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A Reconstructed View of Personalized Medicine

Posted in Amino acids, Anaerobic Glycolysis, Apoptosis, Autophagy, Biochemical pathways, Biological Networks, Gene Regulation and Evolution, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Cell death pathways, Curation, Disease Biology, DNA repair, Enzymes and isoenzymes, Gene Regulation and Evolution, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Genomic Expression, Human aging, Metabolism, Metabolomics, Micronutrients, Nitric Oxide in Health and Disease, Nutrition, Pharmaceutical Analytics, Pharmaceutical Discovery, Protein-energy malnutrition, Proteins, Proteomics, Pyridine nucleotides, Pyruvate Kinase, RNA Biology, Signaling, Signaling & Cell Circuits, Stress Disorders, Ubiquitinylation, Warburg effect, tagged , , , , , , , , , , on February 26, 2016| Leave a Comment »

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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Regulatory DNA engineered

Posted in Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Clinical Genomics, CRISPR/Cas9 & Gene Editing, Curation, Exosomes, Gene Regulation, Genetics & Innovations in Treatment, Genome Biology, Proteins, Proteomics, Translational Science, tagged , , on February 11, 2016| Leave a Comment »

Regulatory DNA Engineered, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Regulatory DNA engineered

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New Type of CRISPR Screen Probes the Regulatory Genome

Aaron Krol    http://www.bio-itworld.com/2016/2/8/new-type-crispr-screen-probes-regulatory-genome.html

February 8, 2016 | When a geneticist stares down the 3 billion DNA base pairs of the human genome, searching for a clue to what’s gone awry in a single patient, it helps to narrow the field. One of the most popular places to look is the exome, the tiny fraction of our DNA―less than 2%―that actually codes for proteins. For patients with rare genetic diseases, which might be fully explained by one key mutation, many studies sequence the whole exome and leave all the noncoding DNA out. Similarly, personalized cancer tests, which can help bring to light unexpected treatment options, often sequence the tumor exome, or a smaller panel of protein-coding genes.

Unfortunately, we know that’s not the whole picture. “There are a substantial number of noncoding regions that are just as effective at turning off a gene as a mutation in the gene itself,” says Richard Sherwood, a geneticist at Brigham and Women’s Hospital in Boston. “Exome sequencing is not going to be a good proxy for what genes are working.”

Sherwood studies regulatory DNA, the vast segment of the genome that governs which genes are turned on or off in any cell at a given time. It’s a confounding area of genetics; we don’t even know how much of the genome is made up of these regulatory elements. While genes can be recognized by the presence of “start” and “stop” codons―sequences of three DNA letters that tell the cell’s molecular machinery which stretches of DNA to transcribe into RNA, and eventually into protein―there are no definite signs like this for regulatory DNA.

Instead, studies to discover new regulatory elements have been somewhat trial-and-error. If you suspect a gene’s activity might be regulated by a nearby DNA element, you can inhibit that element in a living cell, and see if your gene shuts down with it.

With these painstaking experiments, scientists can slowly work their way through potential regulatory regions―but they can’t sweep across the genome with the kind of high-throughput testing that other areas of genetics thrive on. “Previously, you couldn’t do these sorts of tests in a large form, like 4,000 of them at once,” says David Gifford, a computational biologist at MIT. “You would really need to have a more hypothesis-directed methodology.”

Recently, Gifford and Sherwood collaborated on a paper, published in Nature Biotechnology, which presents a new method for testing thousands of DNA loci for regulatory activity at once. Their assay, called MERA (multiplexed editing regulatory assay), is built on the recent technology boom in CRISPR-Cas9 gene editing, which lets scientists quickly and easily cut specific sequences of DNA out of the genome.

So far, their team, including lead author Nisha Rajagopal from Gifford’s lab, has used MERA to study the regulation of four genes involved in the development of embryonic stem cells. Already, the results have defied the accepted wisdom about regulatory DNA. Many areas of the genome flagged by MERA as important factors in gene expression do not fall into any known categories of regulatory elements, and would likely never have been tested with previous-generation methods.

“Our approach allows you to look away from the lampposts,” says Sherwood. “The more unbiased you can be, the more we’ll actually know.”

A New Kind of CRISPR Screen

In the past three years, CRISPR-Cas9 experiments have taken all areas of molecular biology by storm, and Sherwood and Gifford are far from the first to use the technology to run large numbers of tests in parallel. CRISPR screens are an excellent way to learn which genes are involved in a cellular process, like tumor growth or drug resistance. In these assays, scientists knock out entire genes, one by one, and see what happens to cells without them.

This kind of CRISPR screen, however, operates on too small a scale to study the regulatory genome. For each gene knocked out in a CRISPR screen, you have to engineer a strain of virus to deliver a “guide RNA” into the cellular genome, showing the vicelike Cas9 molecule which DNA region to cut. That works well if you know exactly where a gene lies and only need to cut it once—but in a high-throughput regulatory test, you would want to blanket vast stretches of DNA with cuts, not knowing which areas will turn out to contain regulatory elements. Creating a new virus for each of these cuts is hugely impractical.

The insight behind MERA is that, with the right preparation, most of the genetic engineering can be done in advance. Gifford and Sherwood’s team used a standard viral vector to put a “dummy” guide RNA sequence, one that wouldn’t tell Cas9 to cut anything, into an embryonic stem cell’s genome. Then they grew plenty of cells with this prebuilt CRISPR system inside, and attacked each one with a Cas9 molecule targeted to the dummy sequence, chopping out the fake guide.

Normally, the result would just be a gap in the CRISPR system where the guide once was. But along with Cas9, the researchers also exposed the cells to new, “real” guide RNA sequences. Through a DNA repair mechanism called homologous recombination, the cells dutifully patched over the gaps with new guides, whose sequences were very similar to the missing dummy code. At the end of the process, each cell had a unique guide sequence ready to make cuts at a specific DNA locus—just like in a standard CRISPR screen, but with much less hands-on engineering.

By using a large enough library of guide RNA molecules, a MERA screen can include thousands of cuts that completely tile a broad region of the genome, providing an agnostic look at anywhere regulatory elements might be hiding. “It’s a lot easier [than a typical CRISPR screen],” says Sherwood. “The day the library comes in, you just perform one PCR reaction, and the cells do the rest of the work.”

In the team’s first batch of MERA screens, they created almost 4,000 guide RNAs for each gene they studied, covering roughly 40,000 DNA bases of the “cis-regulatory region,” or the area surrounding the gene where most regulatory elements are thought to lie. It’s unclear just how large any gene’s cis-regulatory region is, but 40,000 bases is a big leap from the highly targeted assays that have come before.

“We’re now starting to do follow-up studies where we increase the number of guide RNAs,” Sherwood adds. “Eventually, what you’d like is to be able to tile an entire chromosome.”

Far From the Lampposts

Sherwood and Gifford tried to focus their assays on regions that would be rich in regulatory elements. To that end, they made sure their guide RNAs covered parts of the genome with well-known signs of regulatory activity, like histone markers and transcription factor binding sites. For many of these areas, Cas9 cuts did, in fact, shut down gene expression in the MERA screens.

But the study also targeted regions around each gene that were empty of any known regulatory features. “We tiled some other regions that we thought might serve as negative controls,” explains Gifford. “But they turned out not to be negative at all.”

The study’s most surprising finding was that several cuts to seemingly random areas of the genome caused genes to become nonfunctional. The authors named these DNA regions “unmarked regulatory elements,” or UREs. They were especially prevalent around the genes Tdgf1 and Zfp42, and in many cases, seemed to be every bit as necessary to gene activity as more predictable hits on the MERA screen.

These results caught the researchers so off guard that it was natural to wonder if MERA screens are prone to false positives. Yet follow-up experiments strongly supported the existence of UREs. Switching the guide RNAs from aTdgf1 MERA screen and a Zfp42 screen, for example, produced almost no positive results: the UREs’ regulatory effects were indeed specific to the genes near them.

In a more specific test, the researchers chose a particular URE connected to Tdgf1, and cut it out of a brand new population of cells for a closer look. “We showed that, if we deleted that region from the genome, the cells lost expression of the gene,” says Sherwood. “And then when we put it back in, the gene became expressed again. Which was good proof to us that the URE itself was responsible.”

From these results, it seems likely that follow-up MERA screens will find even more unknown stretches of regulatory DNA. Gifford and Sherwood’s experiments didn’t try to cover as much ground around their target genes as they might have, because the researchers assumed that MERA would mostly confirm what was already known. At best, they hoped MERA would rule out some suspected regulatory regions, and help show which regulatory elements have the biggest effect on gene expression.

“We tended to prioritize regions that had been known before,” Sherwood says. “Unfortunately, in the end, our datasets weren’t ideally suited to discovering these UREs.”

Getting to Basic Principles

MERA could open up huge swaths of the regulatory genome to investigation. Compared to an ordinary CRISPR screen, says Sherwood, “there’s only upside,” as MERA is cheaper, easier, and faster to run.

Still, interpreting the results is not trivial. Like other CRISPR screens, MERA makes cuts at precise points in the genome, but does not tell cells to repair those cuts in any particular way. As a result, a population of cells all carrying the same guide RNA can have a huge variety of different gaps and scars in their genomes, typically deletions in the range of 10 to 100 bases long. Gifford and Sherwood created up to 100 cells for each of their guides, and sometimes found that gene expression was affected in some but not all of them; only sequencing the genomes of their mutated cells could reveal exactly what changes had been made.

By repeating these experiments many times, and learning which mutations affect gene expression, it will eventually be possible to pin down the exact DNA bases that make up each regulatory element. Future studies might even be able to distinguish between regulatory elements with small and large effects on gene expression. In Gifford and Sherwood’s MERA screens, the target genes were altered to produce a green fluorescent protein, so the results were read in terms of whether cells gave off fluorescent light. But a more precise, though expensive, approach would be to perform RNA sequencing, to learn which cuts reduced the cell’s ability to transcribe a gene into RNA, and by how much.

A MERA screen offers a rich volume of data on the behavior of the regulatory genome. Yet, as with so much else in genetics, there are few robust principles to let scientists know where they should be focusing their efforts. Histone markers provide only a very rough sketch of regulatory elements, often proving to be red herrings on closer examination. And the existence of UREs, if confirmed by future experiments, shows that we don’t yet even know which areas of the genome to rule out in the hunt for regulatory regions.

“Every dataset we get comes closer and closer to computational principles that let us predict these regions,” says Sherwood. As more studies are conducted, patterns may emerge in the DNA sequences of regulatory elements that link UREs together, or reveal which histone markers truly point toward regulatory effects. There might also be functional clues hidden in these sequences, hinting at what is happening on a molecular level as regulatory elements turn genes on and off in the course of a cell’s development.

For now, however, the data is still rough and disorganized. For better and for worse, high-throughput tools like MERA are becoming the foundation for most discoveries in genetics—and that means there is a lot more work to do before the regulatory genome begins to come into focus.

CORRECTED 2/9/16: Originally, this story incorrectly stated that only certain cell types could be assayed with MERA for reasons related to homologous recombination. In fact, the authors see no reason MERA could not be applied to any in vitro cell line, and hope to perform screens in a wide range of cell types. The text has been edited to correct the error.

 

 

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Aptamers and Scaffolds

Posted in Clinical Genomics, Immuno-Oncology & Genomics, Immunodiagnostics, Immunology, Immunotherapy, Methods, Proteins, Proteomics, tagged , on February 6, 2016| Leave a Comment »

Aptamers and Scaffolds

Curator: Larry H. Bernstein, MD, FCAP

 

 

 

Antibody Alternatives

Nucleic acid aptamers and protein scaffolds could change the way researchers study biological processes and treat disease.

By Jane McLeod and Paul Ko Ferrigno | February 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45134/title/Antibody-Alternatives/

http://www.the-scientist.com/images/February2016/Antibody.jpg

THE NEW Y: Antibodies, classically depicted as Y-shape molecules, are central elements of the mammalian immune system, flagging bacteria (green) for destruction by phagocytes (large, round cells). Antibodies have also served as key research tools and life-saving therapeutics, but new alternatives are becoming available.© GUNILLA ELAM/SCIENCE SOURCE

There is a growing reproducibility problem across the life sciences. The retraction rate of published papers has increased tenfold over the past decade, and researchers have reported only being able to replicate published results in 11 percent1 or 25 percent2 of attempts. It’s become known as the “reproducibility crisis,” and science is in place to fix it.

One major factor contributing to this problem is the use of poorly validated research antibodies. Lot to lot, antibodies can vary wildly. Some may not bind specifically to their target, or they may bind a different cellular protein altogether. According to one estimate, researchers around the world spend $800 million each year on poorly performing antibodies.3 (See “Exercises for Your Abs” here.)

While many researchers debate the best way to weed out the good antibodies from the bad, others are developing alternatives. Nucleic acid aptamers and protein scaffolds are increasingly being used to detect proteins of interest. Although they currently constitute only a fraction of affinity reagents, with the lion’s share of the market still going to traditional antibodies, these newer options offer an opportunity to rectify the problems stemming from using poorly validated antibodies in research. Researchers can engineer RNA or DNA aptamers and protein scaffolds to a specific target and function, the molecules are consistent from batch to batch, and they can be produced at a fraction of the cost of antibodies. These new reagents can target proteins that remain inaccessible to antibodies. And researchers have designed them to be functional in a wider range of conditions, including intracellular environments that degrade the antibody structure, opening up applications such as super-resolution microscopy and intracellular live-cell imaging to investigate the molecular dynamics of diverse cellular processes.

So, rather than complain about the poor performance of antibodies, perhaps the scientific community should embrace the new antibody alternatives designed to overcome this problem—and, by doing so, begin to resolve the ongoing reproducibility crisis.

