically decreased in brains lacking HSF1 (REF. 13). In contrast to HSF1, HSF2 is already at peak levels during early brain development in mice and is predominantly expressed in the proliferative neuronal progenitors of the ventricular zone and post-mitotic neurons of the cortical plate9699. HSF2-deficient mice have enlarged ventricles and defects in cortical lamination owing to abnormal neuronal migration9799. Incorrect positioning of superficial neurons during cortex formation in HSF2-deficient embryos is caused by decreased expression of the cyclin-dependent kinase 5 (CDK5) activator p35, which is a crucial regulator of the cortical migration signalling pathway100,101. The p35 gene was identified as the first direct target of HSF2 in cortex development99. As correct cortical migration requires the coordination of multiple signalling molecules, it is likely that HSF2, either directly or indirectly, also regulates other components of the same pathway.

 

Cooperativity of HSFs in development

In adult mice, HSF2 is most abundantly expressed in certain cell types of testes, specifically pachytene spermatocytes and round spermatids102. The cell-specific expression of HSF2 in testes is regulated by a microRNA, miR-18, that directly binds to the 3′ untranslated region (UTR) of HSF2 (J.K. Björk, A. Sandqvist, A.N. Elsing, N. Kotaja and L.S., unpublished observations). Targeting of HSF2 in spermatogenesis reveals the first physiological role for miR-18, which belongs to the oncomir-1 cluster associated mainly with tumour progression103. In accordance with the expression pattern during the maturation of male germ cells, HSF2-null male mice display several abnormal features in spermatogenesis, ranging from smaller testis size and increased apoptosis at the pachytene stage to a reduced amount of sperm and abnormal sperm head shape97,98,104. A genome-wide search for HSF2 target promoters in mouse testis revealed the occupancy of HSF2 on the sex chromosomal multi-copy genes spermiogenesis specific transcript on the Y 2 (Ssty2), Sycp3-like Y-linked (Sly) and Sycp3-like X-linked (Slx), which are important for sperm quality104. Compared with the Hsf2-knockout phenotype, disruption of both Hsf1 and Hsf2 results in a more pronounced phenotype, including larger vacuolar structures, more widely spread apoptosis and a complete lack of mature spermatozoa and male sterility105. The hypo thesis that the activities of HSF1 and HSF2 are intertwined and essential for spermatogenesis is further supported by our results that HSF1 and HSF2 synergistically regulate the sex chromosomal multi-copy genes in post-meiotic round spermatids (M.Å., A. Vihervaara, E.S. Christians, E. Henriksson and L.S., unpublished observations). Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are currently the only known transcriptional regulators during post-meiotic repression. These results, together with the earlier findings that HSF2 can also form heterotrimers with HSF1 in testes83, strongly suggest that HSF1 and HSF2 act in a heterocomplex and fine-tune transcription of their common target genes during the maturation of male germ cells.

HSF1 and HSF4 are required for the maintenance of sensory organs, especially when the organs are exposed to environmental stimuli for the first time after birth85,88. During the early postnatal period, Hsf1-knockout mice display severe atrophy of the olfactory epithelium, increased accumulation of mucus and death of olfactory sensory neurons88. Although lens development in HSF4-deficient mouse embryos is normal, severe abnormalities, including inclusion-like structures in lens fibre cells, appear soon after birth and the mice develop cataracts85,106,107. Intriguingly, inherited severe cataracts occurring in Chinese and Danish families have been associated with a mutation in the DBD of HSF4 (REF. 108). In addition to the established target genes, Hsp25Hsp70 and Hsp90, several new targets for HSF1 and HSF4, such as crystallin γF (Crygf), fibroblast growth factor 7 (Fgf7) and leukaemia inhibitory factor (Lif) have been found to be crucial for sensory organs85,88. Furthermore, binding of either HSF1 or HSF4 to the Fgf7 promoter shows opposite effects on gene expression, suggesting competitive functions between the two family members85. In addition to the proximal promoters, HSF1, HSF2 and HSF4 bind to other genomic regions (that is, introns and distal parts of protein-coding genes in mouse lens), and there is also evidence for either synergistic interplay or competition between distinct HSFs occupying the target-gene promoters109. It is possible that the different HSFs are able to compensate for each other to some extent. Thus, the identification of novel functions and target genes for HSFs has been a considerable step forward in understanding their regulatory mechanisms in development.

 

HSFs and lifespan

The lifespan of an organism is directly linked to the health of its tissues, which is a consequence of the stability of the proteome and functionality of its molecular machineries. During its lifetime, an organism constantly encounters environmental and physiological stress and requires an efficient surveillance of protein quality to prevent the accumulation of protein damage and the disruption of proteostasis. Proteotoxic insults contribute to cellular ageing, and numerous pathophysiological conditions, associated with impaired protein quality control, increase prominently with age11. From studies on the molecular basis of ageing, in which a wide range of different model systems and experimental strategies have been used, the insulin and insulin-like growth factor 1 receptor (IGF1R) signalling pathway, which involves the phosphoinositide 3-kinase (PI3K) and AKT kinases and the Forkhead box protein O (FOXO) transcription factors (such as DAF-16 in Caenorhabditis elegans), has emerged as a key process. The downregulation of HSF reduces the lifespan and accelerates the formation of protein aggregates in C. elegans carrying mutations in different components of the IGF1R-mediated pathway. Conversely, inhibition of IGF1R signalling results in HSF activation and promotes longevity by maintaining proteostasis110,111. These results have prompted many laboratories that use other model organisms to investigate the functional relationship between HSFs and the IGF1R signalling pathway.

The impact of HSFs on the lifespan of whole organisms is further emphasized by a recent study, in which proteome stability was examined during C. elegansageing112. The age-dependent misfolding and downregulation of distinct metastable proteins, which display temperature-sensitive missense mutations, was examined in different tissues. Widespread failure in proteostasis occurred rapidly at an early stage of adulthood, coinciding with the severely impaired heat shock response and unfolded protein response112. The age-dependent collapse of proteostasis could be restored by overexpression of HSF and DAF-16, strengthening the evidence for the unique roles of these stress-responsive transcription factors to prevent global instability of the proteome.

Limited food intake or caloric restriction is another process that is associated with an enhancement of lifespan. In addition to promoting longevity, caloric restriction slows down the progression of age-related diseases such as cancer, cardiovascular diseases and metabolic disorders, stimulates metabolic and motor activities, and increases resistance to environmental stress stimuli113. To this end, the dynamic regulation of HSF1 by the NAD+-dependent protein deacetylase SIRT1, a mammalian orthologue of the yeast transcriptional regulator Sir2, which is activated by caloric restriction and stress, is of particular interest. Indeed, SIRT1 directly deacetylates HSF1 and keeps it in a state that is competent for DNA binding. During ageing, the DNA-binding activity of HSF1 and the amount of SIRT1 are reduced. Consequently, a decrease in SIRT1 levels was shown to inhibit HSF1 DNA-binding activity in a cell-based model of ageing and senescence42. Furthermore, an age-related decrease in the HSF1 DNA-binding activity is reversed in cells exposed to caloric restriction114. These results indicate that HSF1 and SIRT1 function together to protect cells from stress insults, thereby promoting survival and extending lifespan. Impaired proteostasis during ageing may at least partly reflect the compromised HSF1 activity due to lowered SIRT1 expression.

 

Impact of HSFs in disease

The heat shock response is thought to be initiated by the presence of misfolded and damaged proteins, and is thus a cell-autonomous response. When exposed to heat, cells in culture, unicellular organisms, and cells in a multicellular organism can all trigger a heat shock response autonomously115117. However, it has been proposed that multicellular organisms sense stress differently to isolated cells. For example, the stress response is not properly induced even if damaged proteins are accumulated in neurodegenerative diseases like Huntington’s disease and Parkinson’s disease, suggesting that there is an additional control of the heat shock response at the organismal level118. Uncoordinated activation of the heat shock response in cells in a multicellular organism could cause severe disturbances of interactions between cells and tissues. In C. elegans, a pair of thermosensory neurons called AFDs, which sense and respond to temperature, regulate the heat shock response in somatic tissues by controlling HSF activity119,120. Moreover, the heat shock response in C. elegans is influenced by the metabolic state of the organism and is reduced under conditions that are unfavourable for growth and reproduction121. Neuronal control may therefore allow organisms to coordinate the stress response of individual cells with the varying metabolic requirements in different tissues and developmental stages. These observations are probably relevant to diseases of protein misfolding that are highly tissue-specific despite the often ubiquitous expression of damaged proteins and the heat shock response.

Elevated levels of HSF1 have been detected in several types of human cancer, such as breast cancer and prostate cancer122,123. Mice deficient in HSF1 exhibit a lower incidence of tumours and increased survival than their wild-type counterparts in a classical chemical skin carcinogenesis model and in a genetic model expressing an oncogenic mutation of p53. Similar results have been obtained in human cancer cells lines, in which HSF1 was depleted using an RNA interference strategy124. HSF1 expression is likely to be crucial for non-oncogene addiction and the stress phenotype of cancer cells, which are attributes given to many cancer cells owing to their high intrinsic level of proteotoxic and oxidative stress, frequent spontaneous DNA damage and aneuploidy125. Each of these features may disrupt proteostasis, raising the need for efficient chaperone and proteasome activities. Accordingly, HSF1 would be essential for the survival of cancer cells that experience constant stress and develop non-oncogene addiction.

