Insights into the Metabolome

Curator: Larry H. Bernstein, MD, FCAP

FCAP

Updated 6/3/2016

Tapping the Metabolome

Genes, Transcripts, Proteins—All Have Come into Their “-Ome”     GEN May 15, 2016 (Vol. 36, No. 10)

• MaryAnn Labant

http://www.genengnews.com/gen-articles/tapping-the-metabolome/5770/

http://www.genengnews.com/Media/images/Article/NationalEyeInst_Weiss_RetinitisPigmentosa1714323106.jpg

The retina is responsible for capturing images from the visual field. Retinitis pigmentosa, which refers to a group of inherited diseases that cause retinal degeneration, causes a gradual decline in vision because retinal photoreceptor cells (rods and cones) die. Images on the left are courtesy of the National Eye Institute, NIH; image on the right is courtesy of Robert Fariss, Ph.D., and Ann Milam, Ph.D., National Eye Institute, NIH.

Metabolomics, the comprehensive evaluation of the products of cellular processes, can provide new findings and insight in a vast array of diseases and dysfunctions. Though promising, metabolomics lacks the standing of genomics or proteomics. It is, in a manner of speaking, the new kid on the “omics” block.

Even though metabolomics is still an emerging discipline, at least some quarters are giving it a warm welcome. For example, metabolomics is being advanced by the Common Fund, an initiate of the National Institutes of Health (NIH). The Common Fund has established six national metabolomics cores. In addition, individual agencies within NIH, such as the National Institute of Environmental Health Sciences (NIEHS), are releasing solicitations focused on growing more detailed metabolomics programs.

Whether metabolomic studies are undertaken with or without public support, they share certain characteristics and challenges. Untargeted or broad-spectrum studies are used for hypotheses generation, whereas targeted studies probe specific compounds or pathways. Reproducibility is a major challenge in the field; many studies cannot be reproduced in larger cohorts. Carefully defined guidance and standard operating procedures for sample collection and processing are needed.

While these challenges are being addressed, researchers are patiently amassing metabolomic insights in several areas, such as retinal diseases, neurodegenerative diseases, and autoimmune diseases. In addition, metabolomic sleuths are availing themselves of a growing selection of investigative tools.

A Metabolomic Eye on Retinal Degeneration

The retina has one of the highest metabolic activities of any tissue in the body and is composed of multiple cell types. This fact suggests that metabolomics might be helpful in understanding retinal degeneration. At least, that’s what occurred to Ellen Weiss, Ph.D., a professor of cell biology and physiology at the University of North Carolina School of Medicine at Chapel Hill. To explore this possibility, Dr. Weiss began collaborating with Susan Sumner, Ph.D., director of systems and translational sciences at RTI International.

Retinal degeneration is often studied through the use of genetic-mouse models that mimic the disease in humans. In the model used by Dr. Weiss, cells with a disease-causing mutation are the major light-sensing cells that degenerate during the disease. Individuals with the same or a similar genetic mutation will initially lose dim-light vision then, ultimately, bright-light vision and color vision.

Wild-type and mutant phenotypes, as well as dark- and light-raised animals, were compared, since retinal degeneration is exacerbated by light in this genetic model. Retinas were collected as early as day 18, prior to symptomatic disease, and analyzed. Although data analysis is ongoing, distinct differences have emerged between the phenotypes as well as between dark- and light-raised animals.

“There is a clear increase in oxidative stress in both light-raised groups but to a larger extent in the mutant phenotype,” reports Dr. Weiss. “There are global changes in metabolites that suggest mitochondrial dysfunction, and dramatic changes in lipid profiles. Now we need to understand how these metabolites are involved in this eye disease and the relevance of these perturbations.”

For example, the glial cells in the retina that upregulate a number of proteins in response to stress to attempt to save the retina are as likely as the light-receptive neurons to undergo metabolic changes.

“One of the challenges in metabolomics studies is assigning the signals that represent the metabolites or compounds in the samples,” notes Dr. Sumner. “Signals may be ‘unknown unknowns,’ compounds that have never been identified before, or ‘known unknowns,’ compounds that are known but that have not yet been assigned in the biological matrix.”

Internal and external libraries, such as the Human Metabolome Dictionary, are used to match signals. Whether or not a match exists, fragmentation patterns are used to characterize the metabolite, and when possible a standard is obtained to confirm identity. To assist with this process, the NIH Common Fund supports Metabolite Standard Synthesis Cores (MSSCs). RTI International holds an MSSC contract in addition to being a NIH-designated metabolomics core.