The rise—and pitfalls—of antibodies

http://www.the-scientist.com/images/February2016/antibody2.jpg

A CLASSIC FIT: Antibodies are large proteins, weighing in at about 150 kDa. Four polypeptides—two heavy chains and two light chains—are linked by disulfide bonds to form a Y-shape molecule. The amino acid sequences at tips of the short ends of the Y vary greatly between antibodies produced by different B cells, while the rest of the molecule is relatively consistent. The variable portion of the antibody binds in a specific region (epitope) on a foreign protein (antigen) and signals the immune system to the presence of an invader.© STEVE GRAEPEL

Antibodies are large protein molecules composed of two heavy and two light chains linked by disulfide bonds. They play a crucial part in the immune system’s ongoing battle to keep our bodies from falling prey to deadly diseases. Through the diversification of gene segments in the antibody sequence, the mammalian immune system produces different combinations of heavy and light chains to bind a wide variety of foreign proteins. When an invader is detected, those B cells that produce the most specific antibodies undergo hypersomatic mutation to fine-tune the antibody’s affinity to a particular antigen, then differentiate into plasma cells that generate the targeted antibody molecules by the million to mark the disease-causing target for destruction. It has been estimated that the human body can create enough different antibodies to recognize 1012 distinct pathogens.4

For decades, life-science researchers have taken advantage of this natural process to develop tags and assays for a wide array of proteins. In the early 1900s, researchers began to cultivate protein-specific antibodies by immunizing rabbits, chickens, goats, donkeys, and other animals with a desired target protein. B cells within the animal host generate antibodies to different antigenic areas (epitopes) on the protein of interest. The antibodies targeting the desired protein can then be isolated and purified for use in biochemical and cell-based assays to document protein expression under different conditions or to identify potential disease biomarkers. But the reliance on an animal host system for production meant lot-to-lot heterogeneity for such polyclonal antibodies. (See illustration adjacent.)

In 1975, Argentine biochemist César Milstein and German biologist Georges Köhler discovered how to generate batches of individual antibodies, produced by a single B cell to target a specific antigen. Once an animal host produces antibodies to a target, the antibody-producing B cells are isolated from the spleen or lymph nodes and fused with tumor cells to generate immortal hybridoma lines. These lines are then screened to identify clones producing antibodies that bind with a high affinity to a specific epitope on the target protein. These cells are then cultured in large-scale bioreactors.

While heterogeneity can arise from drift in the cell line’s antibody expression and downstream production processes, monoclonal antibodies exhibit far less lot-to-lot variation than polyclonal antibodies, and have become the affinity tool of choice in modern research laboratories. Monoclonal antibodies are now routinely employed to localize proteins within tissues, determine protein network interactions, and analyze protein function. They are now being pushed to the limits of their performance in applications such as nanoimmunoassays and in vivo cell imaging. In medicine, antibody therapeutics represents the fastest growing sector of pharmaceutical sales, with 47 monoclonal antibodies currently on the market and a further 300 in clinical trials.5

 

http://www.the-scientist.com/images/February2016/FebAntibody2_640px.jpg

with 47 monoclonal antibodies currently on the market and 300 more in clinical trials. But facing problems of inconsistent, time-consuming, and costly antibody production, some researchers are turning to alternatives—nucleic acid aptamers and protein scaffolds—to target specific proteins of interest, in the lab and in the clinic.
See full infographic: WEB | PDF© STEVE GRAEPEL

 

But there are many examples where the use of antibodies has actually hindered scientific progress, by providing misleading or inaccurate results. Antibodies have evolved to execute their biological function perfectly, but this does not make them foolproof as investigative tools or therapeutic agents. In fact, many of the very characteristics that aid in antibodies’ function as part of the immune system limit their use in research and medicine.

In the context of an immune reaction, for example, not all B cells produce antibodies that are exquisitely specific. So long as the antibodies exceed a certain threshold of binding affinity for the target, they remain part of the immune system’s defense. In the body, this is a good thing: these less-specific antibodies cross-react with a variety of related antigens, making the antibody defense force more versatile.6 If an invading pathogen mutates or a similar pathogen invades, potentially effective antibodies may already be in circulation. As part of an assay to specifically identify a particular protein, however, such cross-reactivity can be the downfall of the experiment or therapy.

Examined in this light, it is easy to see why taking a molecule that is derived for one purpose and applying it to another may not yield the best results. A clear example of the shortcomings of antibody use in life-science research comes from the Human Protein Atlas project. Mathias Uhlén of the Royal Institute of Technology in Stockholm, Sweden, and colleagues set out to catalog protein expression and localization data across 44 normal human tissue types, 20 different cancers, and 46 cell lines. The team sourced antibodies from 51 different commercial vendors for validation. Of the 5,436 antibodies received, about half failed to detect their target in a Western blot or standard immunohistochemistry assay.7

The development of novel antibodies that bind new protein targets continues to face several challenges. For example, using the conventional route of immunizing lab animals to produce an antibody against a toxic target molecule will often kill the host animal prior to the generation of sufficiently specific antibodies. Conversely, if a protein target is highly homologous to a host protein, the immune system may not recognize the target as foreign in order to generate antibodies against it.

Given the rapid pace at which molecular biology proceeds in the modern era, new protein-binding reagents are desperately needed. A review of 20 million published research articles from 1950 to 2009 showed that three-quarters of the research focused on just 10 percent of the proteins that were known prior to the mapping of the human genome.8 Rational design of antibody alternatives will allow us to target a broader swath of proteins and function across more platforms to better investigate the scientific questions at hand.

Nucleic acid aptamers and pro­tein scaffolds can target proteins that remain inaccessible to antibod­ies, and researchers have designed them to be functional in a wider range of con­ditions.

 

http://www.the-scientist.com/images/February2016/FebAntibody4_310px1.jpg

NUCLEIC ACID APTAMERS: Aptamers are short molecules of single-stranded DNA or RNA, typically less than 100 nucleotides in length, that form specific 3-D structures capable of binding target proteins.© STEVE GRAEPEL

 

Engineering a solution

NUCLEIC ACID APTAMERS: Aptamers are short molecules of single-stranded DNA or RNA, typically less than 100 nucleotides in length, that form specific 3-D structures capable of binding target proteins.© STEVE GRAEPELAlternative affinity reagents developed over the past few decades include both nucleic acid– and protein-based molecules. Aptamers are short molecules of single-stranded DNA or RNA, typically less than 100 nucleotides in length, that form 3-D structures capable of binding specific target proteins. Protein scaffolds, formed from polypeptide fragments or whole proteins, have similarly precise interactions with target molecules. Both types of affinity reagents are produced entirely in vitro, so in principle they are not subject to the limitations of antibody production by animal immune systems, allowing researchers to study proteins for which it is impossible to generate antibodies. And even when antibodies do exist, aptamers and protein scaffolds offer more-precise targeting, because they have been engineered for a specific purpose.

These novel affinity reagents also offer other benefits over antibodies. Both nucleic acid aptamers and protein scaffolds are much smaller than natural antibodies, which typically weigh about 150 kDa. Aptamers and scaffolds are as little as one-tenth that size. This means that their distribution is not restricted in the same manner as that of antibodies, opening up new targets that were previously inaccessible, such as epitopes hidden inside molecular grooves and pockets where antibodies simply can’t fit. Labeling target proteins with these smaller tags in cytochemistry experiments reduces the chance of the target protein being dragged around the cell according to the tag’s biochemistry, and increases the chances of identifying the correct protein localization. Additionally, their smaller size increases these affinity reagents’ tissue penetration, enhancing access to epitopes within tissue sections and decreasing false negative immunohistochemistry results. Smaller molecules are also cleared more rapidly from the body, especially when their size is below the renal cut-off of 45 kDa, making these molecules ideal as imaging agents in the clinic.

Researchers first developed nucleic acid aptamers in 1990 as RNA-based molecules, though DNA variants quickly followed to deal with the low stability of the RNA backbone. Aptamers offer simple chemistry that can be easily functionalized, but they lack stability across a range of temperatures and pH, and in the presence of common buffer components or DNases and RNases found in many media and cell environments. Researchers have used various chemical modifications to increase aptamers’ resistance to nuclease activity, to improve aptamer binding, and to increase their structural diversity, but others have turned to yet another option: protein scaffolds.

Developed around the same time as aptamers, protein scaffold affinity reagents were originally designed to identify potential therapeutic targets. Researchers soon began to apply this technology to screening for binders to completely novel proteins, by presenting a random sequence as the binding surface. Because protein scaffolds lack the disulfide bonds of antibodies, they retain their structure in a greater variety of cell culture and assay environments, without being attacked by other proteins that break these bonds and cause antibodies to fall apart. Scaffolds maintain function and target affinity at temperatures up to 80 °C and in solutions with a pH as low as 2 and as high as 13.

Because protein scaffolds can be delivered to the inside of the cell, researchers can use them in live-cell imaging, ultimately allowing use of the same reagent in both biochemical and cell biology assays. Additionally, protein scaffolds could help to deliver drugs directly into cells, improving targeting of pharmaceutical payloads and reducing side effects. And as new protein scaffolds are often engineered to lack cysteine residues, aberrant folding during their production within the cell factory is unlikely, increasing reproducibility within the reagents.

Importantly, both nucleic acid aptamers and protein scaffolds are far easier to consistently produce than antibodies. In addition to requiring animal hosts to provide an antibody-producing B cell, functional antibodies can only be expressed in higher eukaryotic cell systems. Antibodies are extensively glycosylated with a complex range of sugars that are critical to their function. Lower eukaryotic organisms, such as insects and yeast, and prokaryotic cells are not capable of the full range of complex glycosylation. As a result of these complexities, production times for monoclonal antibodies are six months on average, often making the generation of new antibodies the rate-limiting step in the advance of new research. This production process is also extremely expensive, and the use of such intractable biological systems breeds batch-to-batch inconsistencies.

 

http://www.the-scientist.com/images/February2016/FebAntibody5_310px.jpg

PROTEIN SCAFFOLDS: Protein scaffolds, formed from polypeptide fragments or whole proteins, have similarly specific interactions with desired target molecules.© STEVE GRAEPEL

Aptamers and protein scaffolds can be made without a host immune system. Now that robust and scalable methodologies for creating custom DNA and RNA molecules exist, effective aptamer binders can be chemically synthesized at a fraction of the cost of producing protein-based affinity molecules. And because protein scaffolds do not contain any posttranslational modifications, they can be expressed in bacterial cells, which are cheaper and easier to control than the eukaryotic systems used for antibody production. Both aptamers and scaffolds can often be available to researchers in a matter of weeks. (See illustration above)

Alternatives at work

 

http://www.the-scientist.com/images/February2016/antibody4.jpg

A PROTEIN BUILT FOR YOU: Protein scaffolds (orange) can be developed to bind specific protein targets (purple) and can be produced in a matter of weeks. Once the scaffolds bind the protein of interest, fluorescent or color reporter tags (yellow ball) can be used to label and isolate them.© AVACTA LIFE SCIENCES. May not be reproduced without express written permission from the copyright holder.

So far, the majority of the industry attention for antibody alternatives has largely focused on the therapeutic development of antibody alternatives. Many companies now have initiated Phase 2 and 3 clinical trials of candidate molecules to treat conditions from vision problems to cancer, and two such molecules have already been approved for therapeutic use. In 2004, the RNA aptamer–based therapeutic pegaptanib (Macugen), originally developed by NeXstar Pharmaceuticals, became the first antibody alternative to gain US Food and Drug Administration (FDA) approval for the treatment of neovascular age-related macular degeneration. Pegaptanib is a 28-base-long RNA oligonucleotide with modifications to protect the aptamer from endogenous nucleases and extend its half-life in vivo to 10 days.9 Administered directly into the eye, this aptamer selectively binds the most common isoform of vascular endothelial growth factor (VEGF), preventing angiogenesis and the increased permeability of the blood vessels within the eye associated with neovascular age-related macular degeneration.10 Five years later, in 2009, the FDA approved a protein scaffold called ecallantide (Kalbitor) for the treatment of sudden hereditary angioedema attacks.

In a therapeutic context, the most important characteristic of aptamers and scaffolds is that they lack immunogenicity, thus avoiding harmful immune responses in patients. One way to ensure that protein scaffolds do not trigger host immunity is to model one’s scaffolds after proteins found in the human body. For example, the FDA-approved Kalbitor is based on the common Kunitz domain of protease inhibitors, and clinical trial participants have not suffered immunogenic responses. When brought to market, Kalbitor was one of only two approved therapies to treat cardiovascular attacks of this sort, which can cause rapid and serious swelling of the face or other parts of the body that may result in permanent disfigurement, disability, or death; the other is a protein therapeutic derived from human blood.

A major factor holding back the field of antibody alternatives as therapeutics is their small size. While this improves their intracellular function and use in research applications, their low molecular weight means that they are rapidly cleared from the body via the kidneys, reducing their potential therapeutic impact. Various strategies have been employed by the industry to overcome this rapid renal clearance, such as adding an antibody domain or an albumin-binding domain to the scaffold, or increasing the molecular weight of the protein scaffolds (though they still remain significantly smaller than a corresponding antibody). The fusion of an antibody domain to a protein scaffold can also help engage the immune system for improved therapeutic benefit.

While their small size can be a hurdle in developing antibody alternatives in a clinical setting, it is a big advantage in their use as laboratory tools, allowing them to penetrate bodily tissues that are inaccessible to antibodies and offering more-precise molecular labeling. In 2012, for example, Silvio Rizzoli of the European Neuroscience Institute and Center for Molecular Physiology of the Brain in Göttingen, Germany, and colleagues used 15 kDa aptamers to capture the dynamics of endosomal trafficking in live cells using super-resolution imaging.11 Doubling the molecular weight of the aptamer resulted in a substantial reduction in image quality, showing the importance of the small size of intracellular labels in accurate imaging of the intracellular space.

The increased intracellular stability of protein scaffolds as compared with antibodies is also critical to their function as research tools and offers potential therapeutic benefit for intracellular targets. For instance, protein scaffolds have been used to investigate the function of the small intracellular domain of a matrix metalloproteinase, which was shown to determine protein turnover to help regulate protein function in cell movement.12 Using antibodies in the reducing environment of the cell interior in such a study would be impossible.

When developing antibody alternatives for research, scientists are purposefully mimicking natural proteins to avoid interference by the immune system. For example, Janssen produces protein scaffolds called Centyrins that are based on the fibronectin glycoprotein of the extracellular matrix. Our own company, Avacta Life Sciences, recently introduced Affimer scaffolds, which are based on the cystatin protein family of common protease inhibitors. The use of consensus sequences from a number of species may allow these reagents to be used across a variety of different model systems.

Nucleic acid aptamers and protein scaffolds may also help fight emerging outbreaks of acute infectious disease. Examples of recent outbreaks that have caused considerable social, economic, and political stress are not hard to come by—SARS in 2003, the H1N1 flu pandemic in 2009, the Ebola crisis of recent years, and the continued emerging threat of MERS. It is impossible to predict such episodes, and alternative affinity reagents could be crucial tools in quickly stemming the spread of pandemic diseases. Screening libraries of 10 billion sequences can take as little as 7 weeks. And while the processes required for optimization, scale-up, and subsequent culture and validation of substantial quantities of the required affinity reagent remain to be explored, this all may take only a matter of months.