 

HSFs as therapeutic targets

Given the unique role of HSF1 in stress biology and proteostasis, enhanced activity of this principal regulator during development and early adulthood is important for the stability of the proteome and the health of the cell. However, HSF1 is a potent modifier of tumorigenesis and, therefore, a potential target for cancer therapeutics125. In addition to modulating the expression of HSF1, the various PTMs of HSF1 that regulate its activity should be considered from a clinical perspective. As many human, age-related pathologies are associated with stress and misfolded proteins, several HSF-based therapeutic strategies have been proposed126. In many academic and industrial laboratories, small molecule regulators of HSF1 are actively being searched for (see Supplementary information S1 (table)). For example, celastrol, which has antioxidant properties and is a natural compound derived from the Celastreace family of plants, activates HSF1 and induces HSP expression with similar kinetics to heat shock, and could therefore be a potential candidate molecule for treating neurodegenerative diseases127,128. In a yeast-based screen, a small-molecule activator of human HSF1 was found and named HSF1A129. HSF1A, which is structurally distinct from the other known activators, activates HSF1 and enhances chaperone expression, thereby counteracting protein misfolding and cell death in polyQ-expressing neuronal precursor cells129. Triptolide, also from the Celastreace family of plants, is a potent inhibitor of the transactivating capacity of HSF1 and has been shown to have beneficial effects in treatments of pancreatic cancer xenografts130,131. These examples of small-molecule regulators of HSF1 are promising candidates for drug discovery and development. However, the existence of multiple mammalian HSFs and their functional interplay should also be taken into consideration when planning future HSF-targeted therapies.

 

Concluding remarks and future perspectives

HSFs were originally identified as specific heat shock-inducible transcriptional regulators of HSP genes, but now there is unambiguous evidence for a wide variety of HSF target genes that extends beyond the molecular chaperones. The known functions governed by HSFs span from the heat shock response to development, metabolism, lifespan and disease, thereby integrating pathways that were earlier strictly divided into either cellular stress responses or normal physiology.

Although the extensive efforts from many laboratories focusing on HSF biology have provided a richness of understanding of the complex regulatory mechanisms of the HSF family of transcription factors, several key questions remain. For example, what are the initial molecular events (that is, what is the ‘thermometer’) leading to the multistep activation of HSFs? The chromatin-based interaction between HSFs and the basic transcription machinery needs further investigation before the exact interaction partners at the chromatin level can be established. The activation and attenuation mechanisms of HSFs require additional mechanistic insights, and the roles of the multiple signal transduction pathways involved in post-translational regulation of HSFs are only now being discovered and are clearly more complex than anticipated. Although still lacking sufficient evidence, the PTMs probably serve as rheostats to allow distinct forms of HSF-mediated regulation in different tissues during development. Further emphasis should therefore be placed on understanding the PTMs of HSFs during development, ageing and different protein folding diseases. Likewise, the subcellular distribution of HSF molecules, including the mechanism by which HSFs shuttle between the cytoplasm and the nucleus, remains enigmatic, as do the movements of HSF molecules in different nuclear compartments such as NSBs.

Most studies on the impact of HSFs in lifespan and disease have been conducted with model organisms such as D. melanogaster and C. elegans, which express a single HSF. The existence of multiple members of the HSF family in mammals warrants further investigation of their specific and overlapping functions, including their extended repertoire of target genes. The existence of multiple HSFs in higher eukaryotes with different expression patterns suggests that they may have functions that are triggered by distinct stimuli, leading to activation of specific target genes. The impact of the HSF family in the adaptation to diverse biological environments is still poorly understood, and future studies are likely to broaden the prevailing view of HSFs being solely stress-inducible factors. To this end, the crosstalk between distinct HSFs that has only recently been uncovered raises obvious questions about the stoichiometry between the components in different complexes residing in different cellular compartments, and the mechanisms by which the factors interact with each other. Interaction between distinct HSF family members could generate new opportunities in designing therapeutics for protein-folding diseases, metabolic disorders and cancer.

 

  1. Role in the etiology of cancer

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Dan Tang,1 Md Abdul Khaleque,2 Ellen L. Jones,1 Jimmy R. Theriault,2 Cheng Li,3 Wing Hung Wong,3 Mary Ann Stevenson,2 and Stuart K. Calderwood1,2,4
Cell Stress Chaperones. 2005 Mar; 10(1): 46–58. doi:  10.1379/CSC-44R.1

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.

Heat shock proteins (HSPs) were first discovered as a cohort of proteins that is induced en masse by heat shock and other chemical and physical stresses in a wide range of species (Lindquist and Craig 1988Georgopolis and Welch 1993). The HSPs (Table 1) have been subsequently characterized as molecular chaperones, proteins that have in common the property of modifying the structures and interactions of other proteins (Lindquist and Craig 1988Beckmann et al 1990;Gething and Sambrook 1992Georgopolis and Welch 1993Netzer and Hartl 1998). Molecular chaperone function dictates that the HSP often interact in a stoichiometric, one-on-one manner with their substrates, necessitating high intracellular concentrations of the proteins (Lindquist and Craig 1988Georgopolis and Welch 1993). As molecules that shift the balance from denatured, aggregated protein conformation toward ordered, functional conformation, HSPs are particularly in demand when the protein structure is disrupted by heat shock, oxidative stress, or other protein-damaging events (Lindquist and Craig 1988;Gething and Sambrook 1992Georgopolis and Welch 1993). The HSP27, HSP40,HSP70, and HSP110 genes have therefore evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation (Voellmy 1994). HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress (Hickey and Weber 1982Landry et al 1982Li and Werb 1982Subjeck et al 1982Henics et al 1999) (Zhao et al 2002). Heat shock factor (HSF) proteins have been shown to interact with the promoters of many HSP genes and ensure prompt transcriptional activation in stress and equally precipitous switch off after recovery (Sorger and Pelham 1988Wu 1995). The hsf gene family includes HSF1 (hsf1), the molecular coordinator of the heat shock response, as well as 2 less well-characterized genes, hsf2 and hsf4(Rabindran et al 1991Schuetz et al 1991) (Nakai et al 1997). In addition to the class of HSPs induced by heat, cells also contain a large number of constitutively expressed HSP homologs, which are also listed in Table 1. The constitutive HSPs are found in a variety of multiprotein complexes containing both HSPs and cofactors (Buchner 1999). These include HSP10-HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with regulatory proteins within the cell (Netzer and Hartl 1998). HSP90 plays a particularly versatile role in cell regulation, forming complexes with a large number of cellular kinases, transcription factors, and other molecules (Buchner 1999Grammatikakis et al 2002).

 

Table 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1074571/bin/i1466-1268-10-1-46-t01.jpg

 

Heat shock protein family genes studied by microchip array analysis

Many tumor types contain high concentrations of HSP of the HSP28, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca et al 1993Yano et al 1999Cornford et al 2000Strik et al 2000Ricaniadis et al 2001Ciocca and Vargas-Roig 2002). We have concentrated here on HSP gene expression in prostate carcinoma. The progression of prostatic epithelial cells to the fully malignant, metastatic phenotype is a complex process and involves the expression of oncogenes as well as escape from androgen-dependent growth and survival (Cornford et al 2000). There is a molecular link between HSP expression and tumor progression in prostate cancer in that HSP56, HSP70, and HSP90 regulate the function of the androgen receptor (AR) (Froesch et al 1998Grossmann et al 2001). Escape from AR dependence during tumorigenesis may involve altered HSP-AR interactions (Grossmann et al 2001). The role of HSPs in tumor development may also be related to their function in the development of tolerance to stress (Li and Hahn 1981). Thermotolerance is induced in cells preconditioned by mild stress coordinately with the expression of high HSP levels (Landry et al 1982Li and Werb 1982Subjeck et al 1982). Elevated HSP expression appears to be a factor in tumor pathogenesis, and, among other mechanisms, this may involve the ability of individual HSPs to block the pathways of apoptosis and permit malignant cells to arise despite the triggering of apoptotic signals during transformation (Volloch and Sherman 1999). De novo HSP expression may also afford protection of cancer cells from treatments such as chemotherapy and hyperthermia by thwarting the proapoptotic influence of these modalities (Gabai et al 1998Hansen et al 1999Blagosklonny 2001Asea et al 2001Van Molle et al 2002). The mechanisms underlying HSP induction in tumor cells are not known but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment, which would be expected to lead to cellular stress.