Mitochondrial Dysfunction in Alzheimer’s Disease     

Alzheimer’s disease (AD) is difficult to diagnose early due to its asymptomatic phase; accurate diagnosis occurs only in postmortem brain tissue. To evaluate familial AD, a rare inherited form of the disease, the laboratory of Eugenia Trushina, Ph.D., associate professor of neurology and associate professor of pharmacology at the Mayo Clinic, uses mouse models to study the disease’s early molecular mechanisms.

Synaptic loss underlies cognitive dysfunction. The length of neurons dictates that mitochondria move within the cell to provide energy at the site of the synapses. An initial finding was that very early on mitochondrial trafficking was affected reducing energy supply to synapses and distant parts of the cell.

During energy production, the major mitochondrial metabolite is ATP, but the organelle also produces many other metabolites, molecules that are implicated in many pathways. One can assume that changes in energy utilization, production, and delivery are associated with some disturbance.

“Our goal,” explains Dr. Trushina, “was to get a proof of concept that we could detect in the blood of AD patients early changes of mitochondria dysfunction or other changes that could be informative of the disease over time.”

A Mayo Clinic aging study involves a cohort of patients, from healthy to those with mild cognitive impairment (MCI) through AD. Patients undergo an annual battery of tests including cognitive function along with blood and cerebrospinal fluid sampling. Metabolic signatures in plasma and cerebrospinal fluid of normal versus various disease stages were compared, and affected mitochondrial and lipid pathways identified in MCI patients that progressed to AD.

“Last year we published on a new compound that goes through the blood/brain barrier, gets into mitochondria, and very specifically, partially inhibits mitochondrial complex I activity, making the cell resistant to oxidative damage,” details Dr. Trushina. “The compound was able to either prevent or slow the disease in the animal familial models.

“Treatment not only reduced levels of amyloid plaques and phosphorylated tau, it also restored mitochondrial transport in neurons. Now we have additional compounds undergoing investigation for safety in humans, and target selectivity and engagement.”

“Mitochondria play a huge role in every aspect of our lives,” Dr. Trushina continues. “The discovery seems counterintuitive, but if mitochondria function is at the heart of AD, it may provide insight into the major sporadic form of the disease.”

Distinguishing Types of Asthma

In children, asthma generally manifests as allergy-induced asthma, or allergic asthma. And allergic asthma has commonalities with allergic dermatitis/eczema, food allergies, and allergic rhinitis. In adults, asthma is more heterogeneous, and distinct and varied subpopulations emerge. Some have nonallergic asthma; some have adult-onset asthma; and some have obesity-, occupational-, or exercise-induced asthma.

Adult asthmatics may have markers of T_H2 high verus T_H2 low asthma (T helper 2 cell cytokines) and they may respond to various triggers—environmental antigens, occupational antigens, irritants such as perfumes and chlorine, and seasonal allergens. Exercise, too, can trigger asthma.

One measure that can phenotype asthmatics is nitric oxide, an exhaled breath biomarker. Nitric oxide is a smooth muscle relaxant, vasodilator, and bronchodilator that can have anti-inflammatory properties. There is a wide range of values in asthmatics, and a number of values are needed to understand the trend in a particular patient. L-arginine is the amino acid that produces nitric oxide when converted to L-citrulline, a nonessential amino acid.

According to Nicholas Kenyon, M.D., a pulmonary and critical care specialist who is co-director of the University of California, Davis Asthma Network (UCAN), some metabolomic studies suggest that there is a state of L-arginine depletion during asthma attacks or in severe asthma suggesting a lack of substrate to produce nitric oxide. Dr. Kenyon is conducting clinical work on L-arginine supplementation in a double-blind cross-over  intervention trial of L-arginine versus placebo. The 50-subject study in severe asthmatics should be concluded in early 2017.

Many new biologic therapies are coming to market to treat asthma; it will be challenging to determine which advanced therapy to provide to which patient. Therapeutics mostly target severe asthma populations and are for patients with evidence of higher numbers of eosinophils in the blood and lung, which include anti-IL-5 and (soon) anti-IL-13, among others.

Tools Development 

Waters is developing metabolomics applications that use multivariate statistical methods to highlight compounds of interest. Typically these applications combine separation procedures, accomplished by means of liquid chromatography or gas chromatography (LC or GC), with detection methods that rely on mass spectrometry (MS). To support the identification, quantification, and analysis of LC-MS data, the company provides bioinformatics software. For example, Progenesis QI software can interrogate publicly available databases and process information about isotopic patterns, retention times, and collision cross-sections.

Mass spectrometry (MS) is the gold standard in metabolomics and lipidomics. But there is a limit to what accurate mass and resolution can achieve. For example, neither isobaric nor isomeric species are resolvable solely by MS. New orthogonal analytical tools will allow more confident identifications.