Over the past few decades, the rate of advancement of genomic technologies has outpaced proteomics. Yet it is the expressed protein within a cell, not the underlying genetic blueprint, that executes correct or aberrant function. In order to unify and make sense of the numerous data sets being produced, scientists need tools that enable the unraveling of proteomics. This requires affinity reagents that can specifically target individual protein isoforms and glycoforms, and that can tag all the proteins within a cell or organism. While the concerns over antibody irreproducibility are increasing, the solution may already be available.

Jane McLeod is a science writer at Avacta Life Sciences, where Paul Ko Ferrigno is the chief scientific officer. Avacta Life Sciences sells peptide aptamers, one of the main forms of antibody alternative.

References

  1. C.G. Begley, L.M. Ellis, “Drug development: Raise standards for preclinical cancer research,” Nature, 483:531-33, 2012.
  2. F. Prinz et al., “Believe it or not: How much can we rely on published data on potential drug targets?” Nat Rev Drug Discov, 10:712, 2011.
  3. A. Bradbury, A. Plückthun, “Reproducibility: Standardize antibodies used in research,” Nature, 518:27-29, 2015.
  4. B. Alberts et al., Molecular Biology of the Cell, 4th edition (New York: Garland Science, 2002).
  5. D.M. Ecker et al., “The therapeutic monoclonal antibody market,” mAbs, 7:9-14, 2015.
  6. N. Baumgarth, “How specific is too specific? B-cell responses to viral infection reveal the importance of breadth over depth,” Immunol Rev, 255:82-94, 2013.
  7. L. Berglund et al., “A genecentric Human Protein Atlas for expression profiles based on antibodies,” Mol Cell Proteomics, 7:2019-27, 2008.
  8. A.M. Edwards et al., “Too many roads not taken,” Nature, 470:163-65, 2011.
  9. J. Ruckman et al., “2’-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165): Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain,” J Biol Chem, 273:20556-67, 1998.
  10. P. Sundaram et al., “Therapeutic RNA aptamers in clinical trials,” Eur J Pharm Sci, 48:259-71, 2013.
  11. F. Opazo et al., “Aptamers as potential tools for super-resolution microscopy,” Nat Methods, 9:938-39, 2012.
  12. R.D. Wickramasinghe et al., “Peptide aptamers as new tools to modulate clathrin-mediated internalisation—inhibition of MT1-MMP internalisation,” BMC Cell Biol, 11:58, 2010.

Tags  protein scaffolds, monoclonal antibodies, human monoclonal antibodies, aptamers and antibody

February 4, 2016

Nice article. Your statement in the end identifies correctly a potential conflict of interest. It should probably be a bit more clear and obvious than that though. Perhaps at the top of the page, near the title of the article. My 2 cents.

 

 

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Reengineering Therapeutics

Posted in Biological Networks, BioTechnology - Venture Creation, Cancer and Current Therapeutics, Cell Biology, Signaling & Cell Circuits, CRISPR/Cas9 & Gene Editing, Curation, Disease Biology, Genome Biology, Innovations, Metabolism, Proteins, Proteomics, tagged , on February 6, 2016| Leave a Comment »

Reengineering Therapeutics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

The synNotch solution: UCSF scientists engineer a next-gen T-cell immunotherapy

Sunday, January 31, 2016 | By John Carroll

2.2.9

2.2.9   Reengineering Therapeutics, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

CAR-T has been all the rage in cancer R&D for several years now as a slate of biotech upstarts pursue highly promising work reengineering T cells into attack weapons by adding a chimeric antigen receptor that can zero in on particular cancer cells. The approach has been highly effective in acute lymphoblastic leukemia, triggering an attack on B cells by homing in on the CD19 antigen, a breakthrough that has inspired a race to the regulatory finish line with the first CAR-Ts.

That approach, though, has run into some major obstacles when researchers move from the blood cancer to solid tumors. But now a group of scientists working with UC San Francisco’s Wendell Lim say they’ve come up with a new therapeutic model for T-cell engineering that promises to overcome that hurdle and make it a more precise weapon that can tackle solid tumors while avoiding off-target reactions that threaten patients.

The key to this new approach is a new receptor: synNotch. Taking a cue from nature, which relies on a sensor called Notch to perform key functions, the synthetic biology engineers say they can add a receptor that includes one section that sticks out from the cell with one that lies inside. By tinkering with synNotch they can reengineer the immune cell to run down a particular cancer cell target and then issue instructions to turn genes on or off to set up the other half of the therapeutic equation.

In a project described in Cell, the team says they created a synNotch that recognized an antigen on the surface of the cancer cell while the internal mechanism contributed a chimeric antigen receptor that recognized a different antigen. They then tested it on a mouse model that included two different tumor cells: one with both targets recognized by the external synNotch sensor and the CAR and one with just the CAR target.

The newly programmed attack weapon zeroed in on the two targets (which required synNotch activation for it to work) while leaving the other alone, providing preclinical proof-of-concept evidence that they could create a much more efficient tumor cell killing vehicle.

“The kinds of engineered T cells that we can now construct give us the exciting potential to create precision cancer therapeutics that take advantage of all the genomic and proteomic information we are currently gathering on disease,” said Lim, the senior author of the study. “This genomic information now becomes actionable.”

The team was led by Leonardo Morsut and Kole Roybal.

Lim added that it is possible to reengineer a T cell with multiple synNotches to make it even more precise. And there are added applications for autoimmune disease, regenerative medicine, and more.

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

Leonardo  Morsut, Kole T. Roybal, Xin Xiong, Russell M. Gordley, Scott M. Coyle, Matthew Thomson, Wendell A. Lim
J. Cell 2016     http://dx.doi.org/10.1016/j.cell.2016.01.012

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2045482108/2056813637/fx1.jpg

  • •SynNotch receptors with altered extra- and intracellular domains yield new responses
  • •SynNotch pathways drive diverse user-defined responses in many mammalian cell types
  • •Multiple synNotch pathways are functionally orthogonal to one another
  • •SynNotch pathways control differentiation, spatial patterning, and Boolean decisions

The Notch protein is one of the most mechanistically direct transmembrane receptors—the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.

Tricked-Out Immune Cells Could Attack Cancer, Spare Healthy Cells  

New Cell-Engineering Technique May Lead To Precision Immunotherapies

https://www.ucsf.edu/news/2016/01/401471/tricked-out-immune-cells-could-attack-cancer-spare-healthy-cells

UC San Francisco scientists have created a new class of highly customizable biological sensors that can be used to form “logic gates” inside cells of the immune system, giving these cells the capability to home in on and kill a wide range of cancer cells while preventing them from attacking normal tissue.

As reported in two companion papers published Jan. 28 in the online edition of Cell, in addition to their potential to bring much-needed precision and safety to the form of cancer immunotherapy known as CAR T cell therapy, these versatile new sensors, known as synNotch receptors, can also be inserted into cells such as nerve and muscle cells. The receptors may have uses in regenerative medicine, in the treatment of autoimmune diseases, and in basic biological research.

“SynNotch receptors provide us with one of the most flexible ways we have to reprogram the behavior and function of almost any cell,” said Wendell A. Lim, PhD, chair and professor of cellular and molecular pharmacology at UCSF, and senior author of the two new papers. “One of the most exciting applications is in engineering therapeutic cells – we can now provide these cells with highly precise instructions about how to recognize and respond to disease.”

Gene therapy pioneer James Wilson uses CRISPR/Cas9 to target liver disease

Wednesday, February 3, 2016 | By John Carroll

UPenn’s James Wilson

One of the pioneers in the whole gene therapy movement of the past 35 years has combined his knowledge of viral vectors with the hot new CRISPR/Cas9 tech to tackle a rare genetic liver disease. And his work with rodents highlighted both the promise of this new technology as well as an unexpected hurdle.

James Wilson at the University of Pennsylvania is virtually the father of adeno-associated virus–or AAV–vectors used to deliver gene therapies. After years of up and down progress, including an early patient death that slowed research in gene therapies for decades, his technology was in-licensed by ReGenX, which has since outlicensed it to a group of biotechs pursuing new gene therapy programs.

In what is described as a first in research circles, he used an AAV vector to deliver the components of CRISPR/Cas9 gene editing tools into a mouse model of a rare metabolic urea-cycle disorder, triggered by a deficiency in the ornithine transcarbamylase (OTC) enzyme.  When one of a series of enzymes is missing or “deficient,” ammonia can accumulate in the blood and circulate into the brain, where it can badly damage brains. OTC deficiency occurs in one in every 40,000 births.

Wilson’s group used one AAV to deliver a Cas9 enzyme–the cutting tool–specifically into liver cells. Another vector carried a guide RNA to the specified spot and donor DNA to correct the mutation in a cut-and-paste approach that is spreading around academic labs like wild fire.

Wilson says his team achieved a 10% reversion in the liver cells of newborn mice, with an increased survival rate for challenged rodents and a high death rate in the untreated group of newly born mice. In adult mice, though, the team say that the experiment didn’t work as planned, highlighting a needed correction in the process. And now they’re following up with new approaches to see how that might work.

“Correcting a disease-causing mutation following birth in this animal model brings us one step closer to realizing the potential of personalized medicine,” said Wilson, a professor of medicine and director of the Orphan Disease Center at Penn. “Nevertheless, my 35-year career in gene therapy has taught me how difficult translating mouse studies to successful human treatments can be. From this study, we are now adjusting the gene-editing system in the next phases of our investigation to address the unforeseen complications seen in adult animals.”

New CRISPR/Cas9 delivery method could offer a clinical pathway

February 2, 2016 | By

CRISPR/Cas9 gene editing is a rapidly developing technique that is thought to provide revolutionary new ways to manipulate genes for the treatment of a number of diseases. Delivering the CRISPR therapeutic in an efficient, safe and predictable way, though, has been difficult–to this end, researchers at UMass have created a means of administering the gene editor that could help send it to clinical trials.

CRISPR/Cas9 is a natural immune system in bacteria used to protect them from foreign genetic material, and scientists have used its components to cut and repair DNA sequences to replace faulty, disease-causing portions with corrected versions.

The difficulty comes about in getting the separate components (the Cas9 “scalpel,” the DNA repair template and an RNA guide) to the genetic material in a target cell. Previous attempts at delivering the system through disruptive, high-powered injection has caused damage to the liver and required too much volume when treating mice with metabolic diseases.

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Nanosensors for Protein Recognition, and gene-proteome intertaction

Posted in Biological Networks, Cancer - General, Cell Biology, Curation, Disease Biology, Gene Regulation, Proteins, Proteomics, RNA Biology, tagged , , , , , , , , , , , , , , , , on January 30, 2016| Leave a Comment »

Nanosensors for Protein Recognition and Gene Proteome Interaction

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

Synthetic Antibody Detects Proteins

http://www.technologynetworks.com/Proteomics/news.aspx?ID=187242

Research could lead to nanosensors that recognize fibrinogen, insulin, or other biomarkers

Using carbon nanotubes, MIT chemical engineers have devised a new method for detecting proteins, including fibrinogen, one of the coagulation factors critical to the blood-clotting cascade.

This approach, if developed into an implantable sensor, could be useful for monitoring patients who are taking blood thinners, allowing doctors to make sure the drugs aren’t interfering too much with blood clotting.

The new method is the first to create synthetic recognition sites (similar to natural antibodies) for proteins and to couple them directly to a powerful nanosensor such as a carbon nanotube. The researchers have also made significant progress on a similar recognition site for insulin, which could enable better monitoring of patients with diabetes. It may also be possible to use this approach to detect proteins associated with cancer or heart disease, says Michael Strano, the Carbon P. Dubbs Professor in Chemical Engineering at MIT.

A targeted search

The new sensor is the latest example of a method developed in Strano’s lab, known asCorona Phase Molecular Recognition (CoPhMoRe).

This technique takes advantage of the interactions between a given polymer and a nanoparticle surface such as that of a fluorescent single-walled carbon nanotube, when the polymer is wrapped around the nanotube.

Certain regions of the polymers latch onto the nanoparticle surface like anchors, while other regions extend outwards into their environment. This outward-facing region, also known as the adsorbed phase or corona, has a 3-D structure that depends on the composition of the polymer.

CoPhMoRe works when a specific polymer adsorbs to the nanoparticle surface and creates a corona that recognizes the target molecule. These interactions are very specific, just like the binding between an antibody and its target. Binding of the target alters the carbon nanotubes’ natural fluorescence, allowing the researchers to measure how much of the target molecule is present.

Strano’s lab has previously used this approach to find recognition sites and develop nansensors for estradiol and riboflavin, among other molecules. The new paper represents their first attempt to identify corona phases that can detect proteins, which are larger, more complex, and more fragile than the molecules identified by their previous sensors.

For this study, Bisker began by screening carbon nanotubes wrapped in 20 different polymers including DNA, RNA, and polyethylene glycol (PEG), a polymer often added to drugs to increase their longevity in the bloodstream.

On their own, none of the polymers had any affinity for the 14 proteins tested, all taken from human blood. However, when the researchers tested polymer-wrapped nanotubes against the same proteins, they turned up a match between one of the modified nanotubes and fibrinogen.

“A chemist or a biologist would not be able to predict ahead of time that there should be any kind of affinity between fibrinogen and this corona phase,” Strano says. “It really is a new kind of molecular recognition.”

Fibrinogen, one of the most abundant proteins in human blood, is part of the blood-clotting cascade. When a blood vessel is damaged, an enzyme called thrombin converts fibrinogen into fibrin, a stringy protein that forms clots to seal the wound.

A sensor for fibrinogen could help doctors determine if patients who are taking blood thinners still have enough clotting capability to protect them from injury, and could allow doctors to calculate more finely tuned dosages. It could also be used to test patients’ blood clotting before they go into surgery, or to monitor wound healing, Bisker says.

Synthetic antibodies

The researchers believe their synthetic molecular recognition agents are an improvement over existing natural systems based on antibodies or DNA sequences known as aptamers, which are more fragile and tend to degrade over time.

“One of the advantages of this is that it’s a completely synthetic system that can have a much longer lifetime within the body,” Bisker says.