Here, we have examined expression of hsf and HSP genes in immortalized normal human prostate epithelial cells and a range of prostate carcinoma cells obtained from human tumors at the mRNA and protein levels. Our aim was to determine whether hsf-HSP expression profiles are conserved in cells that express varying degrees of malignancy, under resting conditions and after heat and ionizing radiation. In addition, we have compared HSP expression profiles of a metastatic human prostate carcinoma cell line growing either in monolayer culture or as a tumor xenograft in nude mice. These studies were prompted by findings in our laboratory that prostate carcinoma cells are considerably more sensitive to heat-induced apoptosis in vivo growing as tumors compared with similar cells growing in tissue culture in vitro. Our studies show that, although the hsf-HSP expression profiles are similar in normal and malignant prostate-derived cells at the mRNA level, expression at the protein level was very different. HSF1 and HSP protein expression was highest in the 3 aggressively metastatic prostate cancer cell lines (PC-3, DU-145, and CA-HPV-10). Although the gene expression patterns of constitutive HSP differ enormously in PC-3 cells in vitro and in xenografts in vivo, stress induction of HSP genes is not markedly altered by exposure to the tumor microenvironment, indicating the hierarchical rank of the stress response that permits it to override other forms of regulation. ……

The experiments described here are largely supportive of the notion that HSP gene expression and HSF activity and expression are increased in more advanced stages of cancer (Fig 4). The most striking finding in the study was the elevation of HSF1 and HSP levels in aggressively malignant prostate carcinoma cell lines (Fig 4). It is significant that these changes in HSF and HSP levels would not have been predicted from microarray studies of HSF (Fig 3) and HSP (Fig 1) mRNA levels. The increased HSF levels observed in the metastatic prostate carcinoma cell lines in particular appear to be due to altered regulation of either mRNA translation or protein turnover (or both) (Figs 3 and ​and4).4). Although we do not at this stage know the mechanisms involved, 1 candidate could be differential activity of the proteosome in the metastatic cell lines: both HSF1 and HSF2 are targets for proteosomal degradation (Mathew et al 1998). Despite these differences in HSP expression between cells of varying degrees of malignancy under growth conditions, stress caused a major shift in HSP gene expression and activation of HSP40-1, HSP70-1A, HSP70-1B, HSP70-6 (HSP70B), DNA-J2–like, and HSP105 in all cells (Fig 2). Even in LnCap cells with minimal HSF1 and HSF2 expression, heat-inducible HSP70 protein expression was observed (Fig 4). Interestingly, we observed minimal induction of the HSP70B gene in LnCap cells: because the HSP70B promoter is known to be almost exclusively induced by stress through the HSE in its promoter, the findings may suggest that a mechanism for HSP70 induction alternative to HSF1 activation may be operative in LnCap cells (Schiller et al 1988). Increased HSP expression in cancer patients has been shown to signal a poor response to treatment by a number of modalities, suggesting that HSP expression is involved with development of resistance to treatment in addition to being involved in the mechanisms of malignant progression (Ciocca et al 1993Cornford et al 2000Yamamoto et al 2001Ciocca and Vargas-Roig 2002;Mese et al 2002). In addition, subpopulations of LnCap-derived cells, selected for enhanced capacity to metastasize, have been shown to express elevated levels of HSF1, HSP70, and HSP27 compared with nonselected controls (Hoang et al 2000). This may be highly significant because our studies indicate minimal levels of HSF1 and HSP in the poorly metastatic parent LnCap cells (Figs 1 and ​and4).4). Previous studies have also indicated that elevated HSP70 expression occurs at an early stage in cellular immortalization from embryonic stem cells (Ravagnan et al 2001). We had to use immortalized prostatic epithelial cells for our normal controls and may have missed a very early change in HSP expression during the immortalization process.

As indicated by the kinetic studies (Figs 5–7), HSPs are activated at a number of regulatory levels by stress in addition to transcriptional activation, and these may include stress-induced mRNA stabilization, differential translation, and protein stabilization (Hickey and Weber 1982Zhao et al 2002). HSF1 activity and HSP expression appear to be subject to differential regulation by a number of pathways at normal temperatures but are largely independent of such regulation when exposed to heat shock, which overrides constitutive regulation and permits prompt induction of this emergency response.

Growth of PC-3 cells in vivo as tumor xenografts was accompanied by a marked decrease in constitutive HSP expression (Figs 8 and ​and11).11). Decreased HSP expression was part of a global switch in gene expression that accompanies the switch of PC-3 cells from growth as monolayers in tissue culture to growth as tumors in vivo (D. Tang and S.K. Calderwood, in preparation). Many reports indicate changes in a wide range of cellular properties as cells grow as tumors, and these properties may reflect the remodeling of gene expression patterns. These changes may reflect adaptation to the chemical nature of the tumor microenvironment and the alterations in cell-cell interaction in growth as a tumor in vivo. Our studies also indicate the remarkable sturdiness of the heat shock response that remains intact in the PC-3 cells growing in vivo despite the global rearrangements in other gene expressions mentioned above (Figs 10 and ​and1111).

The elevation in HSF1 and HSP levels in cancer shown in our studies and in those of others and its association with a poor prognosis and inferior response to therapy suggests the strategy of targeting HSP in cancer therapy. Treatment with HSP70 antisense oligonucleotides, for instance, can cause tumor cell apoptosis on its own and can synergize with heat shock in cell killing (Jones et al 2004). Indeed, it has been shown that antagonizing heat-inducible HSP expression with quercitin, a bioflavonoid drug that inhibits HSF1 activation, or by using antisense oligonucleotides directed against HSP70 mRNA further sensitizes PC-3 cells to heat-induced apoptosis in vitro and leads to tumor regression in vivo (Asea et al 2001Lepchammer et al 2002Jones et al 2004) (A. Asea et al, personal communication). The strategy of targeting HSP expression or function in cancer cells may thus be indicated. Such a strategy might prove particularly effective because constitutive HSP expression is reduced in tumors, and this might be related to increased killing of PC-3 tumor cells by heat (Fig 12).

 

  1. Molecular chaperones in aging

Aging and molecular chaperones

Csaba So˝ti*, Pe´ter Csermely
Exper Geront 2003; 38:1037–1040  http://195.111.72.71/docs/pcs/03exger.pdf

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Molecular chaperones Chaperones are ubiquitous, highly conserved proteins (Hartl, 1996), either assisting in the folding of newly synthesized or damaged proteins in an ATP-dependent active process or working in an ATP-independent passive mode sequestering damaged proteins for future refolding or digestion. Environmental stress leads to proteotoxic damage. Damaged, misfolded proteins bind to chaperones, and liberate the heat shock factor (HSF) from its chaperone complexes. HSF is activated and transcription of chaperone genes takes place (Morimoto, 2002). Most chaperones, therefore, are also called stress or (after the archetype of experimental stress) heat shock proteins (Hsp-s).

Aging proteins—proteins of aging organisms During the life-span of a stable protein, various posttranslational modifications occur including backbone and side chain oxidation, glycation, etc. In aging organisms, the disturbed cellular homeostasis leads to an increased rate of protein modification: in an 80-year old human, half of all proteins may become oxidized (Stadtman and Berlett, 1998). Susceptibility to various proteotoxic damages is mainly increased due to dysfunction of mitochondrial oxidation of starving yeast cells (Aguilaniu et al., 2001). In prokaryotes, translational errors result in folding defects and subsequent protein oxidation (Dukan et al., 2000), which predominantly takes place in growth arrested cells (Ballesteros et al., 2001). Additionally, damaged signalling networks loose their original stringency, and irregular protein phosphorylation occurs (e.g.: the Parkinson disease-related a-synuclein also becomes phosphorylated, leading to misfolding and aggregation; Neumann et al., 2002).

Aging protein degradation Irreversibly damaged proteins are recognized by chaperones, and targeted for degradation. Proteasome level and function decreases with aging, and some oxidized, aggregated proteins exert a direct inhibition on proteasome activity. Chaperones also aid in lysosomal degradation. The proteolytic changes are comprehensively reviewed by Szweda et al. (2002). Due to the degradation defects, damaged proteins accumulate in the cells of aged organisms, and by aggregation may cause a variety of protein folding diseases (reviewed by So˝ti and Csermely, 2002a).

Aging chaperones I: defects in chaperone induction Damaged proteins compete with the HSF in binding to the Hsp90-based cytosolic chaperone complex, which may contribute to the generally observed constitutively elevated chaperone levels in aged organisms (Zou et al., 1998; So˝ti and Csermely, 2002b). On the contrary, the majority of the reports showed that stress-induced synthesis of chaperones is impaired in aged animals. While HSF activation does not change, DNA binding activity may be reduced during aging (Heydari et al., 2000). A number of signaling events use an overlapping network of chaperones not only to establish the activation-competent state of different transcription factors (e.g. steroid receptors), but also as important elements in the attenuation of respective responses. HSF transcriptional activity is also negatively influenced by higher levels of chaperones (Morimoto, 2002). Differential changes of these proteins in various organisms and tissues may lead to different extents of (dys)regulation. More importantly, the cross-talk between different signalling pathways through a shared pool of chaperones may have severe consequences during aging when the cellular conformational homeostasis is deranged (see below).

Aging chaperones II: defects in chaperone function   Direct studies on chaperone function in aged organisms are largely restricted to a-crystallin having a decreased activity in aged human lenses (Cherian and Abraham, 1995; Cherian-Shaw et al., 1999). In a recent study, an initial test of passive chaperone function of whole cytosols was assessed showing a decreased chaperone capacity in aged rats compared to those of young counterparts (Nardai et al., 2002). What can be the mechanism behind these deleterious changes in chaperone function? Chaperones may also be prone to oxidative damage, as GroEL is preferentially oxidized in growth-arrested E. coli (Dukan and Nystro¨m, 1999). Macario and Conway de Macario (2002) raised the idea of ‘sick chaperones’ in aged organisms in a recent review. Indeed, chaperones are interacting with a plethora of other proteins (Csermely, 2001a), which requires rather extensive binding surfaces. These exposed areas may make chaperones a preferential target for proteotoxic damage: chaperones may behave as ‘suicide proteins’ during aging, sacrificing themselves instead of ‘normal’ proteins. The high abundance of chaperones (which may constitute more than 5% of cellular proteins), and their increased constitutive expression in aged organisms makes them a good candidate for this ‘altruistic courtesy.’ It may be especially true for mitochondrial Hsp60, the role of which would deserve extensive studies.