In 2013, researchers in Strano’s lab demonstrated that carbon nanotube sensors can remain active in mice for more than a year after being embedded in a polymer gel and surgically implanted under the skin.

In addition to insulin, the researchers are also interested in detecting troponin, a protein that is released by dying heart cells, or detecting proteins associated with cancer, which would be useful for monitoring the success of chemotherapy. These and other protein sensors could become critical components of devices that deliver drugs in response to a sign of illness.

“By measuring therapeutic markers in the human body in real time, we can enable drug delivery systems that are much smarter, and release drugs in precise quantities,” Strano says. “However, measurement of those biomarkers is the first step.”

 

New Device Uses Carbon Nanotubes to Snag Molecules
Nanotube “forest” in a microfluidic channel may help detect rare proteins and viruses.
Tuesday, December 22, 2015

Nanotube “forest” in a microfluidic channel may help detect rare proteins and viruses.

Engineers at MIT have devised a new technique for trapping hard-to-detect molecules, using forests of carbon nanotubes.

The team modified a simple microfluidic channel with an array of vertically aligned carbon nanotubes — rolled lattices of carbon atoms that resemble tiny tubes of chicken wire. The researchers had previously devised a method for standing carbon nanotubes on their ends, like trees in a forest. With this method, they created a three-dimensional array of permeable carbon nanotubes within a microfluidic device, through which fluid can flow.

Now the researchers have given the nanotube array the ability to trap certain particles. To do this, the team coated the array, layer by layer, with polymers of alternating electric charge.

“You can think of each nanotube in the forest as being concentrically coated with different layers of polymer,” says Brian Wardle, professor of aeronautics and astronautics at MIT. “If you drew it in cross-section, it would be like rings on a tree.”

Depending on the number of layers deposited, the researchers can create thicker or thinner nanotubes and thereby tailor the porosity of the forest to capture larger or smaller particles of interest.

The nanotubes’ polymer coating may also be chemically manipulated to bind specific bioparticles flowing through the forest. To test this idea, the researchers applied an established technique to treat the surface of the nanotubes with antibodies that bind to prostate specific antigen (PSA), a common experimental target. The polymer-coated arrays captured 40 percent more antigens, compared with arrays lacking the polymer coating.

Wardle says the combination of carbon nanotubes and multilayer coatings may help finely tune microfluidic devices to capture extremely small and rare particles, such as certain viruses and proteins.

“There are smaller bioparticles that contain very rich amounts of information that we don’t currently have the ability to access in point-of-care [medical testing] devices like microfluidic chips,” says Wardle, who is a co-author on the paper. “Carbon nanotube arrays could actually be a platform that could target that size of bioparticle.”

The paper’s lead author is Allison Yost, a former graduate student who is currently an engineer at Accion Systems. Others on the paper include graduate student Setareh Shahsavari; postdoc Roberta Polak; School of Engineering Professor of Teaching Innovation Gareth McKinley; professor of materials science and engineering Michael Rubner, and Raymond A. And Helen E. St. Laurent Professor of Chemical Engineering Robert Cohen.

A porous forest

Carbon nanotubes have been a subject of intense scientific study, as they possess exceptional electrical, mechanical, and optical properties. While their use in microfluidics has not been well explored, Wardle says carbon nanotubes are an ideal platform because their properties may be manipulated to attract certain nanometer-sized molecules. Additionally, carbon nanotubes are 99 percent porous, meaning a nanotube is about 1 percent carbon and 99 percent air.

“Which is what you need,” Wardle says. “You need to flow quantities of fluid through this material to shed all the millions of particles you don’t want to find and grab the one you do want to find.”

What’s more, Wardle says, a three-dimensional forest of carbon nanotubes would provide much more surface area on which target molecules may interact, compared with the two-dimensional surfaces in conventional microfluidics.

“The capture efficiency would scale with surface area,” Wardle notes.

A versatile array

The team integrated a three-dimensional array of carbon nanotubes into a microfluidic device by using chemical vapor deposition and photolithography to grow and pattern carbon nanotubes onto silicon wafers. They then grouped the nanotubes into a cylinder-shaped forest, measuring about 50 micrometers tall and 1 millimeter wide, and centered the array within a 3 millimeter-wide, 7-millimeter long microfluidic channel.

The researchers coated the nanotubes in successive layers of alternately charged polymer solutions in order to create distinct, binding layers around each nanotube. To do so, they flowed each solution through the channel and found they were able to create a more uniform coating with a gap between the top of the nanotube forest and the roof of the channel. Such a gap allowed solutions to flow over, then down into the forest, coating each individual nanotube. In the absence of a gap, solutions simply flowed around the forest, coating only the outer nanotubes.

After coating the nanotube array in layers of polymer solution, the researchers demonstrated that the array could be primed to detect a given molecule, by treating it with antibodies that typically bind to prostate specific antigen (PSA). They pumped in a solution containing small amounts of PSA and found that the array captured the antigen effectively, throughout the forest, rather than just on the outer surface of a typical microfluidic element.

Wardle says that the nanotube array is extremely versatile, as the carbon nanotubes may be manipulated mechanically, electrically, and optically, while the polymer coatings may be chemically altered to capture a wide range of particles. He says an immediate target may be biomarkers called exosomes, which are less than 100 nanometers wide and can be important signals of a disease’s progression.

“Science is really picking up on how much information these particles contain, and they’re sort of everywhere, but really hard to find, even with large-scale equipment,” Wardle says. “This type of device actually has all the characteristics and functionality that would allow you to go after bioparticles like exosomes and things that really truly are nanometer scale.”

This research was funded, in part, by the National Science Foundation.

 

A Natural Light Switch

MIT scientists identify and map the protein behind a light-sensing mechanism.

MIT scientists, working with colleagues in Spain, have discovered and mapped a light-sensing protein that uses vitamin B12 to perform key functions, including gene regulation.

The result, derived from studying proteins from the bacterium Thermus thermophilus, involves at least two findings of broad interest. First, it expands our knowledge of the biological role of vitamin B12, which was already understood to help convert fat into energy, and to be involved in brain formation, but has now been identified as a key part of photoreceptor proteins — the structures that allow organisms to sense and respond to light.

Second, the research describes a new mode of gene regulation, in which the light-sensing proteins play a key role. In so doing, the scientists observe, the bacteria have repurposed existing protein structures that use vitamin B12, and put them to work in new ways.

MIT-Proteins-Light-1_0.jpg

http://www.technologynetworks.com/images/videos/News%20Images/CR/MIT-Proteins-Light-1_0.jpg

“Nature borrowed not just the vitamin, but really the whole enzyme unit, and modified it … and made it a light sensor,” says Catherine Drennan, a professor of chemistry and biology at MIT

 

The paper describes the photoreceptors in three different states: in the dark, bound to DNA, and after being exposed to light.

“It’s wonderful that we’ve been able to get all the series of structures, to understand how it works at each stage,” Drennan says.

The paper has nine co-authors, including Drennan; graduate students Percival Yang-Ting Chen, Marco Jost, and Gyunghoon Kang of MIT; Jesus Fernandez-Zapata and S. Padmanabhan of the Institute of Physical Chemistry Rocasolano, in Madrid; and Monserrat Elias-Arnanz, Juan Manuel Ortiz-Guerreo, and Maria Carmen Polanco, of the University of Murcia, in Murcia, Spain.

The researchers used a combination of X-ray crystallography techniques and in-vitro analysis to study the bacteria. Drennan, who has studied enzymes that employ vitamin B12 since she was a graduate student, emphasizes that key elements of the research were performed by all the co-authors.

Jost performed crystallography to establish the shapes of the structures, while the Spanish researchers, Drennan notes, “did all of the control experiments to show that we were really thinking about this right,” among other things.

MIT-Proteins-Light-2.jpg

By studying the structures of the photoreceptor proteins in their three states, the scientists developed a more thorough understanding of the structures, and their functions, than they would have by viewing the proteins in just one state.

Microbes, like many other organisms, benefit from knowing whether they are in light or darkness. The photoreceptors bind to the DNA in the dark, and prevent activity pertaining to the genes of Thermus thermophilus. When light hits the microbes, the photoreceptor structures cleave and “fall apart,” as Drennan puts it, and the bacteria start producing carotenoids, which protect the organisms from negative effects of sunlight, such as DNA damage.

The research also shows that the exact manner in which the photoreceptors bind to the DNA is novel. The structures contain tetramers, four subunits of the protein, of which exactly three are bound to the genetic material — something Drennan says surprised her.

“That’s the best part about science,” Drennan says. “You see something novel, then you think it’s not really going to be that novel, but you do the experiments [and it is].”

Other scientists say the findings are significant. “It’s a very exciting development,” says Rowena Matthews, a professor emerita of biological chemistry at the University of Michigan, who has read the paper. Of the newly discovered use of vitamin B12 and a derivative of it, adenosylcobalamin, Matthews adds, “There was very limited knowledge of its versatility.”

Drennan adds that in the long run, the finding could have practical applications, such as the engineering of light-directed control of DNA transcription, or the development of controlled interactions between proteins.

“I would be very interested in … thinking about whether there could be practical applications of this,” Drennan says.

 

HIV Protein Manipulates Hundreds of Human Genes

Findings search for new or improved treatments for patients with AIDS.

UT Southwestern Medical Center researchers have deciphered how a small protein made by the human immunodeficiency virus (HIV) that causes AIDS manipulates human genes to further its deadly agenda.

The findings, published in the online journal eLife, could aid in the search for new or improved treatments for patients with AIDS, or to the development of preventive strategies.

“We have identified the molecular mechanisms by which the Tat protein made by HIV interacts with the host cell to activate or repress several hundred human genes,” said Dr. Iván D’Orso, Assistant Professor of Microbiology at UT Southwestern and senior author of the study. “The findings clearly suggest that blocking Tat activity may be of therapeutic value to HIV patients.”

It has long been known that HIV causes AIDS by hijacking the body’s immune cells, transforming them into HIV factories and killing other immune cells that normally fight disease. HIV also hides in cells and continues to undermine the host’s immune system despite antiretroviral therapy that has improved the outlook of those with AIDS.

The latest data from the Centers for Disease Control and Prevention (CDC), in 2012, estimated 1.2 million Americans were living with HIV, including 156,300 whose infections had not been diagnosed. About 50,000 people in the U.S. are newly infected with HIV annually, the CDC projects. In 2013, the CDC estimated that over 26,000 Americans had the advanced form of HIV infection, AIDS.

Like all retroviruses, HIV has very few genes of its own and must take over the host’s cellular machinery in order to propagate and spread throughout the body. Although the broad aspects of that cellular hijacking were known, the nuances remain to be explored, Dr. D’Orso said.

“We observed that HIV methodically and precisely manipulates the host’s genes and cellular machinery. We also observed that HIV rewires cellular defensive pathways to benefit survival of the virus,” he added.

The study provides insights into HIV’s ability to survive despite antiretroviral therapy, findings that could lead to new therapeutic targets or ways to make current therapies more effective, he said.

“Our study indicates that this small viral protein, Tat, directly binds to about 400 human genes to generate an environment in which HIV can thrive. Then, this protein precisely turns off the body’s immune defense. It is striking that such a small viral protein has such a large impact,” Dr. D’Orso said. “The human genes and pathways that Tat manipulates correlate well with symptoms observed in these patients, such as immune system hyperactivation, then weakening, and accelerated aging,” Dr. D’Orso said, describing the situation in which HIV infection leads to AIDS.

Italy’s National Institute of Health in Rome recently completed a phase II clinical trial of an experimental vaccine that targets the Tat protein. That trial, which followed 87 HIV-positive patients for up to three years, reported that the vaccine was well-tolerated without significant side effects. However, it will take several years to determine if the vaccine works, Dr. D’Orso said.

Although someone can have HIV for years without showing symptoms, AIDS occurs when HIV blocks the body’s ability to fight off illness. The person then becomes overrun by the opportunistic infections and specific cancers that are hallmarks of AIDS.

 

New Light Shed on Genetic Regulation

A team of scientists has uncovered greater intricacy in protein signaling than was previously understood, shedding new light on the nature of genetic production.

Christine Vogel, an assistant professor in New York University’s Department of Biology and one of the study’s senior authors, explains that “to make a protein, we need to make a messenger RNA molecule from the gene encoded in the DNA, and then, in a second process, make proteins from these RNA molecules. Both processes are highly regulated and coupled.”

This coupling is similar to the coupling between a moving escalator and a person walking on it at the same time.

The research takes a closer look at how the two coupled processes change in the cell responding to an outside stimulus.

“Until recently, it has been very difficult to study these systems and researchers have thought that the movement of the escalator is most important during the cellular response,” Vogel explains. “We now show that is not necessarily the case, and under some circumstances, the person’s walking determines the overall outcome.”

In biology, this means that both of the processes—to make RNAs and proteins—play important roles, but with different patterns.

In their study, the scientists, who also included researchers from National University Singapore and Berlin’s Max Delbruck Center, took a closer look at how the two processes exactly respond over time.

Their results showed notable distinctions between DNA and mRNA in the nature of their signaling. Notably, the process of making RNA from DNA was pulse-like—a brief messaging over the studied period that returned to the normal levels by the end of the measurements. By contrast, the process of making a protein from RNA was akin to an on/off switch: once started, levels remained constant for consistent periods before reverting back to long stretches of dormancy.

While the reasons for these differences in cell behavior remain unknown, the researchers believe the answer may lie in the nature of the two tasks.

“It is very costly for the cell to make proteins, but making RNA messages from DNA is a relatively low-energy and simple process, so it makes sense that we see frequent, or pulsating, signaling at this stage,” observes Vogel. “By contrast, creating proteins is an intricate undertaking, requiring a great deal of time and energy. This may be why, once you decided to stop production of proteins, you do not turn it back on that easily—and the other way around.”

 

Where Cancer Cells May Begin

Scientists use fruit fly genetics to understand how things could go wrong in cancer.

Cancer cells are normal cells that go awry by making bad developmental decisions during their lives. In a study involving the fruit fly equivalent of an oncogene implicated in many human leukemias, Northwestern University researchers have gained insight into how developing cells normally switch to a restricted, or specialized, state and how that process might go wrong in cancer.

The fruit fly’s eye is an intricate pattern of many different specialized cells, such as light-sensing neurons and cone cells. Because flies share with humans many of the same cancer-causing genes, scientists use the precisely made compound eye of Drosophila melanogaster (the common fruit fly) as a workhorse to study what goes wrong in human cancer.

retina638.jpg

http://www.technologynetworks.com/images/videos/News%20Images/GT/retina638.jpg

A multidisciplinary team co-led by biologist Richard W. Carthew and engineer Luís A.N. Amaral studied normal cell behavior in the developing eye. The researchers were surprised to discover that the levels of an important protein called Yan start fluctuating wildly when the cell is switching from a more primitive, stem-like state to a more specialized state. If the levels don’t or can’t fluctuate, the cell doesn’t switch and move forward.

“This mad fluctuation, or noise, happens at the time of cell transition,” said Carthew, professor of molecular biosciences in Northwestern’s Weinberg College of Arts and Sciences. “For the first time, we see there is a brief time period as the developing cell goes from point A to point B. The noise is a state of ‘in between’ and is important for cells to switch to a more specialized state. This limbo might be where normal cells take a cancerous path.”

The researchers also found that a molecular signal received by a cell receptor called EGFR is important for turning the noise off. If that signal is not received, the cell remains in an uncontrolled state.

By pinpointing this noise and its “off” switch as important points in the normal process of cell differentiation, the Northwestern researchers provide targets for scientists studying how cells can go out of control and transform into cancer cells.

The “noisy” protein the Northwestern researchers studied is called Yan in the fly and Tel-1 in humans. (The protein is a transcription factor.) The Tel-1 protein instructs cells to turn into white blood cells; the gene that produces the protein, oncogene Tel-1, is frequently mutated in leukemia.

The EGFR protein that turns off the noise in flies is called Her-2 in humans. Her-2 is an oncogene that plays an important role in human breast cancer.

“On the surface, flies and humans are very different, but we share a remarkable amount of infrastructure,” said Carthew, a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. “We can use fruit fly genetics to understand how humans work and how things go wrong in cancer and other diseases.”

Fruit fly cells are small and closely packed together, making study of them challenging. Carthew and Amaral’s team of biologists, chemical and biological engineers, computer scientists and chemists together figured out how to identify and analyze thousands and thousands of individual cells in the flies’ eyes.

“In the past, people have built models of regulatory networks that control cell differentiation mostly by genetically perturbing one or two components of the network at a time and then compiling those results into models,” said Amaral, professor of chemical and biological engineering at the McCormick School of Engineering. “We instead measured the retina as it developed and found the unexpected behavior of the key regulatory factors Yan and EGFR.”

Nicolás Peláez, first author of the study and a Ph.D. candidate in interdisciplinary biological sciences working with Amaral and Carthew, built new tools to study this strange feature of noise in developing flies. His methods enabled the researchers to easily measure both the concentration of the Yan protein and its fluctuation (noise).

It takes 15 to 20 hours for a fruit fly cell to go from being an unrestricted cell to a restricted cell, Carthew said. Peláez determined the Yan protein is noisy, or fluctuating, for six to eight of those hours.

“Studying the dynamics of molecules regulating fly-eye patterning can inform us about human disease,” Peláez said. “Using model organisms such as fruit flies will help us understand quantitatively the basic biological principles governing differentiation in complex animals.”

 

Mechanism of Tumor Suppressing Gene Uncovered

The most commonly mutated gene in cancer,p53, works to prevent tumor formation by keeping mobile elements in check that otherwise lead to genomic instability, UT Southwestern Medical Center researchers have found.

The p53 gene long has been known to suppress tumor formation, but the mechanisms behind this function – and why disabling the gene allows tumors to form – were not fully understood.

Findings from the study answer some of these questions and could one day lead to new ways of diagnosing and treating cancer, said the study’s senior author, Dr. John Abrams, Professor of Cell Biology at UT Southwestern.

The investigators found that normal p53 gene action restrains transposons, mobile genetic elements called retroelements that can make copies of themselves and move to different positions on chromosomes. But, they discovered, when p53 is disabled by mutation, dramatic eruptions of these mobile elements occur. The study revealed that in mice with cancer and in human samples of two types of cancer (Wilms’ tumors and colon tumors) disabled for p53, transposons became very active.

In a healthy state, certain mechanisms work to keep these retroelements quiet and inactive, explained Dr. Abrams. One of those mechanisms is p53 action. Conversely, when p53 is mutated, retroelements can erupt.

“If you take the gene away, transposons can wreak havoc throughout the genome by causing it to become highly dysregulated, which can lead to disease,” Dr. Abrams said. “Our findings help explain why cancer genomes are so much more fluid and destabilized than normal genomes. They also provide a novel framework for understanding how normal cells become tumors.”

Although much more research is needed, Dr. Abrams said, the potential clinical implications of the team’s findings are significant.

“Understanding how p53 prevents tumors raises the prospect of therapeutic interventions to correct cases in which p53 is disabled,” Dr. Abrams said. “If retroelements are at the heart of certain p53-driven cancers, finding ways to suppress them could potentially allow us to prevent those cancers or intervene to keep them from progressing.”

This understanding also could lead to advances in diagnosing some cancers through biomarkers related to p53 and transposon activity.

“One possibility is that perhaps blood or urine tests could detect dysregulated retroelements that could be indicative of certain types of cancer,” Dr. Abrams said.

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The transport of molecules across membranes

Posted in Ca2+ triggered activation, Genome Biology, Metabolomics, Microvesicle, Neuroscience, Proteins, Proteomics, tagged , , , , on January 23, 2016| Leave a Comment »

The transport of molecules across membranes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Cellular Transport and the Nobel Prize for Medicine

Extracted  from October 8, 2013 | by

The 2013 Nobel Prize in Physiology or Medicine was  awarded to  to Randy W. Schekman, at the University of California at Berkeley; James E. Rothman,  at Yale University in New Haven, Connecticut; and Thomas C. Südhof,  at Stanford University, for their discoveries of machinery regulating vesicle traffic, a major transport system in cells.three U.S. scientists for their work on how the cell coordinates its transport system to shuttle proteins and other molecules from one location to another.

The organization and transport of molecules across cellular mmembranes is accomplished via vesicles that shuttle cargo between organelles or fuse to other structures to release their cargo outside the cell. The vesicle transport system is critical for a variety of physiological processes, ranging from signaling in the brainto release of hormones and immune cytokines.

Schekman identified three classes of genes that control different facets of the cell’s transport system.

Vesicle fusion

http://www.highlighthealth.com/wp-content/uploads/2013/10/vesicle-fusion.jpg

This was followed by James Rothman’s discovery that a protein complex enables vesicles to fuse with their target membranes (pictured in orange above). This lock and key mechanism ensures that the vesicle fuses at the right location and that cargo molecules are delivered to the correct destination.

Also in the 1990s, Thomas Südhof was studying how nerve cells communicate in the brain. Calcium ions were known to be involved in vesicle cargo release, and Südhof searched for calcium sensitive proteins in nerve cells. He identified the molecular machinery (pictured in purple above) that responds to an influx of calcium ions (Ca2+) and triggers vesicle fusion.

Extracellular vesicles are  participate in the pathogenesis of various diseases, most notably neurodegenerative disorders, and extracellular vesicles are likely to have therapeutic applications in large-molecule drug delivery.

References

  1. The Nobel Prize in Physiology or Medicine 2013 – Press Release. Nobelprize.org. 7 Oct 2013.
  2. Andaloussi et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nature Reviews Drug Discovery 2013 Vol: 12(5):347-357. DOI: 10.1038/nrd3978
    View abstract
  3. Anderson et al. Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis. Lab Invest. 2010 Nov;90(11):1549-57. DOI: 10.1038/labinvest.2010.152. Epub 2010 Aug 30.
    View abstract

 

Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells

http://www.nobelprizemedicine.org/wp-content/uploads/2013/10/Scientific-Background-Zierath-and-Lindahl.pdf

Together, Rothman, Schekman and Südhof have transformed the way we view transport of molecular cargo to specific destinations inside and outside the cell. Their discoveries explain a long-standing enigma in cell biology and also shed new light on how disturbances in this machinery can have deleterious effects and contribute to conditions such as neurological diseases, diabetes, and immunological disorders.

Eukaryotic cells differ from prokaryotic cells by their more complex intracellular organization. In eukaryotes, specific cellular functions are compartmentalized into the cell nucleus and organelles surrounded by intracellular membranes. This compartmentalization vastly improves the efficiency of many cellular functions and prevents potentially dangerous molecules from roaming freely within the cell. But when distinct cellular processes are compartmentalized, a problem emerges. Different compartments need to exchange specific molecules (Figure 1). Furthermore, certain molecules need to be exported to the cell exterior. Most molecules are too large to directly pass through membranes, thus a mechanism that ensures specific delivery of this molecular cargo is required.

Figure 1: Each cell in the body has a complex organization where specific cellular functions are separated into different compartments called organelles. Molecules produced in the cell are packaged in vesicles and transported with special and temporal precision to the correct locations within and outside the cell.

Mysteries of cellular compartmentalization have long intrigued scientists. Improved light microscopy techniques aided in the understanding of intracellular organization in eukaryotic cells, but the advent of electron microscopy and new staining techniques, combined with subcellular fractionation assays using differential ultracentrifugation procedures, led to a deeper understanding of the cell’s inner life. Albert Claude, George Palade and Christian de Duve, who received the Nobel Prize in Physiology or Medicine 1974*, were pioneers in this area and have shed light on how the cell is organized and compartmentalized. Secretory proteins were shown to be produced on ribosomes in the endoplasmic reticulum (ER) and trafficked to the Golgi complex (named after the 1906 Nobel Laureate Camillo Golgi) (Figure 1). Progress was also made in deciphering how proteins find their appropriate destination. Günter Blobel was awarded the 1999 Nobel Prize in Physiology or Medicine* for his discoveries that proteins have intrinsic signals that govern their transport and localization in the cell. Yet, a lingering question remained. How are molecules, including hormones, transport proteins, and neurotransmitters, correctly routed to their appropriate destination? From the work of Palade, the traffic of secretory proteins from the ER was understood to be carried out using small membrane-surrounded vesicles that bud from one membrane and fuse with another, but how precision could be acquired in this process remained enigmatic.

The work of  Rothman, Schekman and Südhof represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes the routing of molecules packaged in vesicles to various intracellular destinations, as well as to the outside of the cell. Specificity in the delivery of molecular cargo is essential for cell function and survival. This specificity is required for the release of neurotransmitters into the presynaptic region of a nerve cell to transmit a signal to a neighboring nerve cell. Likewise, specificity is required for the export of hormones such as insulin to the cell surface. While vesicles within the cell were long known to be critical components of this transportation scheme, the precise mechanism by which these vesicles found their correct destination and how they fused with organelles or the plasma membrane to deliver the cargo remained mysterious. The work of the three 2013 Laureates radically altered our understanding of this aspect of cell physiology. Randy W. Schekman used yeast genetics to identify a set of genes critical for vesicular trafficking. He showed that these genes were essential for life and could be classified into three categories regulating different aspects of vesicle transport. James E. Rothman embarked on a biochemical approach and identified proteins that form a functional complex controlling cell fusion. Proteins on the vesicle and target membrane sides bind in specific combinations, ensuring precise delivery of molecular cargo to the right destination. Thomas C. Südhof became interested in how vesicle fusion machinery was controlled. He unraveled the mechanism by which calcium ions trigger release of neurotransmitters, and identified key regulatory components in the vesicle fusion machinery.

Schekman discovered genes encoding proteins that are key regulators of vesicle traffic. Comparing normal with genetically mutated yeast cells in which vesicle traffic was disturbed, he identified genes that control transport to different compartments and to the cell surface

Rothman published a series of papers where he reconstituted the intracellular transport of the VSV-G protein within the Golgi complex. He then used the assay to study both vesicle budding and fusion, and purified proteins from the cytoplasm that were required for transport. The first protein to be purified was the Nethylmaleimide-sensitive factor (NSF). Rothman’s discovery of NSF paved the way for the subsequent identification of other proteins important for the control of vesicle fusion, and the next one in line was SNAP (soluble NSFattachment protein). SNAPs bind to membranes and assist in the recruitment of NSF.

One of the yeast mutants, sec18, corresponded to NSF, which also revealed that the vesicle fusion machinery was evolutionarily ancient. Furthermore, Rothman and Schekman collaboratively cloned sec17 and provided evidence of its functional equivalence to SNAP. Other sec genes were shown to correspond to genes encoding fusion proteins were identified by other methods.

Using the NSF and SNAP proteins as bait, Rothman next turned to brain tissue, from which he purified proteins that he later named SNAREs (soluble NSF-attachment protein receptors). Intriguingly, three SNARE proteins, VAMP/Synaptobrevin, SNAP-25 and syntaxin, were found in stoichiometric amounts, which suggested to Rothman that they functioned together in the vesicle and target membranes. The three proteins had previously been identified by several scientists, including Richard Scheller, Kimio Akagawa, Reinhard Jahn and Pietro de Camilli, and localized to the presynaptic region, but their function was largely unknown. VAMP/Synaptobrevin resided on the vesicle, whereas SNAP-25 and syntaxin were found at the plasma membrane. This prompted Rothman to propose a hypothesis – the SNARE hypothesis – which stipulated that target and vesicle SNAREs (t-SNAREs and v-SNAREs) were critical for vesicle fusion through a set of sequential steps of synaptic docking, activation and fusion.

Thomas C. Südhof originally trained at the Georg-August-Universität and the Max-Planck Institute for Biophysical Sciences in Göttingen, Germany, and was a postdoctoral fellow with Michael Brown and Joseph Goldstein (Nobel Prize 1985) at University of Texas Southwestern Medical School in Dallas. As a junior group leader, he set out to study how synaptic vesicle fusion was controlled. Rothman and Schekman had provided fundamental machinery for vesicle fusion, but how vesicle fusion was temporally controlled remained enigmatic. Vesicular fusions in the body need to be kept carefully in check, and in some cases vesicle fusion has to be executed with high precision in response to specific stimuli. This is the case for example for neurotransmitter release in the brain and for insulin secretion from the endocrine pancreas.

The neurophysiology field was electrified by the discoveries of Bernard Katz, Ulf von Euler and Julius Axelrod who received the Nobel Prize in Physiology or Medicine 1970* for their discoveries concerning the humoral transmittors in the nerve terminals and the mechanism for their storage, release and inactivation. Südhof was intrigued by the rapid exocytosis of synaptic vesicles, which is under tight temporal control and regulated by the changes in the cytoplasmic free calcium concentration. Südhof elucidated how calcium regulates neurotransmitter release in neurons and discovered that complexin and synaptotagmin are two critical proteins in calcium-mediated vesicle fusion.