Aging chaperones III: defects in capacity, the chaperone overload Another possible reason of decreased chaperone function is chaperone overload (Csermely, 2001b). In aging organisms, the balance between misfolded proteins and available free chaperones is grossly disturbed: increased protein damage, protein degradation defects increase the amount of misfolded proteins, while chaperone damage, inadequate synthesis of molecular chaperones and irreparable folding defects (due to posttranslational changes) decrease the amount of available free chaperones. Chaperone overload occurs, where the need for chaperones may greatly exceed the available chaperone capacity (Fig. 1). Under these conditions, the competition for available chaperones becomes fierce and the abundance of damaged proteins may disrupt the folding assistance to other chaperone targets, such as: (1) newly synthesized proteins; (2) ‘constantly damaged’ (mutant) proteins; and (3) constituents of the cytoarchitecture (Csermely, 2001a). This may cause defects in signal transduction, protein transport, immune recognition, cellular organization as well as the appearance of previously buffered, hidden mutations in the phenotype of the cell (Csermely, 2001b). Chaperone overload may significantly decrease the robustness of cellular networks, as well as shift their function towards a more stochastic behavior. As a result of this, aging cells become more disorganized, their adaptation is impaired.

Fig. 1. Chaperone overload: a shift in the balance between misfolded proteins and available free chaperones in aging organisms. The accumulation of chaperone substrates along with an impaired chaperone function may exhaust the folding assistance to specific chaperone targets and leads to deterioration in vital processes. Chaperone overload may significantly decrease the robustness of cellular networks, and compromise the adaptative responses. See text for details.

Senescent cells and chaperones The involvement of chaperones in aging at the cellular level is recently reviewed (So˝ti et al., 2003). Non-dividingsenescent-peripheral cells tend to have increased chaperone levels (Verbeke et al., 2001), and cannot preserve the induction of several chaperones (Liu et al., 1989), similarly to cells from aged animals. Activation and binding of HSF to the heat shock element is decreased in aged cells (Choi et al., 1990). Interestingly, cellular senescence seems to unmask a proteasomal activity leading to the degradation of HSF (Bonelli et al., 2001). Chaperone induction per se seems to counteract senescence. Repeated mild heat shock (a kind of hormesis) has been reported to delay fibroblast aging (Verbeke et al., 2001), though it does not seem to extend replicative lifespan. A major chaperone, Hsp90 is required for the correct function of telomerase, an important enzyme to extend the life-span of cells (Holt et al., 1999). Mortalin (mtHsp70/Grp75), a member of the Hsp70 family, produces opposing phenotypic effects related to its localization. In normal cells, it is pancytoplasmically distributed, and its expression causes senescence. Its upregulation and perinuclear distribution, however, is connected to transformation, probably via p53 inactivation. Mortalin also induces life-span extension in human fibroblasts or in C. elegans harboring extra copies of the orthologous gene (Kaul et al., 2002).

Aging organisms and chaperones: age-related diseases Unbalanced chaperone requirement and chaperone capacity in aged organisms helps the accumulation of aggregated proteins, which often cause folding diseases, mostly of the nervous system, due to the very limited proliferation potential of neurons. Over expression of chaperones often delays the onset or diminishes the symptoms of the disease (So˝ti and Csermely, 2002b). Other aging diseases, such as atherosclerosis and cancer are also related to chaperone action. Here space limitation precludes a detailed description of these rapidly developing fields, however, numerous recent reviews were published on these subjects, where the interested readers may find a good summary and several hints for further readings (Ferreira and Carlos, 2002; Neckers, 2002; Sarto et al., 2000; Wick and Xu, 1999).

 

Chaperones and Longevity

Increased chaperone induction leads to increased longevity (Tatar et al., 1997). Moreover, a close correlation exists between stress resistance and longevity in several long-lived C. elegans and Drosophila mutants (Lithgow and Kirkwood, 1996). As the other side of the same coin, damaged HSF has been found as an important gene to cause accelerated aging in C. elegans (Garigan et al., 2002). Caloric restriction, the only effective experimental manipulation known to retard aging in rodents and primates (Ramsey et al., 2000), restores age-impaired chaperone induction, while reversing the age-induced changes in constitutive Hsp levels (see So˝ti and Csermely, 2002a,b). These examples confirm the hypothesis that a better adaptation capacity to various stresses greatly increases the chances to reach longevity. 10. Conclusions and perspectives Aging can be defined as a multicausal process leading to a gradual decay of self-defensive mechanisms, and an exponential accumulation of damage at the molecular, cellular and organismal level. The protein oxidation, damage, misfolding and aggregation together with the simultaneously impaired function and induction of chaperones in aged organisms disturb the balance between chaperone requirement and availability. There are several important aspects for future investigation of this field: † the measurement of active chaperone function (i.e. chaperone-assisted refolding of damaged proteins) in cellular extracts does not have a well-established method yet; † we have no methods to measure free chaperone levels; † among the consequences of chaperone overload, changes in signal transduction, protein transport, immune recognition and cellular organization have not been systematically measured and/or related to the protein folding homeostasis of aging organisms and cells.

 

  1. Extracellular HSPs in inflammation and immunity

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1

Amir Kol,* Andrew H. Lichtman,† Robert W. Finberg,‡ Peter Libby,*† and Evelyn A. Kurt-Jones2‡
J  Immunol 2000; 164: 13–17.  https://www.researchgate.net/profile/Robert_Finberg/publication/12696457_Cutting_Edge_Heat_Shock_Protein_(HSP)_60_Activates_the_Innate_Immune_Response_CD14_Is_an_Essential_Receptor_for_HSP60_Activation_of_Mononuclear_Cells/links/53ee00460cf23733e80b21c0.pdf

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

There is increasing interest in the role of nontraditional mediators of inflammation in atherosclerosis (1). Recent studies from our laboratory have shown that chlamydial and human heat shock protein 60 (HSP60)3 colocalize in human atheroma (2), and either HSP60 induces adhesion molecule and cytokine production by human vascular cells and macrophages, in a pattern similar to that induced by Escherichia coli LPS (3, 4). These results suggested that HSP60 and LPS might share similar signaling mechanisms. CD14 is the major high-affinity receptor for bacterial LPS on the cell membrane of mononuclear cells and macrophages (5, 6). In addition to LPS, CD14 functions as a signaling receptor for other microbial products, including peptidoglycan from Gram-positive bacteria and mycobacterial lipoarabinomann (7, 8). CD14 is considered a pattern recognition receptor for microbial Ags and, with Toll-like receptor (TLR) proteins, an important mediator of innate immune responses to infection (9–14). We have examined the role of CD14 in the response of human monocytes and macrophages to HSP60.  …..

HSP may play a central role in the innate immune response to microbial infections. Because both microbes and stressed or injured host cells produce abundant HSP (36), and dying cells likely release these proteins, it is conceivable that HSP furnish signals that inform the innate immune system of the presence of infection and cell damage. The findings reported here, that human HSP60 induces IL-6 production by mononuclear cells and macrophages via the CD14, supports this hypothesis, suggesting that human HSP60 may act together with LPS or other microbial products to provoke innate immune responses.

Inflammation and immunity can contribute to the pathogenesis and complications of atherosclerosis (37). Moreover, the search for novel risk factors for atherosclerosis has revived the concept that microbial products might substantially contribute to the inflammatory reaction in the atheromatous vessel wall (38, 39). We have shown that chlamydial HSP60 colocalizes with human HSP60 in the macrophages of human atheroma (2). Therefore, bacterial and human HSP60, released from dying or injured cells during atherogenesis (40) or myocardial injury (41), may further promote local inflammation and possibly activate the innate immune system. Previous reports that immunization with mycobacterial HSP65 enhances atheroma formation in rabbits (42), have suggested an important role for HSPs in atherogenesis, particularly because the high degree of homology between HSPs of the same m.w. among different species might stimulate autoimmunity (43).

In conclusion, our findings, that CD14 mediates cellular activation induced by human HSP60 provide further insight into the molecular mechanisms by which HSP may activate the innate immune system and participate in atherogenesis and other inflammatory disorders.

DAMPs, PAMPs and alarmins: all we need to know about danger

Marco E. Bianchi1
J. Leukoc. Biol. 81: 1–5; 2007.   http://aerozon.ru/documents/publications/37_Bianche.pdf

Multicellular animals detect pathogens via a set of receptors that recognize pathogen associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term “alarmin” is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage associated molecular patterns (DAMPs).

Multicellular animals must distinguish whether their cells are alive or dead and detect when microorganisms intrude, and have evolved surveillance/defense/repair mechanisms to this end. How these mechanisms are activated and orchestrated is still incompletely understood, and I will argue that that these themes define a unitary field of investigation, of both basic and medical interest.

A complete system for the detection, containment, and repair of damage caused to cells in the organism requires warning signals, cells to respond to them via receptors and signaling pathways, and outputs in the form of physiological responses. Classically, a subset of this system has been recognized and studied in a coherent form: pathogen-associated molecular patterns (PAMPs) are a diverse set of microbial molecules which share a number of different recognizable biochemical features (entire molecules or, more often, part of molecules or polymeric assemblages) that alert the organism to intruding pathogens [1]. Such exogenous PAMPs are recognized by cells of the innate and acquired immunity system, primarily through toll-like receptors (TLRs), which activate several signaling pathways, among which NF-kB is the most distinctive. As a result, some cells are activated to destroy the pathogen and/or pathogen-infected cells, and an immunological response is triggered in order to produce and select specific T cell receptors and antibodies that are best suited to recognize the pathogen on a future occasion. Most of the responses triggered by PAMPs fall into the general categories of inflammation and immunity.

However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Tissues can be ripped, squashed, or wounded by mechanical forces, like falling rocks or simply the impact of one’s own body hitting the ground. Animals can be wounded by predators. In addition, tissues can be damaged by excessive heat (burns), cold, chemical insults (strong acids or bases, or a number of different cytotoxic poisons), radiation, or the withdrawal of oxygen and/or nutrients. Finally, humans can also be damaged by specially designed drugs, such as chemotherapeutics, that are meant to kill their tumor cells with preference over their healthy cells. Very likely, we would not be here to discuss these issues if evolution had not incorporated in our genetic program ways to deal with these damages, which are not caused by pathogens but are nonetheless real and common enough. Tellingly, inflammation is also activated by these types of insults. A frequently quoted reason for the similarity of the responses evoked by pathogens and trauma is that pathogens can easily breach wounds, and infection often follows trauma; thus, it is generally effective to respond to trauma as if pathogens were present. In my opinion, an additional reason is that pathogens and trauma both cause tissue and cell damage and thus trigger similar responses.

None of these considerations is new; however, a new awareness of the close relationship between trauma- and pathogenevoked responses emerged from the EMBO Workshop on Innate Danger Signals and HMGB1, which was held in February 2006 in Milano (Italy); many of the findings presented at the meeting are published in this issue of the Journal of Leukocyte Biology. At the end of the meeting, Joost Oppenheim proposed the term “alarmin” to differentiate the endogenous molecules that signal tissue and cell damage. Together, alarmins and PAMPs therefore constitute the larger family of damage-associated molecular patterns, or DAMPs.

Extranuclear expression of HMGB1 has been involved in a number of pathogenic conditions: sepsis [44], arthritis [45, 46], atherosclerosis [10], systemic lupus erythematosus (SLE) [47], cancer [48] and hepatitis [49, this issue]. Uric acid has been known to be the aethiologic agent for gout since the 19th century. S100s may be involved in arthritis [31, this issue] and psoriasis [50]. However, although it is clear that excessive alarmin expression might lead to acute and chronic diseases, the molecular mechanisms underlying these effects are still largely unexplored.

The short list of alarmins presented above is certainly both provisional and incomplete and serves only as an introduction to the alarmin concept and to the papers published in this issue of JLB. Other molecules may be added to the list, including cathelicidins, defensins and eosinophil-derived neurotoxin (EDN) [51], galectins [52], thymosins [53], nucleolin [54], and annexins [55; and 56, this issue]; more will emerge with time. Eventually, the concept will have to be revised and adjusted to the growing information. Indeed, I have previously argued that any misplaced protein in the cell can signal damage [57], and Polly Matzinger has proposed that any hydrophobic surface (“Hyppo”, or Hydrophobic protein part) might act as a DAMP [58]. As most concepts in biology, the alarmin category serves for our understanding and does not correspond to a blueprint or a plan in the construction of organisms. Biology proceeds via evolution, and evolution is a tinkerer or bricoleur, finding new functions for old molecules. In this, the reuse of cellular components as signals for alerting cells to respond to damage and danger, is a prime example.

 

  1. Role of heat shock and the heat shock response in immunity and cancer

 

Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

Stuart K. Calderwood,1,* Ayesha Murshid,1 and Jianlin Gong1
Front Immunol. 2012; 3: 75.  doi:  10.3389/fimmu.2012.00075

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock protein, vaccine, inflammation, antigen presentation

Heat shock proteins (HSP) were first discovered as a group of polypeptides whose level of expression increases to dominate the cellular proteome after stress (Lindquist and Craig, 1988). These increases in HSPs synthesis correlate with a marked resistance to potentially toxic stresses such as heat shock (Li and Werb,1982). The finding that such proteins have extracellular immune functions suggested that, as highly abundant intracellular proteins they could be prime candidates as danger signals to the immune response (Srivastava and Amato,2001). There are several human HSP gene families with known immune significance and their classification is reviewed in Kampinga et al. (2009). These include the HSPA (Hsp70) family, which includes the HPA1A and HSPA1B genes encoding the two major stress-inducible Hsp70s, that together are often referred to as Hsp72. When referring to Hsp70 in this chapter, we generally refer to the products of these two genes. The Hsp70 family also includes two other members with immune function – HSPA8 and HSPA5 genes, whose protein products are known as Hsc70 the major constitutive Hsp70 family member and Grp78, a key ER-resident protein. In addition two more Hsp70 related genes have immune significance and these include HSPH2 (Hsp110) and HSPH4 the ER-resident class H protein Grp170. The Hsp90 family also has major functions in tumor immunity and these include HSPC2 and HSPC3, which encode the major cytoplasmic proteins Hsp90a and Hsp90b, and HSPC4 that encodes ER chaperone Grp94. In addition, the product of the HSPD1 gene, the mitochondrial chaperone Hsp60 has some immunological functions. Mice have been shown to encode orthologs of each of these genes (Kampinga et al., 2009).

It has been suggested that many of the HSPs have the property of damage associated molecular patterns (DAMPs), inducers of sterile inflammation and innate immunity (Kono and Rock, 2008). The additional discovery that intracellular HSPs function as molecular chaperones and can bind to a wide spectrum of intracellular polypeptides further indicated that they could play a broad role in the immune response and might mediate both innate immunity due to their status as DAMPs and adaptive immunity by chaperoning antigens.

Heat shock proteins are currently employed as vaccines in cancer immunotherapy (Tamura et al., 1997; Murshid et al., 2011a). The rationale behind the approach is that if HSPs can be extracted from tumor tissue bound to the polypeptides which they chaperone during normal metabolism, they may retain antigenic peptides specific to the tumor (Noessner et al., 2002; Srivastava, 2002; Wang et al., 2003; Enomoto et al., 2006; Gong et al., 2010). Indeed, vaccines based on Hsp70, Hsp90, Grp94, Hsp110, and Grp170 polypeptide complexes have been used successfully to immunize mice to a range of tumor types and Hsp70 and Grp94 vaccines have undergone recent clinical trials (rev: Murshid et al., 2011a). These effects of the HSP vaccines on tumor immunity appear to be mediated largely to the associated, co-isolated tumor polypeptides, although in the case of Grp94 this question is still controversial and tumor regression was observed in mice treated with the chaperone devoid of its peptide binding domain (Udono and Srivastava, 1993; Srivastava, 2002; Nicchitta, 2003; Chandawarkar et al., 2004; Nicchitta et al.,2004). Use of such HSP vaccines is potentially a powerful approach to tumor immunotherapy as the majority of the antigenic repertoire of most individual tumor cells is unknown (Srivastava and Old, 1988; Srivastava, 1996). Individual cancer cells are likely to take a lone path in accumulating a spectrum of random mutations. Although some mutations are functional, permitting cells to become transformed and to progress into a highly malignant state, many such changes are likely to be passenger mutations not required to drive tumor growth (Srivastava and Old, 1988; Srivastava, 1996). Some of these individual mutant sequences will be novel antigenic epitopes and together with the few known shared tumor antigens comprise an “antigenic fingerprint” for each individual tumor (Srivastava,1996). Accumulation of mutations in cancer appears to be related to, and may drive the increases in HSPs observed in many tumors (Kamal et al., 2003; Whitesell and Lindquist, 2005; Trepel et al., 2010). As the mutant conformations of tumor proteins are “locked in” due to the covalent nature of the alterations, cancer cells appear to be under permanent proteotoxic stress and rich in HSP expression (Ciocca and Calderwood, 2005). For tumor immunology these conditions may offer a therapeutic opportunity as individual HSPs, whose expression is expanded in cancer will chaperone a cross-section of the “antigenic fingerprint” of the individual tumors (Murshid et al., 2011a). This approach was first utilized by Srivastava (20002006) and led to the development of immunotherapy using HSP–peptide complexes.

In addition to using HSP–peptide complexes extracted from tumors, in cases where tumor antigens are known, these can be directly loaded onto purified or recombinant HSPs and the complex used as a vaccine. This procedure has been carried out successfully in the case of the “large HSPs,” Hsp110 and Grp170 (Manjili et al., 20022003). A variant of this approach employs the molecular engineering of tumor antigens in order to produce molecular chaperone-fusion genes which encode products in which the HSP is fused covalently to the antigen. The fusion proteins are then employed as vaccines. This approach was pioneered by Young et al. who showed that a fusion between mycobacterial Hsp70 and ovalbumin could induced cytotoxic lymphocytes (CTL) in mice with the capacity to kill Ova-expressing cancer cells (Suzue et al., 1997). The vaccines could be used effectively without adjuvant and adjuvant properties were ascribed to the molecular chaperone component of the fusion protein. Subsequent studies have confirmed the utility of the approach in targeting common tumor antigens such as the melanoma antigen Mage3 (Wang et al., 2009).