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Hybrid lipid bioelectronic membranes

Posted in Bioengineering & reverse engineering design, BioTechnology - Venture Creation, Cell Biology, Signaling & Cell Circuits, Curation, Electrophysiology, Innovations, Innovations in Neurophysiology & Neuropsychology, Ionic Transporters Na+, Lipid metabolism, Lipidomics, Lipids, Liposome, Metabolism, Microvesicle, Na-K transport, Nephrology, Neurohumoral Transmission, Physics, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, Synaptic vesicle, tagged , , , , , on December 14, 2015| Leave a Comment »

Hybrid lipid bioelectronic membranes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Hybrid solid-state chips and biological cells integrated at molecular level

Biological ion channels combine with solid-state transistors to create a new kind of hybrid bioelectronics. Imagine chips with dog-like capability to taste and smell, or even recognize specific molecules.
http://www.kurzweilai.net/hybrid-solid-state-chips-and-biological-cells-integrated-at-molecular-level
http://www.kurzweilai.net/images/biocell-CMOS-.jpg
Illustration depicting a biocell attached to a CMOS integrated circuit with a membrane containing sodium-potassium pumps in pores. Energy is stored chemically in ATP molecules. When the energy is released as charged ions (which are then converted to electrons to power the chip at the bottom of the experimental device), the ATP is converted to ADP + inorganic phosphate. (credit: Trevor Finney and Jared Roseman/Columbia Engineering)

Columbia Engineering researchers have combined biological and solid-state components for the first time, opening the door to creating entirely new artificial biosystems.

In this experiment, they used a biological cell to power a conventional solid-state complementary metal-oxide-semiconductor (CMOS) integrated circuit. An artificial lipid bilayer membrane containing adenosine triphosphate (ATP)-powered ion pumps (which provide energy for cells) was used as a source of ions (which were converted to electrons to power the chip).

The study, led by Ken Shepard, Lau Family Professor of Electrical Engineering and professor of biomedical engineering at Columbia Engineering, was published online today (Dec. 7, 2015) in an open-access paper in Nature Communications.

How to build a hybrid biochip

Living systems achieve this functionality with their own version of electronics based on lipid membranes and ion channels and pumps, which act as a kind of “biological transistor.” Charge in the form of ions carry energy and information, and ion channels control the flow of ions across cell membranes.

Solid-state systems, such as those in computers and communication devices, use electrons; their electronic signaling and power are controlled by field-effect transistors.

To build a prototype of their hybrid system, Shepard’s team packaged a CMOS integrated circuit (IC) with an ATP-harvesting “biocell.” In the presence of ATP, the system pumped ions across the membrane, producing an electrical potential (voltage)* that was harvested by the integrated circuit.

“We made a macroscale version of this system, at the scale of several millimeters, to see if it worked,” Shepard notes. “Our results provide new insight into a generalized circuit model, enabling us to determine the conditions to maximize the efficiency of harnessing chemical energy through the action of these ion pumps. We will now be looking at how to scale the system down.”

While other groups have harvested energy from living systems, Shepard and his team are exploring how to do this at the molecular level, isolating just the desired function and interfacing this with electronics. “We don’t need the whole cell,” he explains. “We just grab the component of the cell that’s doing what we want. For this project, we isolated the ATPases because they were the proteins that allowed us to extract energy from ATP.”

The capability of a bomb-sniffing dog, no Alpo required

Next, the researchers plan to go much further, such as recognizing specific molecules and giving chips the potential to taste and smell.

The ability to build a system that combines the power of solid-state electronics with the capabilities of biological components has great promise, they believe. “You need a bomb-sniffing dog now, but if you can take just the part of the dog that is useful — the molecules that are doing the sensing — we wouldn’t need the whole animal,” says Shepard.

The technology could also provide a power source for implanted electronic devices in ATP-rich environments such as inside living cells, the researchers suggest.

*  “In general, integrated circuits, even when operated at the point of minimum energy in subthreshold, consume on the order of 10−2 W mm−2 (or assuming a typical silicon chip thickness of 250 μm, 4 × 10−2 W mm−3). Typical cells, in contrast, consume on the order of 4 × 10−6 W mm−3. In the experiment, a typical active power dissipation for the IC circuit was 92.3 nW, and the active average harvesting power was 71.4 fW for the biocell (the discrepancy is managed through duty-cycled operation of the IC).” — Jared M. Roseman et al./Nature Communications

 

references:

Jared M. Roseman et al. Hybrid integrated biological–solid-state system powered with adenosine triphosphate. Nature Communications 6, 10070 doi:10.1038/ncomms10070. 07 December 2015 (open access)

Topics: Biotech | Electronics | Synthetic Biology

Hybrid integrated biological–solid-state system powered with adenosine triphosphate

Jared M. Roseman, Jianxun Lin, Siddharth Ramakrishnan, Jacob K. Rosenstein & Kenneth L. Shepard
Nature Communications 7 Dec 2015; 6(10070)     http://dx.doi.org:/10.1038/ncomms10070

There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na+/K+ adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 106 mm−2) are able to sustain a short-circuit current of 32.6 pA mm−2 and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm−2 from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

 

Figure 1: Fully hybrid biological–solid-state system.

 

 

Fully hybrid biological-solid-state system.

http://www.nature.com/ncomms/2015/151207/ncomms10070/images/ncomms10070-f1.jpg

(a) Illustration depicting biocell attached to CMOS integrated circuit. (b) Illustration of membrane in pore containing sodium–potassium pumps. (c) Circuit model of equivalent stacked membranes, =2.1 pA, =98.6 GΩ, =575 GΩ and =75 pF, Ag/AgCl electrode equivalent resistance RWE+RCE<20 kΩ, energy-harvesting capacitor CSTOR=100 nF combined with switch as an impedance transformation network (only one switch necessary due to small duty cycle), and CMOS IC voltage doubler and resistor representing digital switching load. RL represents the four independent ring oscillator loads. (d) Equivalent circuit detail of stacked biocell. (e) Switched-capacitor voltage doubler circuit schematic.

 

The energetics of living systems are based on electrochemical membrane potentials that are present in cell plasma membranes, the inner membrane of mitochondria, or the thylakoid membrane of chloroplasts1. In the latter two cases, the specific membrane potential is known as the proton-motive force and is used by proton adenosine triphosphate (ATP) synthases to produce ATP. In the former case, Na+/K+-ATPases hydrolyse ATP to maintain the resting potential in most cells.

While there have been recent efforts to harness power from some naturally occurring potentials in living systems that are the result of ion pump action both in plants2 and animals3, 4 to power complementary metal-oxide semiconductor (CMOS) integrated circuits (ICs), this work is the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitroenvironment to power such an artificial system. Prior efforts to harness power from in vitromembrane systems incorporating ion-pumping ATPases5, 6, 7, 8, 9 and light-activated bacteriorhodopsin9, 10, 11 have been limited by difficulty in incorporating these proteins in sufficient quantity to attain measurable current and in achieving sufficiently large membrane resistances to harness these currents. Both problems are solved in this effort to power an IC from ATP in an in vitro environment. The resulting measurements provide new insight into a generalized circuit model, which allows us to determine the conditions to maximize the efficiency of harnessing chemical energy through the action of electrogenic ion pumps.

 

ATP-powered IC

Figure 1a shows the complete hybrid integrated system, consisting of a CMOS IC packaged with an ATP-harvesting ‘biocell’. The biocell consists of two series-stacked ATPase bearing suspended lipid bilayers with a fluid chamber directly on top of the IC. Series stacking of two membranes is necessary to provide the required start-up voltage for IC and eliminates the need for an external energy source, which is typically required to start circuits from low-voltage supplies2, 3. As shown inFig. 1c, a matching network in the form of a switched capacitor allows the load resistance of the IC to be matched to that presented by the biocell. In principle, the switch S can be implicit. The biocell charges CSTOR until the self start-up voltage, Vstart, is reached. The chip then operates until the biocell voltage drops below the minimum supply voltage for operation, Vmin. Active current draw from the IC stops at this point, allowing the charge to build up again on CSTOR. In our case, however, the IC leakage current exceeds 13.5 nA at Vstart, more than can be provided by the biocell. As a result, an explicit transistor switch and comparator (outside of the IC) are used for this function in the experimental results presented here, which are not powered by the biocell and not included in energy efficiency calculations (see Supplementary Discussion for additional details). The energy from the biocell is used to operate a voltage converter (voltage doubler) and some simple inverter-based ring oscillators in the IC, which receive power from no other sources.

Figure 1: Fully hybrid biological–solid-state system.

http://www.nature.com/ncomms/2015/151207/ncomms10070/images/ncomms10070-f1.jpg

 

……..   Prior to the addition of ATP, the membrane produces no electrical power and has an Rm of 280 GΩ. A 1.7-pA short-circuit (SC) current (Fig. 2b) through the membrane is observed upon the addition of ATP (final concentration 3 mM) to the cis chamber where functional, properly oriented enzymes generate a net electrogenic pump current. To perform these measurements, currents through each membrane of the biocell are measured using a voltage-clamp amplifier (inset of Fig. 2b) with a gain of 500 GΩ with special efforts taken to compensate amplifier leakage currents. Each ATPase transports three Na+ ions from the cis chamber to the trans chamber and two K+ ions from thetrans chamber to the cis chamber (a net charge movement of one cation) for every molecule of ATP hydrolysed. At a rate of 100 hydrolysis events per second under zero electrical (SC) bias13, this results in an electrogenic current of ~16 aA. The observed SC current corresponds to about 105 active ATPases in the membrane or a concentration of about 2 × 106 mm−2, about 5% of the density of channels occurring naturally in mammalian nerve fibres14. It is expected that half of the channels inserted are inactive because they are oriented incorrectly.

Figure 2: Single-cell biocell characterization.

http://www.nature.com/ncomms/2015/151207/ncomms10070/images_article/ncomms10070-f2.jpg

(a)…Pre-ATP data linear fit (black line) slope yield Rm=280 GΩ. Post ATP data fit to a Boltzmann curve, slope=0.02 V (blue line). Post-ATP linear fit (red line) yields Ip=−1.8 pA and Rp=61.6 GΩ, which corresponds to a per-ATP source resistance of 6.16 × 1015. The current due to membrane leakage through R_{m} is subtracted in the post-ATP curve…. (b)…

 

Current–voltage characteristics of the ATPases

Figure 2a shows the complete measured current–voltage (I–V) characteristic of a single ATPase-bearing membrane in the presence of ATP. The current due to membrane leakage through Rm is subtracted in the post-ATP curve. The I–V characteristic fits a Boltzmann sigmoid curve, consistent with sodium–potassium pump currents measured on membrane patches at similar buffer conditions13, 15, 16. This nonlinear behaviour reflects the fact that the full ATPase transport cycle (three Na+ ions from cis to trans and two K+ ions from trans to cis) time increases (the turn-over rate, kATP, decreases) as the membrane potential increases16. No effect on pump current is expected from any ion concentration gradients produced by the action of the ATPases (seeSupplementary Discussion). Using this Boltzmann fit, we can model the biocell as a nonlinear voltage-controlled current source IATPase (inset Fig. 2a), in which the current produced by this source varies as a function of Vm. In the fourth quadrant, where the cell is producing electrical power, this model can be linearized as a Norton equivalent circuit, consisting of a DC current source (Ip) in parallel with a current-limiting resistor (Rp), which acts to limit the current delivered to the load at increasing bias (IATPase~Ip−Vm/Rp). Figure 2c shows the measured and simulated charging of Cm for a single membrane (open-circuited voltage). A custom amplifier with input resistance Rin>10 TΩ was required for this measurement (see Electrical Measurement Methods).

 

Reconciling operating voltage differences

The electrical characteristics of biological systems and solid-state systems are mismatched in their operating voltages. The minimum operating voltage of solid-state systems is determined by the need for transistors to modulate a Maxwell–Boltzmann (MB) distribution of carriers by several orders of magnitude through the application of a potential that is several multiples of kT/q (where kis Boltzmann’s constant, T is the temperature in degrees Kelvin and q is the elementary charge). Biological systems, while operating under the same MB statistics, have no such constraints for operating ion channels since they are controlled by mechanical (or other conformational) processes rather than through modulation of a potential barrier. To bridge this operating voltage mismatch, the circuit includes a switched-capacitor voltage doubler (Fig. 1d) that is capable of self-startup from voltages as low Vstart=145 mV (~5.5 kT/q) and can be operated continuously from input voltages from as low as Vmin=110 mV (see Supplementary Discussion)…..

 

Maximizing the efficiency of harvesting energy from ATP

Solid-state systems and biological systems are also mismatched in their operating impedances. In our case, the biocell presents a source impedance, =84.2 GΩ, while the load impedance presented by the complete integrated circuit (including both the voltage converter and ring oscillator loads) is approximately RIC=200 kΩ. (The load impedance, RL, of the ring oscillators alone is 305 kΩ.) This mismatch in source and load impedance is manifest in large differences in power densities. In general, integrated circuits, even when operated at the point of minimum energy in subthreshold, consume on the order of 10−2 W mm−2 (or assuming a typical silicon chip thickness of 250 μm, 4 × 10−2 W mm−3) (ref. 17). Typical cells, in contrast, consume on the order of 4 × 10−6 W mm−3 (ref. 18). In our case, a typical active power dissipation for our circuit is 92.3 nW, and the active average harvesting power is 71.4 fW for the biocell. This discrepancy is managed through duty-cycled operation of the IC in which the circuit is largely disabled for long periods of time (Tcharge), integrating up the power onto a storage capacitor (CSTOR), which is then expended in a very brief period of activity (Trun), as shown in Fig. 3a.

The overall efficiency of the system in converting chemical energy to the energy consumed in the load ring oscillator (η) is given by the product of the conversion efficiency of the voltage doubler (ηconverter) and the conversion efficiency of chemical energy to electrical energy in the biocell (ηbiocell), η=ηconverter × ηbiocell. ηconverter is relatively constant over the range of input voltages at ~59%, as determined by various loading test circuits included in the chip design (Supplementary Figs 1–6). ηbiocell, however, varies with transmembrane potential Vm. η is the efficiency in transferring power to the power ring oscillator loads from the ATP harvested by biocell.