HSPs and Immunosurveillance in Cancer

The question next arises as to the role of endogenous HSPs, with or without bound antigens in immunosurveillance of cancer cells. Although the immune system can recognize tumor antigens and generate a CTL response, most cancers evade immune cell killing by a range of strategies (van der Bruggen et al., 1991; Pardoll,2003). These include the down-regulation of surface MHC class I molecules by individual tumor cells and release of immunosuppressive IL-10 by tumors (Moller and Hammerling, 1992; Chouaib et al., 2002). Tumors in vivo also appear to attract a range of hematopoietic cells with immunosuppressive action including regulatory CD4+CD25+FoxP3+ T cells (Treg), M2 macrophages, myeloid-derived suppressor cells (MDSC) and some classes of natural killer cells (Pekarek et al.,1995; Terabe et al., 2005; Mantovani et al., 2008; Marigo et al., 2008). The tumor milieu also contain a small fraction of cells of mesenchymal origin identified by surface fibroblast activation protein-a (FAP cells) that suppress antitumor immune responses (Kraman et al., 2010). Endogenous tumor HSPs may also participate in immune suppression. Although the majority of the HSPs function as intracellular molecular chaperones, a fraction of these proteins can be released from cells even under unstressed conditions and may participate in immune functions (rev: Murshid and Calderwood, 2012). Intracellular Hsp70 can be actively secreted from tumor cells in either free form or packaged into lipid-bounded structures called exosomes (Mambula and Calderwood, 2006b; Chalmin et al., 2010). In addition Hsp70 and Hsp90 can also be found associated with the surfaces of tumor cells where they can function as molecular chaperones or as recognition structures for immune cells (Sidera et al., 2008; Qin et al., 2010; Multhoff and Hightower, 2011). As Hsp70 was shown in a number of earlier studies to be pro-inflammatory due to its interaction with pattern recognition receptors such as Toll-like receptors 2 and 4 (TLR2 and TLR4), these findings might suggest, as mentioned above, that Hsp70 released by tumors could be pro-inflammatory and possess the properties of DAMPs (Asea et al., 20002002; Vabulas et al., 2002). However, subsequent studies indicated that a portion of the TLR4 activation detected in the earlier reports, involving exposure of monocytes, macrophages, or dendritic cells (DC) to HSPs in vitro may be due to trace contamination with bacterial pathogen associated molecular patterns (PAMPs), potent TLR activators (Tsan and Gao,2004). In spite of these drawbacks, an overwhelming amount of evidence now seems to indicate the interaction of Hsp70 and other HSPs with TLRs (particularly TLR4) in vivo – in a wide range of physiological and pathological conditions, leading to acute inflammation in many conditions (Chase et al., 2007; Wheeler et al., 2009; see Appendix for a full list of references). Thus both TLR2 and TLR4 appear to be important components of inflammatory responses to Hsp70 under many pathophysiological conditions. In cancer therapy it has been shown that autoimmunity can be triggered in mice through necrotic killing of melanocytes engineered to overexpress Hsp70; such treatment led to the concomitant immune destruction of B16 melanoma tumors that share patterns of antigen expression with the killed melanocytes (Sanchez-Perez et al., 2006). Hsp70 appears to play an adjuvant role in this form of therapy through its interaction with TLR4 and induction of the cytokine TNF-a (Sanchez-Perez et al., 2006). However, despite these findings it has also been shown that depletion of Hsp70 in cancer cells can, in the absence of other treatments lead to tumor regression by inducing antitumor immunity (Rerole et al., 2011). This effect appears to be due to the secretion by cancer cells of immunosuppressive exosomes containing Hsp70 that activate MDSC and lead to local immunosuppression (Chalmin et al., 2010). Under normal circumstances therefore, release of endogenous Hsp70 into the extracellular microenvironment may be a component of the tumor defenses against immunosurveillance. Extracellular Hsp60 has also been shown be immunomodulatory and can increase levels of FoxP3 Treg in vitro and suppress T cell-mediated immunity (de Kleer et al., 2010; Aalberse et al., 2011).

The pro-inflammatory properties of extracellular HSPs may be more evident underin vivo situations particularly in the context of tissue damage (Sanchez-Perez et al.,2006). For instance when elevated temperatures were used to boost Hsp70 release from Lewis Lung carcinoma cells in vivo, antitumor immunity was activated along with release of chemokines CCL2, CCL5, and CCL10, in a TLR4-dependent manner, leading to attraction of DC and T cells into the tumor (Chen et al., 2009). Thus under resting conditions, the tumor milieu appears to be a specialized immunosuppressive environment, rich in inhibitory cells such as Treg, MDSC, and M2 macrophages and inaccessible to “exhausted” CD8+ T cells that often fail to penetrate the tumor microcirculation. However, under inflammatory conditions involving necrotic cell killing of tumor cells, extracellular HSPs may be able to amplify the anticancer immune response, intracellular HSPs may be released to further increase such a response and CTL may triggered to penetrate the tumor milieu, inducing antigen-specific cancer cell killing (Evans et al., 2001; Mambula and Calderwood, 2006a; Sanchez-Perez et al., 2006; Chen et al., 2009).

 

HSP-Based Anticancer Vaccines

It is apparent that a number of HSP types, conjugated to peptide complexes (HSP.PC) from cancer cells form effective bases for immunotherapy approaches with unique properties, as mentioned above (Calderwood et al., 2008; Murshid et al., 2011a). The immunogenicity of most HSP.PC appears to involve the ability of the HSPs to sample the tumor “antigenic fingerprint,” deliver the antigens to antigen presenting cells (APC) such as DC and stimulate activation of CTL (Tamura et al., 1997; Singh-Jasuja et al., 2000b; Wang et al., 2003; Murshid et al.,2010). A number of studies show that HSPs can chaperone tumor antigens and deliver them to the appropriate destination – MHC class I molecules on the DC surface (Singh-Jasuja et al., 2000a,b; Srivastava and Amato, 2001; Delneste et al.,2002; Enomoto et al., 2006; Gong et al., 2009). In addition, Hsp70 has been shown to chaperone viral antigenic peptides and increase cross priming of human CTL under ex vivo conditions (Tischer et al., 2011). However, it is still far from clear how the process of HSP-mediated cross priming unfolds. For instance, the CD8+ expressing DC subpopulation in lymph nodes is regarded as the primary cross-presenting APC (Heath and Carbone, 2009). It is not however currently known whether the CD8+ DC subset or other peripheral or lymph-node resident, DC interact with HSP vaccines to induce cross presentation. HSPs appear to be able to enter APC, such as mouse bone marrow derived DC (BMDC) and human DC in a receptor-mediated manner (Basu et al., 2001; Delneste et al., 2002; Gong et al.,2009; Murshid et al., 2010). However, no unique endocytosing HSP receptor has emerged and HSP–antigen complexes appear instead to be taken up by proteins with “scavenger” function such as LOX-1, SRECI, and CD91 that can each take up a wide range of extracellular ligands (Basu et al., 2001; Delneste et al., 2002; Theriault et al., 2006; Murshid et al., 2010). A pathway for Hsp90–peptide (Hsp90.PC) uptake has been characterized in mouse BMDC by scavenger receptor SRECI (Murshid et al., 2010). SRECI is able to mediate the whole process of Hsp90.PC endocytosis, trafficking through the cytoplasm to the sites of antigen processing and presentation of antigens to CD8+ T lymphocytes on MHC class I molecules (Murshid et al., 2010). This process is known as antigen cross presentation (Kurts et al., 2010). It is not currently clear what the relative contribution to antigen cross presentation of the various HSP receptors might be under in vivo conditions. It may be that each receptor class contributes to an individual aspect of CTL activation by HSP peptide complexes although a definitive understanding may await studies in mice deficient in each receptor class.

 

HSPs and CTL Programming

It is evident that that HSPs can mediate antigen cross presentation and activate CD8+ T lymphocytes. However, presentation of tumor antigens by DC is not sufficient for CTL programming and, in the absence of co-stimulatory molecules and innate immunity, the “helpless” CD8+ cells will cease to proliferate abundantly and will most likely undergo apoptosis (Schurich et al., 2009; Kurts et al., 2010). One mechanism for enhancing CTL programming involves activation of the TLR pathways that lead to synthesis of co-stimulatory molecules (Rudd et al.,2009; Yamamoto and Takeda, 2010). The co-stimulatory molecules, including CD80 and CD86 then become expressed on the DC cell surface and amplify the signals induced by binding of the T cell receptor on CD8+ T cells to MHC class I peptide complexes on the presenting DC (Parra et al., 1995; Rudd et al., 2009). This process is important in pathogen infection in which microbially derived antigens are encountered in the presence of inflammatory PAMPs that can activate innate immune transcriptional networks. Originally it had been thought that HSPs could provide analogous stimulation through their suspected activity as DAMPs and their inbuilt ability to trigger innate immunity through TLR2 and TLR4 on DC (Asea et al., 20002002; Vabulas et al., 2002). (The potential role of HSPs as DAMPs has been the subject of a recent review: van Eden et al., 2012). Subsequent studies on the capacity of HSPs to bind TLRs do not indicate avid binding of Hsp70 to either TLR2 or TLR4 when expressed in cells deficient in HSP receptors in vitro (Theriault et al., 2006). In vivo however, TLR signaling is essential for Hsp70 vaccine-induced tumor cell killing. Studies of tumor-bearing mice treated with an Hsp70 vaccine in vivo indicated that vaccine function is depleted by knockout of the TLR signaling intermediate Myd88 and completely abrogated by double knockout of TLR2 and TLR4 (Gong et al., 2009). These findings were somewhat complicated by the fact that TLR4 is involved in upstream regulation of the expression of Hsp70 receptor SRECI, but do strongly implicate a role for these receptors in amplifying immune signaling by Hsp70 vaccines and Hsp70-based immunotherapy (Sanchez-Perez et al., 2006; Gong et al., 2009). It is still not clear to what degree HSPs are capable of providing a sturdy DC maturing signal through TLR2/TLR4. The potency of HSP anticancer vaccines could potentially be improved by addition of PAMPs such as CpG DNA shown to activate TLR9, or double stranded RNA that can activate TLR3 (Murshid et al., 2011a). As mentioned, one contradictory factor in the earlier studies was that, although TLR2 and TLR4 are required for a sturdy Hsp70 vaccine-mediated immune response, direct binding of Hsp70 to these receptors was not observed (Theriault et al., 2006; Gong et al., 2009; Murshid et al., 2012). A rationale for these findings might be that HSPs can activate TLR signaling indirectly through primary binding to established HSP receptors such as LOX-1 and SRECI which secondarily recruit and activate the TLRs (Murshid et al., 2011b). Both of these scavenger receptors bind to TLR2 upon stimulation and activate TLR2-based signaling (Jeannin et al., 2005; A. Murshid and SK Calderwood, in preparation). In addition, we have found that Hsp90–SRECI complexes move to the lipid raft compartment of the cell, an environment highly enriched in TLR2 and TLR4 (Triantafilou et al., 2002; Murshid et al., 2010).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342006/bin/fimmu-03-00075-g001.jpg