…….

To first order, the energy made available to the Na+/K+-ATPase by the hydrolysis of ATP is independent of the chemical or electric potential of the membrane and is given by |ΔGATP|/(qNA), where ΔGATP is the Gibbs free energy change due to the ATP hydrolysis reaction per mole of ATP at given buffer conditions and NA is Avogadro’s number. Since every charge that passes through IATPase corresponds to a single hydrolysis event, we can use two voltage sources in series with IATPase to independently account for the energy expended by the pumps both in moving charge across the electric potential difference and in moving ions across the chemical potential difference. The dependent voltage source Vloss in this branch fixes the voltage across IATPase, and the total power produced by the pump current source is (|ΔGATP|/NA)(NkATP), which is the product of the energy released per molecule of ATP, the number of active ATPases and the ATP turnover rate. The power dissipated in voltage source Vchem models the work performed by the ATPases in transporting ions against a concentration gradient. In the case of the Na+/K+ ATPase,Vchem is given by . The power dissipated in this source is introduced back into the circuit in the power generated by the Nernst independent voltage sources, and . The power dissipated in the dependent voltage source Vloss models any additional power not used to perform chemical or electrical work. ……

 

Integration of ATP-harvesting ion pumps could provide a means to power future CMOS microsystems scaled to the level of individual cells22. In molecular diagnostics, the integration of pore-forming proteins such as alpha haemolysin23 or MspA porin24 with CMOS electronics is already finding application in DNA sequencing25. Exploiting the large diversity of function available in transmembrane proteins in these hybrid systems could, for example, lead to highly specific sensing platforms for airborne odorants or soluble molecular entities26, 27. Heavily multiplexed platforms could become high-throughput in vitro drug-screening platforms against this diversity of function. In addition, integration of transmembrane proteins with CMOS may become a convenient alternative to fluorescence for coupling to synthetic biological systems28.

 

Roseman, J. M. et al. Hybrid integrated biological–solid-state system powered with adenosine triphosphate. Nat. Commun. 6:10070      http://dx.doi.org:/10.1038/ncomms10070 (2015).

 

 

  • Rottenberg, H. The measurement of membrane potential and deltapH in cells, organelles, and vesicles. Methods Enzymol. 55, 547–569 (1979).
  • Himes, C., Carlson, E., Ricchiuti, R. J., Otis, B. P. & Parviz, B. A. Ultralow voltage nanoelectronics powered directly, and solely, from a tree. IEEE Trans. Nanotechnol. 9, 2–5(2010).
  • Mercier, P. P., Lysaght, A. C., Bandyopadhyay, S., Chandrakasan, A. P. & Stankovic, K. M.Energy extraction from the biologic battery in the inner ear. Nat. Biotechnol. 30, 1240–1243(2012).
  • Halámková, L. et al. Implanted Biofuel Cell Operating in a Living Snail. J. Am. Chem. Soc.134, 5040–5043 (2012).

 

 

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Topology of Protein Complexes

Posted in Physics, Proteins, Proteomics, Sensors & Analytics, Signaling, Translational Science, Unfolded Protein Response (UPR), tagged , , , , , , on December 11, 2015| Leave a Comment »

Topology of Protein Complexes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Periodic Table of Protein Complexes Unveiled

http://www.genengnews.com/gen-news-highlights/periodic-table-of-protein-complexes-unveiled/81252091/

 

 

http://www.genengnews.com/Media/images/GENHighlight/thumb_Dec11_2015_EMBLEBI_ProteinComplexesPeriodicTable1692141643.jpg

A new periodic table presents a systematic, ordered view of protein assembly, providing a visual tool for understanding biological function. [EMBL-EBI / Spencer Phillips]

 

Move over Mendeleev, there’s a new periodic table in science. Unlike the original periodic table, which organized the chemical elements, the new periodic table organizes protein complexes, or more precisely, quaternary structure topologies. Though there are other differences between the old and new periodic tables, they share at least one important feature—predictive power.

When Mendeleev introduced his periodic table, he predicted that when new chemical elements were discovered, they would fill his table’s blank spots. Analogous predictions are being ventured by the scientific team that assembled the new periodic table. This team, consisting of scientists from the Wellcome Genome Campus and the University of Cambridge, asserts that its periodic table reveals the regions of quaternary structure space that remain to be populated.

The periodic table of protein complexes not only offers a new way of looking at the enormous variety of structures that proteins can build in nature, it also indicates which structures might be discovered next. Moreover, it could point protein engineers toward entirely novel structures that never occurred in nature, but could be engineered.

The new table appeared December 11 in the journal Science, in an article entitled, “Principles of assembly reveal a periodic table of protein complexes.” The “principles of assembly” referenced in this title amount to three basic assembly types: dimerization, cyclization, and heteromeric subunit addition. In dimerization, one protein complex subunit doubles, and becomes two; in cyclization, protein complex subunits from a ring of three or more; and in heteromeric subunit addition, two different proteins bind to each other.

These steps, repeated in different combinations, gives rise to enormous number of proteins of different kinds. “Evolution has given rise to a huge variety of protein complexes, and it can seem a bit chaotic,” explained Joe Marsh, Ph.D., formerly of the Wellcome Genome Campus and now of the MRC Human Genetics Unit at the University of Edinburgh. “But if you break down the steps proteins take to become complexes, there are some basic rules that can explain almost all of the assemblies people have observed so far.”

The authors of the Science article noted that many protein complexes assemble spontaneously via ordered pathways in vitro, and these pathways have a strong tendency to be evolutionarily conserved. “[There] are strong similarities,” the authors added, “between protein complex assembly and evolutionary pathways, with assembly pathways often being reflective of evolutionary histories, and vice versa. This suggests that it may be useful to consider the types of protein complexes that have evolved from the perspective of what assembly pathways are possible.”

To explore this rationale, the authors examined the fundamental steps by which protein complexes can assemble, using electrospray mass spectrometry experiments, literature-curated assembly data, and a large-scale analysis of protein complex structures. Ultimately, they derived their approach to explaining the observed distribution of known protein complexes in quaternary structure space. This approach, they insist, provides a framework for understanding their evolution.

“In addition, it can contribute considerably to the prediction and modeling of quaternary structures by specifying which topologies are most likely to be adopted by a complex with a given stoichiometry, potentially providing constraints for multi-subunit docking and hybrid methods,” the authors concluded. “Lastly, it could help in the bioengineering of protein complexes by identifying which topologies are most likely to be stable, and thus which types of essential interfaces need to be engineered.”

The rows and columns of the periodic table of the elements, called periods and groups, were originally determined by each element’s atomic mass and chemical properties, later by atomic number and electron configuration. In contrast, the rows and columns of the periodic table of protein complexes correspond to the number of different subunit types and the number of times these subunits are repeated. The new table is not, it should be noted, periodic in the same sense as the periodic table of the elements. It is in principle open-ended.

Although there are no theoretical limitations to quaternary structure topology space in either dimension, the abridged version of the table presented in the Science article can accommodate the vast majority of known structures. Moreover, when the table’s creators compared the large variety of countenanced topologies to observed structures, they found that about 92% of known protein complex structures were compatible with their model.

“Despite its strong predictive power, the basic periodic table model does not account for about 8% of known protein complex structures,” the authors conceded. “More than half of these exceptions arise as a result of quaternary structure assignment errors.

“A benefit of this approach is that it highlights likely quaternary structure misassignments, particularly by identifying nonbijective complexes with even subunit stoichiometry. However, this still leaves about 4% of known structures that are correct but are not compatible with the periodic table.” The authors added that the exceptions to their model are interesting in their own right, and are the subject of ongoing studies.

 

 

http://phys.org/news/2015-12-periodic-table-protein-complexes.html

The Periodic Table of Protein Complexes, published today in Science, offers a new way of looking at the enormous variety of structures that proteins can build in nature, which ones might be discovered next, and predicting how entirely novel structures could be engineered. Created by an interdisciplinary team led by researchers at the Wellcome Genome Campus and the University of Cambridge, the Table provides a valuable tool for research into evolution and protein engineering.

Different ballroom dances can be seen as an endless combination of a small number of basic steps. Similarly, the ‘dance’ of assembly can be seen as endless variations on dimerization (one doubles, and becomes two), cyclisation (one forms a ring of three or more) and subunit addition (two different proteins bind to each other). Because these happen in a fairly predictable way, it’s not as hard as you might think to predict how a novel protein would form.

“We’re bringing a lot of order into the messy world of protein complexes,” explains Sebastian Ahnert of the Cavendish Laboratory at the University of Cambridge, a physicist who regularly tangles with biological problems. “Proteins can keep go through several iterations of these simple steps, , adding more and more levels of complexity and resulting in a huge variety of structures. What we’ve made is a classification based on these underlying principles that helps people get a handle on the complexity.”

The exceptions to the rule are interesting in their own right, adds Sebastian, as are the subject of on-going studies.

“By analysing the tens of thousands of protein complexes for which three-dimensional structures have already been experimentally determined, we could see repeating patterns in the assembly transitions that occur – and with new data from we could start to see the bigger picture,” says Joe.

“The core work for this study is in theoretical physics and computational biology, but it couldn’t have been done without the mass spectrometry work by our colleagues at Oxford University,” adds Sarah Teichmann, Research Group Leader at the European Bioinformatics Institute (EMBL-EBI) and the Wellcome Trust Sanger Institute. “This is yet another excellent example of how extremely valuable interdisciplinary research can be.”

Read more at: http://phys.org/news/2015-12-periodic-table-protein-complexes.html#jCp

 

More information: “Principles of assembly reveal a periodic table of protein complexes” www.sciencemag.org/lookup/doi/10.1126/science.aaa2245

PRINCIPLES OF ASSEMBLY REVEAL A PERIODIC TABLE OF PROTEIN COMPLEXES

Sebastian E. Ahnert1,*, Joseph A. Marsh2,3,*, Helena Hernández4, Carol V. Robinson4, Sarah A. Teichmann1,3,5,†
Science 11 Dec 2015; 350(6266): aaa2245         DOI:http://dx.doi.org:/10.1126/science.aaa2245      

INTRODUCTION

The assembly of proteins into complexes is crucial for most biological processes. The three-dimensional structures of many thousands of homomeric and heteromeric protein complexes have now been determined, and this has had a broad impact on our understanding of biological function and evolution. Despite this, the organizing principles that underlie the great diversity of protein quaternary structures observed in nature remain poorly understood, particularly in comparison with protein folds, which have been extensively classified in terms of their architecture and evolutionary relationships.

RATIONALE

In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization. Our approach was to consider protein complexes in terms of their assembly. Many protein complexes assemble spontaneously via ordered pathways in vitro, and these pathways have a strong tendency to be evolutionarily conserved. Furthermore, there are strong similarities between protein complex assembly and evolutionary pathways, with assembly pathways often being reflective of evolutionary histories, and vice versa. This suggests that it may be useful to consider the types of protein complexes that have evolved from the perspective of what assembly pathways are possible.

RESULTS

We first examined the fundamental steps by which protein complexes can assemble, using electrospray mass spectrometry experiments, literature-curated assembly data, and a large-scale analysis of protein complex structures. We found that most assembly steps can be classified into three basic types: dimerization, cyclization, and heteromeric subunit addition. By systematically combining different assembly steps in different ways, we were able to enumerate a large set of possible quaternary structure topologies, or patterns of key interfaces between the proteins within a complex. The vast majority of real protein complex structures lie within these topologies. This enables a natural organization of protein complexes into a “periodic table,” because each heteromer can be related to a simpler symmetric homomer topology. Exceptions are mostly the result of quaternary structure assignment errors, or cases where sequence-identical subunits can have different interactions and thus introduce asymmetry. Many of these asymmetric complexes fit the paradigm of a periodic table when their assembly role is considered. Finally, we implemented a model based on the periodic table, which predicts the expected frequencies of each quaternary structure topology, including those not yet observed. Our model correctly predicts quaternary structure topologies of recent crystal and electron microscopy structures that are not included in our original data set.

CONCLUSION

This work explains much of the observed distribution of known protein complexes in quaternary structure space and provides a framework for understanding their evolution. In addition, it can contribute considerably to the prediction and modeling of quaternary structures by specifying which topologies are most likely to be adopted by a complex with a given stoichiometry, potentially providing constraints for multi-subunit docking and hybrid methods. Lastly, it could help in the bioengineering of protein complexes by identifying which topologies are most likely to be stable, and thus which types of essential interfaces need to be engineered.

http://www.sciencemag.org/content/350/6266/aaa2245/F1.small.gif

Protein assembly steps lead to a periodic table of protein complexes and can predict likely quaternary structure topologies.

Three main assembly steps are possible: cyclization, dimerization, and subunit addition. By combining these in different ways, a large set of possible quaternary structure topologies can be generated. These can be arranged on a periodic table that describes most known complexes and that can predict previously unobserved topologies.

Ahnert SE, et. al. ‘Principles of assembly reveal a periodic table of protein complexes.’
Science (2015).   DOI: http://dx.doi.org:/10.1126/science.aaa2245    http://www.cam.ac.uk/research/news/the-periodic-table-of-proteins

 

Evolution, classification and dynamics of protein complexes

This talk is included in these lists:

This talk is part of the Biological and Statistical Physics discussion group (BSDG) series.