Heat shock protein–peptide complexes extracted from tumor cells interact with endocytosing receptors (HSP-R) such as SRECI or signaling receptors (TLR) such as TLR4 on DC. SREC1 mediates uptake and intracellular processing of antigens and the presentation of resulting peptides on surface MHC class I and MHC class II proteins. MHC class II receptor–peptide complexes then bind to T cell receptors on CD4+ cells. One consequence of binding is interaction of CD40 ligand on the MHC class II cell with CD40 on the DC leading to the licensing interaction that results in enhanced expression of co-stimulatory proteins on the DC cell surface. The licensed DC may then interact with CD8+ T cells through T cell interaction with MHC class I peptide complexes. This effect will be enhanced by simultaneous interaction of CD80 or Cd86 co-stimulatory complexes on the DC with CD28 on the CD8+ cells, leading to effective CD8+ CTL that can lyse tumor cells. T cell programming can also be amplified by signals emanating from activated TLR that can boost levels of CD80 and CD86 as well as inflammatory cytokines (not shown).

 

Hsp70, Cell Damage, and Inflammation

The question of whether Hsp70 acts as DAMP and could by itself induce an inflammatory response in cancer patients in vivo is still open. However, some recent studies by Vile et al. using a gene therapy approach may shed some light on the inflammatory role of Hsp70 in tumor therapy. In this approach, as mentioned above, normal murine tissues were engineered to express high Hsp70 levels then subjected to treatments that lead to necrotic killing. The aim was to stimulate an autoimmune response that could lead to bystander immune killing of tumor cells that share the antigenic repertoire as the killed normal cells (Sanchez-Perez et al.,2006). In the initial studies, normal melanocytes were preloaded with Hsp70 plasmids and then necrotic cell death was triggered (Daniels et al., 2004). This treatment led to T cell-mediated immune killing of syngeneic B16 melanoma cells transplanted at a distant site in the mouse, presumably in response to antigens shared by the killed normal melanocytes and melanoma cell (Daniels et al., 2004). This effect only occurred when melanocytes were induced to undergo necrosis and Hsp70 levels were elevated, indicating a role for high levels of Hsp70 in the tumor specific immune response. Interestingly, these conditions did not lead to a prolonged autoimmune response, an effect mediated by the induction of a delayed Treg response (Srivastava, 2003; Daniels et al., 2004). It is notable that some early studies of chaperone-based tumor vaccines in animal models demonstrated a primary CTL response to tumors in response to treatment followed by delayed activation of a Treg reaction, and that chaperone levels must be carefully titrated for effective induction of tumor immunity (Udono and Srivastava, 1993; Liu et al.,2009). The role of Hsp70 in autoimmune rejection of tumors was also investigated in prostate cancer (Kottke et al., 2007). Ablation of normal prostate cells by necrotic killing with fusogenic viruses in the absence of Hsp70 elevation led to the induction of the cytokines IL-10 and TGF-b in the mouse prostate and a Treg response. However, when Hsp70 levels were elevated in these cells, IL-10, TGF-b, and IL-6 were induced simultaneously, the IL-6 component leading to further induction of IL-17, a profound Th17 response and tumor rejection (Kottke et al.,2007). Thus elevated levels of Hsp70, presumably released from cells undergoing necrosis can influence the local cytokine patterns and lead to an inflammatory statein vivo. Interestingly, these results seem to be tissue specific as inflammatory killing of pancreatic cells even in the presence of elevated Hsp70 did not provoke IL-6 release, a Th17 response or tumor rejection and the Treg response dominated under these conditions (Kottke et al., 2009). Thus the role of Hsp70 in tissue inflammation and tumor rejection seems to require elevated concentrations of extracellular chaperones, significant levels of necrotic cell killing, and tissue specific cytokine release.

Conclusion

  • Earlier studies investigating HSP vaccines considered such structures to be the “Swiss penknives” of immunology able to deliver antigens directly to APC and confer a maturing signal that could render DC able to effectively program CTL (Srivastava and Amato, 2001; Noessner et al., 2002). It is well established now that Hsp70, Hsp90, Hsp110, and GRP170 can chaperone tumor antigens and activate antigen cross presentation (Murshid et al., 2011a). In addition, HSPs were thought to be DAMPs with ability to strongly activate TLR signaling and innate immunity (Asea et al., 2000). However, although there is compelling evidence to indicate that Hsp70, for instance can interact with TLR4 under a number of pathological situations (see Appendix, Sanchez-Perez et al., 2006), it remains unclear whether free Hsp70 binds directly to the Toll-like receptor and induces innate immunity in the absence of other treatments in vitro(Tsan and Gao, 2004).
  • Elevated levels of extracellular HSPs appear to have the capacity to amplify the effects of inflammatory signals emanating from necrotic cells in vivoin a TLR4-dependent manner (Daniels et al., 2004; Sanchez-Perez et al., 2006; Kottke et al., 2007). In the presence of cell injury and death, elevated levels of Hsp70 appear to increase the production of inflammatory signals that involve cytokines such as IL-6 and IL-17 and lead to a specific T cell-mediated immune response to tumor cells sharing antigens with the dying cells (Kottke et al., 2007). The mechanisms involved in these processes are not clear although one possibility is that HSPs can induce the engulfment of necrotic cells. Hsp70 has been shown to increase bystander engulfment of a variety of structures (Wang et al., 2006a,b). In addition, tumor cells treated with elevated temperatures release inflammatory chemokines in an Hsp70 and TLR4-dependent mechanisms and this effect may be significant in CTL programming and tumor cell killing (Chen et al., 2009). Our studies indicate that CTL induction by Hsp70 vaccines in vivo has an absolute requirement for TLR2 and TLR4 suggesting that at least in vivo HSPs can trigger innate immunity through TLR signaling (Gong et al., 2009).
  • HSPs appear also to be able to direct antigen presentation through the class II pathway in DC and may stimulate T helper cells (Gong et al., 2009). It may thus be possible that HSPs participate in DC licensing and reinforce CTL programming during exposure to HSP vaccines. Future studies will address these questions.
  • A further interesting consideration is whether HSPs released from untreated tumor cells enhance or depress tumor immunity. One initial study shows that Hsp70 released from tumor cells in exosomes can strongly decrease tumor immunity through effects on MDSC (Chalmin et al., 2010). Further studies will be required to make a definitive statement on these questions.

 

  1. Protein aggregation disorders and HSP expression

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Christopher J. Cummings1,5, Michael A. Mancini3, Barbara Antalffy4, Donald B. DeFranco7, Harry T. Orr8 & Huda Y. Zoghbi1,2,6
Nature Genetics 19, 148 – 154 (1998) http://dx.doi.org:/10.1038/502

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease

Andreas Wyttenbach, Jenny Carmichael, Jina Swartz, Robert A. Furlong, Yolanda Narain, Julia Rankin, and David C. Rubinsztein*
https://www.researchgate.net/profile/David_Rubinsztein/publication/24447892_Effects_of_heat_shock_heat_shock_protein_40_(HDJ2)_and_proteasome_inhibition_on_protein_aggregation_in_cellular_models_of_Huntington’s_disease/links/00b7d528b80aab69bb000000.pdf

Huntington’s disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGypolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2yHSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2yHSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2yHSDJ in protein folding.

 

  1. Hsp70 in blood cell differentiation.

 

Apoptosis Versus Cell Differentiation -Role of Heat Shock Proteins HSP90, HSP70 and HSP27

David Lanneau, Aurelie de Thonel, Sebastien Maurel, Celine Didelot, and Carmen Garrido
Prion. 2007 Jan-Mar; 1(1): 53–60.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs

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Introduction

Stress or heat shock proteins (HSPs) were first discovered in 19621 as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.2 Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.3 Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α’s functions.4 HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.7 HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.8 This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.9

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.10 Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.11 One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.12Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases15 (Fig. 1).

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/figure/F1/

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and 

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.16 It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.

 

HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.2,1921 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.2224 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).

 

HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.25

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.26 A direct interaction between Akt and HSP90 was reported later.27 Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.26 Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.30 When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.27 Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.26 Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.31 HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.32 Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.33 The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p5334 and the transcription factors Her2 and Hif1α.35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.37

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,23 suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.38

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.

 

HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.39Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.40 In cancer cells, depletion of HSP70 results in spontaneous apoptosis.41

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation44 in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.45

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.46

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.47However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.48

HSP70 also prevents cell death in conditions in which caspase activation does not occur.49 Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.52 AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.53 HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation54 but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.55 During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.56

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).57 Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-150 and AIF binding,51 it is dispensable for JNK60 or GATA-161binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.61

 

HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.6466 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.65 In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.67

HSP27 can also interfere with caspase activation upstream of the mitochondria.66This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin68preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release66 (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,69 to decrease reactive oxygen species cell content,19 and to neutralize the toxic effects of oxidized proteins.70 These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.71

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.72 HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.73 In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).74 Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival75 while that of p27kip1facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.76

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.77 Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.78 In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary73,79 and it is its phosphorylated form that protects against neurotoxicity.80

 

HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.81 In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.82 In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.83

In leukemic cells HSP27 has been described as a pre-differentiation marker84because its induction occurs early during differentiation.8588 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes89 and for C2C12 muscle cells.90 This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G0/G1).19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells’ formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.91 HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.61 HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.18

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate92 and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.93 This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,94 RIP32 or Rb.95 Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.96 In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.97 In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.98 In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.4

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.99 In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G1 block. These G1 arrested cells do not differentiate but instead die by apoptosis.100

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.101Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.102 In breast cancer cells, 17-AAG-induced G1 block is accompanied by differentiation followed by apoptosis.103 The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.104

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.105 Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.16,17Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.61 Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.106It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.18 Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.

 

Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells’ aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells’ tumorigenicty and their depletion can induce a spontaneous regression of the tumors.24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.108 However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.

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HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death inC. elegans

A new pathway for non-apoptotic cell death

The results presented here allow us to construct a model for the initiation and execution of LCD in C. elegans (Figure 7). The logic of the LCD pathway may be similar to that of developmental apoptotic pathways. In C. elegans and Drosophila, where the control of specific cell deaths has been primarily examined, cell lineage or fate determinants control the expression of specific transcription factors that then impinge on proteins regulating caspase activation (Fuchs and Steller, 2011). Likewise, LCD is initiated by redundant determinants that require a transcription factor to activate protein degradation genes.

Figure 7.

https://elife-publishing-cdn.s3.amazonaws.com/12821/elife-12821-fig7-v3-480w.jpg

Figure 7. Model for linker cell death.

Green, upstream regulators. Orange, HSF-1. Purple, proteolytic components.    DOI: http://dx.doi.org/10.7554/eLife.12821.016

 

Our data suggest that three partially redundant signals control LCD initiation. The antagonistic Wnt pathways we describe may provide positional information to the linker cell, as the relevant ligands are expressed only near the region where the linker cell dies. The LIN-29 pathway, which controls timing decisions during the L4-adult molt, may ensure that LCD takes place only at the right time. Finally, while the TIR-1/SEK-1 pathway could act constitutively in the linker cell, it may also respond to specific cues from neighboring cells. Indeed, MAPK pathways are often induced by extracellular ligands. We propose that these three pathways, together, trigger activation of HSF-1. Our data support a model in which HSF-1 is present in two forms, HSF-1LC, promoting LCD, and HSF-1HS, protecting cells from stresses, including heat shock. We postulate that the redundant LCD initiation pathways tip the balance in favor of HSF-1LC, allowing this activity to bind to promoters and induce transcription of key LCD effectors, including LET-70/UBE2D2 and other components of the ubiquitin proteasome system (UPS), functioning through E3 ligase complexes consisting of CUL-3, RBX-1, BTBD-2, and SIAH-1.

Importantly, the molecular identification of LCD components and their interactions opens the door to testing the impact of this cell death pathway on vertebrate development. For example, monitoring UBE2D2 expression during development could reveal upregulation in dying cells. Likewise, genetic lesions in pathway components we identified may lead to a block in cell death. Double mutants in apoptotic and LCD genes would allow testing of the combined contributions of these processes.

The proteasome and LCD

As is the case with caspase proteases that mediate apoptosis (Pop and Salvesen, 2009), how the UPS induces LCD is not clear, and remains an exciting area of future work. That loss of BTBD-2, a specific E3 ligase component, causes extensive linker cell survival suggests that a limited set of targets may be required for LCD. Previous work demonstrated that BTBD2, the vertebrate homolog of BTBD-2, interacts with topoisomerase I (Khurana et al., 2010; Xu et al., 2002), raising the possibility that this enzyme may be a relevant target, although other targets may exist.

The UPS has been implicated in a number of cell death processes in which it appears to play a general role in cell dismantling, most notably, perhaps, in intersegmental muscle remodeling during metamorphosis in moths (Haas et al., 1995). However, other studies suggest that the UPS can have specific regulatory functions, as with caspase inhibition by IAP E3 ligases (Ditzel et al., 2008).

During Drosophila sperm development, caspase activity is induced by the UPS to promote sperm individualization, a process that resembles cytoplasm-specific activation of apoptosis (Arama et al., 2007). While C. elegans caspases are dispensible for LCD, it remains possible that they participate in linker cell dismantling or serve as a backup in case the LCD program fails.

Finally, the proteasome contains catalytic domains with target cleavage specificity reminiscent of caspases; however, inactivation of the caspase-like sites does not, alone, result in overt cellular defects (Britton et al., 2009), suggesting that this activity may be needed to degrade only specific substrates. Although the proteasome generally promotes proteolysis to short peptides, site-specific cleavage of proteins by the proteasome has been described (Chen et al., 1999). It is intriguing to speculate, therefore, that caspases and the proteasome may have common, and specific, targets in apoptosis and LCD.

A pro-death developmental function for HSF-1

Our discovery that C. elegans heat-shock factor, HSF-1, promotes cell death is surprising. Heat-shock factors are thought to be protective proteins, orchestrating the response to protein misfolding induced by a variety of stressors, including elevated temperature. Although a role for HSF1 has been proposed in promoting apoptosis of mouse spermatocytes following elevated temperatures (Nakai et al., 2000), it is not clear whether this function is physiological. In this context, HSF1 induces expression of the gene Tdag51 (Hayashida et al., 2006). Both pro- and anti-apoptotic activities have been attributed to Tdag51 (Toyoshima et al., 2004), and which is activated in sperm is not clear. Recently, pathological roles for HSF1 in cancer have been detailed (e.g. Mendillo et al., 2012), but in these capacities HSF1 still supports cell survival.

Developmental functions for HSF1 have been suggested in which HSF1 appears to act through transcriptional targets different from those of the heat-shock response (Jedlicka et al., 1997), although target identity remains obscure. Here, we have shown that HSF-1 has at least partially non-overlapping sets of stress-induced and developmental targets. Indeed, typical stress targets of HSF-1, such as the small heat-shock gene hsp-16.49 as well as genes encoding larger chaperones, likehsp-1, are not expressed during LCD, whereas let-70, a direct transcriptional target for LCD, is not induced by heat shock. Interestingly, the yeast let-70 homologs ubc4 and ubc5 are induced by heat shock (Seufert and Jentsch, 1990), supporting a conserved connection between HSF and UBE2D2-family proteins. However, the distinction between developmental and stress functions is clearly absent in this single-celled organism, raising the possibility that this separation of function may be a metazoan innovation.

What distinguishes the stress-related and developmental forms of HSF-1? One possibility is that whereas the stress response appears to be mediated by HSF-1 trimerization, HSF-1 monomers or dimers might promote LCD roles. Although this model would nicely account for the differential activities in stress responses and LCD of the HSF-1(R145A) transgenic protein, which would be predicted to favor inactivation of a larger proportion of higher order HSF-1 complexes, the identification of conserved tripartite HSEs in the let-70 and rpn-3 regulatory regions argues against this possibility. Alternatively, selective post-translational modification of HSF-1 could account for these differences. In mammals, HSF1 undergoes a variety of modifications including phosphorylation, acetylation, ubiquitination, and sumoylation (Xu et al., 2012), which, depending on the site and modification, stimulate or repress HSF1 activity. In this context, it is of note that p38/MAPK-mediated phosphorylation of HSF1 represses its stress-related activity (Chu et al., 1996), and the LCD regulator SEK-1 encodes a MAPKK. However, no single MAPK has been identified that promotes LCD (E.S.B., M.J.K. unpublished results), suggesting that other mechanisms may be at play.

Our finding that POP-1/TCF does not play a significant role in LCD raises the possibility that Wnt signaling exerts direct control over HSF-1 through interactions with β-catenin. However, we have not been able to demonstrate physical interactions between these proteins to date (M.J.K, unpublished results).

Finally, a recent paper (Labbadia and Morimoto, 2015) demonstrated that in young adult C. elegans, around the time of LCD, global binding of HSF-1 to its stress-induced targets is reduced through changes in chromatin modification. Remarkably, we showed that chromatin regulators play a key role in let-70 induction and LCD (J.A.M., M.J.K and S.S., manuscript in preparation), suggesting, perhaps, that differences in HSF-1 access to different loci may play a role in distinguishing its two functions.

LCD and neurodegeneration

Previous studies from our lab raised the possibility that LCD may be related to degenerative processes that promote vertebrate neuronal death. Nuclear crenellation is evident in dying linker cells and in degenerating cells in polyQ disease (Abraham et al., 2007) and the TIR-1/Sarm adapter protein promotes LCD in C. elegans as well as degeneration of distal axonal segments following axotomy in Drosophila and vertebrates (Osterloh et al., 2012). The studies we present here, implicating the UPS and heat-shock factor in LCD, also support a connection with neurodegeneration. Indeed, protein aggregates found in cells of patients with polyQ diseases are heavily ubiquitylated (Kalchman et al., 1996). Chaperones also colocalize with protein aggregates in brain slices from SCA patients, and HSF1 has been shown to alleviate polyQ aggregation and cellular demise in both polyQ-overexpressing flies and in neuronal precursor cells (Neef et al., 2010). While the failure of proteostatic mechanisms in neurodegenerative diseases is generally thought to be a secondary event in their pathogenesis, it is possible that this failure reflects the involvement of a LCD-like process, in which attempts to engage protective measures instead result in activation of a specific cell death program.

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A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP

 

There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.  Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another,  demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression.  Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described concluding that induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site. Another example is the inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  We have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.

 

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