Classification of protein structure has had a broad impact on our understanding of biological function and evolution, yet this work has largely focused on individual protein domains and their pairwise interactions. In contrast, the assembly of individual polypeptides into protein complexes, which are ubiquitous in cells, has received comparatively little attention. The periodic table of protein complexes is a new framework for analysis of complexes based on the principles of self-assembly. This reveals that sequence-identical subunits almost always have identical assembly roles within a complex and allows us to unify the vast majority of complexes of known structure (~32,000) into about 120 topologies. This facilitates the exhaustive enumeration of unobserved protein complex topologies and has significant practical applications for quaternary structure prediction, modelling and engineering.

http://talks.cam.ac.uk/talk/index/61632

 

 

Genome-wide analysis of thylakoid-bound ribosomes in maize reveals principles of cotranslational targeting to the thylakoid membrane

Reimo Zoschke1 and Alice Barkan2
http://www.pnas.org/content/112/13/E1678.full.pdf

Chloroplast genomes encode ∼37 proteins that integrate into the thylakoid membrane. The mechanisms that target these proteins to the membrane are largely unexplored. We used ribosome profiling to provide a comprehensive, high-resolution map of ribosome positions on chloroplast mRNAs in separated membrane and soluble fractions in maize seedlings. The results show that translation invariably initiates off the thylakoid membrane and that ribosomes synthesizing a subset of membrane proteins subsequently become attached to the membrane in a nucleaseresistant fashion. The transition from soluble to membraneattached ribosomes occurs shortly after the first transmembrane segment in the nascent peptide has emerged from the ribosome. Membrane proteins whose translation terminates before emergence of a transmembrane segment are translated in the stroma and targeted to the membrane posttranslationally. These results indicate that the first transmembrane segment generally comprises the signal that links ribosomes to thylakoid membranes for cotranslational integration. The sole exception is cytochrome f, whose cleavable N-terminal cpSecA-dependent signal sequence engages the thylakoid membrane cotranslationally. The distinct behavior of ribosomes synthesizing the inner envelope protein CemA indicates that sorting signals for the thylakoid and envelope membranes are distinguished cotranslationally. In addition, the fractionation behavior of ribosomes in polycistronic transcription units encoding both membrane and soluble proteins adds to the evidence that the removal of upstream ORFs by RNA processing is not typically required for the translation of internal genes in polycistronic chloroplast mRNAs.

 

Significance Proteins in the chloroplast thylakoid membrane system are derived from both the nuclear and plastid genomes. Mechanisms that localize nucleus-encoded proteins to the thylakoid membrane have been studied intensively, but little is known about the analogous issues for plastid-encoded proteins. This genome-wide, high-resolution analysis of the partitioning of chloroplast ribosomes between membrane and soluble fractions revealed that approximately half of the chloroplast encoded thylakoid proteins integrate cotranslationally and half integrate posttranslationally. Features in the nascent peptide that underlie these distinct behaviors were revealed by analysis of the position on each mRNA at which elongating ribosomes first become attached to the membrane.

 

 

Structures of the HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of the AIM2 Inflammasome and IFI16 Receptor

Tengchuan Jin, Andrew Perry, Jiansheng Jiang, Patrick Smith, James A. Curry, et al.
Immunity 20 Apr 2012; 36(4):561–571    http://dx.doi.org/10.1016/j.immuni.2012.02.014

Figure thumbnail fx1
http://www.cell.com/cms/attachment/2007963858/2030721291/fx1.jpg
Highlights
  • Electrostatic attraction underlies innate dsDNA recognition by the HIN domains
  • Both OB folds and the linker between them engage the dsDNA backbone
  • An autoinhibited state of AIM2 is activated by DNA that liberates the PYD domain
  • DNA serves as an oligomerization platform for the inflammasome assembly

 

Summary

Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.

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P13K delta-gamma anticancer agent

Posted in Biochemical pathways, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Drug Toxicity, FDA, CE Mark & Global Regulatory Affairs: process management and strategic planning - GCP, GLP, ISO 14155, Gene Regulation and Evolution, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genomic Expression, Hematology, Human Immune System in Health and in Disease, Innovations, Intellectual Property, Innovations, Commercialization, Investment in technological breakthrough, Kinase, Lymphoma, Metabolism, Patents, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Pharmacologic toxicities, Proteins, Proteomics, Pyruvate Kinase, tagged , , on December 10, 2015| Leave a Comment »

P13K delta-gamma anticancer agent

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RP 6350, Rhizen Pharmaceuticals S.A. and Novartis tieup for Rhizen’s inhaled dual Pl3K-delta gamma inhibitor

by DR ANTHONY MELVIN CRASTO Ph.D

 

(A)           and                         (Al)                  and                (A2)

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one (Compound A1 is RP 6350).

 

str1

 

RP 6350, RP6350, RP-6350

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

mw 415

Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation  In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016

PATENTS……..WO 11/055215 ,  WO 12/151525.

  • Antineoplastics; Small molecules
  • Mechanism of Action Phosphatidylinositol 3 kinase delta inhibitors; Phosphatidylinositol 3 kinase gamma inhibitors
  • Phase I Haematological malignancies
  • Preclinical Multiple myeloma

 

Swaroop K. V. S. Vakkalanka,
COMPANY Rhizen Pharmaceuticals Sa

https://clinicaltrials.gov/ct2/show/NCT02017613

 

PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)

Company Rhizen Pharmaceuticals S.A.
Description Dual phosphoinositide 3-kinase (PI3K) delta and gamma inhibitor
Molecular Target Phosphoinositide 3-kinase (PI3K) delta ; Phosphoinositide 3-kinase (PI3K) gamma
Mechanism of Action Phosphoinositide 3-kinase (PI3K) delta inhibitor; Phosphoinositide 3-kinase (PI3K) gamma inhibitor
Therapeutic Modality Small molecule

 

Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
 Swaroop Vakkalanka, PhD*,1, Srikant Viswanadha, Ph.D.*,2, Eugenio Gaudio, PhD*,3, Emanuele Zucca, MD4, Francesco Bertoni, MD5, Elena Bernasconi, B.Sc.*,3, Davide Rossi, MD, Ph.D.*,6, and Anastasios Stathis, MD*,7
 1Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland, 2Incozen Therapeutics Pvt. Ltd., Hyderabad, India, 3Lymphoma & Genomics Research Program, IOR-Institute of Oncology Research, Bellinzona, Switzerland, 4IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 5Lymphoma Unit, IOSI-Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 6Italian Multiple Myeloma Network, GIMEMA, Italy, 7Oncology Institute of Southern Switzerland, Bellinzona, Switzerland

RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

POSTER SESSIONS
Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
Blood 2013 122:4411; published ahead of print December 6, 2013
Swaroop Vakkalanka, Srikant Viswanadha, Eugenio Gaudio, Emanuele Zucca, Francesco Bertoni, Elena Bernasconi, Davide Rossi, Anastasios Stathis
  • Dual PI3K delta/gamma Inhibition By RP6530 Induces Apoptosis and Cytotoxicity
  • RP6530, a novel, small molecule PI3K delta/gamma
  • Activity and selectivity of RP6530 for PI3K delta and gamma isoforms

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas.

Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining.

Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples.

Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Disclosures:Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership.Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.

 

PI3K Dual Inhibitor (RP-6530)

Therapeutic Area Respiratory , Oncology – Liquid Tumors , Rheumatology Molecule Type Small Molecule
Indication Peripheral T-cell lymphoma (PTCL) , Non-Hodgkins Lymphoma , Asthma , Chronic Obstructive Pulmonary Disease (COPD) , Rheumatoid Arthritis
Development Phase Phase I Rt. of Administration Oral

Description

Rhizen is developing dual PI3K gamma/delta inhibitors for liquid tumors and inflammatory conditions.

Situation Overview

Dual Pl3K inhibition is strongly implicated as an intervention treatment in allergic and non-allergic inflammation of the airways and autoimmune diseases manifested by a reduction in neutrophilia and TNF in response to LPS. Scientific evidence for PI3-kinase involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches, which makes it a potential target for treatment of respiratory disease. Resistance to conventional therapies such as corticosteroids in several patients has been attributed to an up-regulation of the PI3K pathway; thus, disruption of PI3K signaling provides a novel strategy aimed at counteracting the immuno-inflammatory response. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the ? and ? isoforms would be expected to attenuate the progression of immune response encountered in most variations of airway inflammation and arthritis.

Mechanism of Action

While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.

Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.

 

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.png

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.pngtps:/

Clinical Trials

Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.

In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.

 

Partners by Region

Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.

In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of hematological malignancies.

PATENT
WO2014195888, DUAL SELECTIVE PI3 DELTA AND GAMMA KINASE INHIBITORS

This scheme provides a synthetic route for the preparation of compound of formula wherein all the variables are as described herein in above

Figure imgf000094_0001

15 14 10 12 12a

REFERENCES
April 2015, preclinical data were presented at the 106th AACR Meeting in Philadelphia, PA. RP-6530 had GI50 values of 17,028 and 22,014 nM, respectively
December 2014, data were presented at the 56th ASH Meeting in San Francisco, CA.
December 2013, preclinical data were presented at the 55th ASH Meeting in New Orleans, LA.
June 2013, preclinical data were presented at the 18th Annual EHA Congress in Stockholm, Sweden. RP-6530 inhibited PI3K delta and gamma isoforms with IC50 values of 24.5 and 33.2 nM, respectively.
  • 01 Sep 2015 Phase-I clinical trials in Hematological malignancies (Second-line therapy or greater) in USA (PO) (NCT02567656)
  • 18 Nov 2014 Preclinical trials in Multiple myeloma in Switzerland (PO) prior to November 2014
  • 18 Nov 2014 Early research in Multiple myeloma in Switzerland (PO) prior to November 2014

 

WO2011055215A2 Nov 3, 2010 May 12, 2011 Incozen Therapeutics Pvt. Ltd. Novel kinase modulators
WO2012151525A1 May 4, 2012 Nov 8, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO2013164801A1 May 3, 2013 Nov 7, 2013 Rhizen Pharmaceuticals Sa Process for preparation of optically pure and optionally substituted 2- (1 -hydroxy- alkyl) – chromen – 4 – one derivatives and their use in preparing pharmaceuticals
US20110118257 May 19, 2011 Rhizen Pharmaceuticals Sa Novel kinase modulators
US20120289496 May 4, 2012 Nov 15, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO 2011055215

 

 

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Effect of Heat Shock on Protein Folding

Posted in Cell Biology, Signaling & Cell Circuits, Curation, Disease Biology, Proteins, Proteomics, tagged , , , , , on December 8, 2015| Leave a Comment »

Effect of Heat Shock on Protein Folding

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Getting Back in Shape

Contrary to years of research suggesting otherwise, most aggregated proteins regain their shape and functionality following heat shock.

By Karen Zusi | Dec 1, 2015    http://www.the-scientist.com//?articles.view/articleNo/44506/title/Getting-Back-in-Shape/

http://www.the-scientist.com/Dec2015/DecMediLit1_640px.jpg

SHOCKED OUT OF SHAPE: Exogenous proteins in a cell denature and aggregate in misfolded clumps when heat-shocked. During cell recovery, specialized molecular chaperone proteins degrade and dispose of the aggregates. A small portion of the exogenous proteins may refold, escaping degradation.© EVAN OTO/SCIENCE SOURCE

http://www.the-scientist.com/Dec2015/DecMediLit2_640px.jpg

Mature endogenous proteins aggregate in an organized fashion when heat-shocked, remodeling the cell’s protein synthesis machinery to facilitate survival. During recovery, molecular chaperones free the mature proteins to resume their normal activity.© EVAN OTO/SCIENCE SOURCE

EDITOR’S CHOICE IN CELL & MOLECULAR BIOLOGY

The paper
E.W.J. Wallace et al., “Reversible, specific, active aggregates of endogenous proteins assemble upon heat stress,” Cell, 162:1286-98, 2015.

Like cooking an egg, heating up a purified protein enough will denature it, destroying the 3-D structure key to its functionality. The protein unfolds in a one-way trip to a fried state. Previous studies of this phenomenon in cells often used exogenous proteins, which clumped together in response to heat and were largely degraded by the cell’s internal cleanup machinery—a set of molecular chaperones known as heat-shock proteins—if the cell survived.

“That, and other examples, had convinced people that what they were seeing inside cells, the clumps of proteins, represented a disaster—these giant piles of damaged proteins shoved together inside the cell so they can ultimately be cleaned up,” reflects Allan Drummond, a molecular biologist at the University of Chicago. “Nobody had really looked systematically at what happens to the proteins that are native to the cell.”

To do so, Drummond and colleagues tagged proteins in yeast cells with a set of stable isotope labels. They subjected the cells to temperatures that would stress, but not kill, them and then flash-froze them within minutes to capture a snapshot of protein aggregation at different intervals. The researchers then analyzed the clumped proteins using mass spectrometry.

Drummond’s group identified 982 proteins in the yeast cells, 177 of which aggregated in the cytosol and nucleus after heat shock. However, the researchers’ isotope labels revealed something unexpected: aggregated proteins were unclumping and returning to their previous states during the cell’s recovery period. “We had no cases that we were able to detect where proteins go into aggregates and then are degraded,” says Drummond.

“It’s really challenging this long-held assumption that heat stress causes terminal aggregation. They’ve shown that we get these aggregates or assemblies that are reversible and that may actually be pro-survival,” says Kevin Morano, a microbiologist at the University of Texas Health Science Center at Houston who studies cellular responses to stress. “It is paradigm-shifting in a sense. We thought they were destroyed.” But in fact, the researchers found, the yeast cells began growing again without making substantial numbers of new proteins, suggesting that the proteins coming out of the aggregates were still functional.

Drummond’s group figures that heat stress, and the ensuing aggregation, may trigger the cell to produce more of its molecular chaperones to aid in recovery. The team observed a three-protein complex, needed to synthesize chaperones, that was active even while clumped with other proteins. “There are many different things that aggregation seems to be doing,” says Drummond. “It’s stopping the synthesis of most proteins, but promoting synthesis of a small set of proteins that are called in response to heat shock.”

Postdoc Edward Wallace, the lead author on the study, says halting the production of most other proteins may protect newly synthesized ones. Before they have folded into their mature shapes, new proteins are more susceptible to heat shock—and may still be degraded following stress. “We speculate that [the aggregated complexes] are remodeling protein synthesis, stopping the majority of new proteins from being made, and thus preventing the aggregation of these newly synthesized proteins,” says Wallace.

Editor’s Note (December 2): The sub-headline for this article was changed to emphasize the finding that most proteins regain functionality.

Tags

protein structure, protein aggregating, protein, literature, heat shock proteins and cell & molecular biology

Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress

Edward W.J. Wallace, Jamie L. Kear-Scott, Evgeny V. Pilipenko, Michael H. Schwartz,…, Edoardo M. Airoldi, Tao Pan, Bogdan A. Budnik, D. Allan Drummond
Cell 10 Sept 2015; 162(6):1286–1298   DOI: http://dx.doi.org/10.1016/j.cell.2015.08.041
Figure thumbnail fx1
http://www.cell.com/cms/attachment/2037405845/2051884402/fx1.jpg
Highlights
  • •Mass spectrometry quantifies aggregation of endogenous proteins during heat stress
  • •Aggregates form rapidly in specific subcellular compartments
  • •Endogenous protein aggregates are disassembled without degradation during recovery
  • •In vitro, a heat-aggregated enzyme complex retains activity and fidelity

 

Summary

Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.

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