To improve metabolomics separations before MS detection, a post-ionization separation tool, like ion mobility, which is currently used to support traditional UPLC-MS and MS imaging metabolomics protocols, becomes useful. The collision-cross section (CCS), which measures the shape of molecules, can be derived, and it can be used as an additional identification coordinate.

Other new chromatographic tools are under development, such as microflow devices and UltraPerformance Convergence Chromatography (UPC2), which uses liquid CO2 as its mobile phase, to enable new ways of separating chiral metabolites. Both UPC2 and microflow technologies have decreased solvent consumption and waste disposal while maintaining UPLC-quality performance in terms of chromatographic resolution, robustness, and reproducibility.

Informatics tools are also improving. In the latest versions of Waters’ Progenesis software, typical metabolomics identification problems are resolved by allowing interrogation of publicly available databases and scoring according to accurate mass, isotopic pattern, retention time, CCS, and either theoretical or experimental fragments.

MS imaging techniques, such as MALDI and DESI, provide spatial information about the metabolite composition in tissues. These approaches can be used to support and confirm traditional analyses without sample extraction, and they allow image generation without the use of antibodies, similar to immunohistochemistry.

“Ion-mobility tools will soon be implemented for routine use, and the use of extended CCS databases will help with metabolite identification,” comments Giuseppe Astarita Ph.D., principal scientist, Waters. “More applications of ambient ionization MS will emerge, and they will allow direct-sampling analyses at atmospheric pressure with little or no sample preparation, generating real-time molecular fingerprints that can be used to discriminate among phenotypes.”

Microflow Technology   

Microflow technology offers sensitivity and robustness. For example, at the Proteomics and Metabolomics Facility, Colorado State University, peptide analysis was typically performed using nanoflow chromatography; however, nanoflow chromatography is slow and technically challenging. Moving to microflow offered significant improvements in robustness and ease-of-use and resulted in improved chromatography without sacrificing sensitivity.

Conversely, small molecule applications were typically performed with analytical-scale chromatography. While this flow regime is extremely robust and fast, it can sometimes be limited in sensitivity. Moving to microflow offered significant improvements in sensitivity, 5- to 10-fold depending on the compound, without sacrificing robustness.

But broad-scale microflow adoption is hampered by a lack of available column chemistries and legacy HPLC or UPLC infrastructure that is not conducive to low-flow operation.

“We utilize microflow technology on all of our tandem quadrupole instruments for targeted quantitative assays,” says Jessica Prenni, Ph.D., director, Proteomics and Metabolomics Facility, Colorado State University. “All of our peptide quantitation is exclusively performed with microflow technology, and many of our small molecule assays. Application examples include endocannabinoids, bile acids and plant phytohormone panels.”

Compound annotation and comparability and transparency in data processing and reporting is a challenge in metabolomics research. Multiple groups are actively working on developing new tools and strategies; common best practices need to be adopted.

The continued growth of open-source spectral databases and new tools for spectral prediction from compound databases will dramatically impact the ability for metabolomics to result in novel discoveries. The move to a systems-level understanding through the combination of various omics data also will have a huge influence and be enabled by the continued development of open-source and user-friendly pathway-analysis tools.

• Kate Marusina, Ph.D.

http://www.genengnews.com/gen-articles/paving-the-road-for-clinical-biomarkers/5757/

http://www.genengnews.com/Media/images/Article/thumb_ArcherDX_AnalyticalSensitivity2362411344.jpg

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Fusion detection can be carried out with traditional opposing primer-based library preparation methods, which require target- and fusion-specific primers that define the region to be sequenced. With these methods, primers are needed that flank the target region and the fusion partner, so only known fusions can be detected. An alternative method, ArcherDX’ Anchored Multiplex PCR (AMP), can be used to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific unidirectional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification.

In time, the narrow, tortuous paths followed by pioneers become wider and straighter, whether the pioneers are looking to settle new land or bring new biomarkers to the clinic.

In the case of biomarkers, we’re still at the stage where pioneers need to consult guides and outfitters or, in modern parlance, consultants and technology providers. These hardy souls tend to congregate at events like the Biomarker Conference, which was held recently in San Diego.

At this event, biomarker experts discussed ways to avoid unfortunate detours on the trail from discovery and development to clinical application and regulatory approval. Of particular interest were topics such as the identification of accurate biomarkers, the explication of disease mechanisms, the stratification of patient groups, and the development of standard protocols and assay platforms. In each of these areas, presenters reported progress.

Another crucial subject is the integration of techniques such as next-generation sequencing (NGS). This particular technique has been instrumental in advancing clinical cancer genomics and continues to be the most feasible way of simultaneously interrogating multiple genes for driver mutations.

Enriching nucleic acid libraries for target genes of interest prior to NGS greatly enhances the sensitivity of detecting mutations, as the enriched regions are sequenced multiple times. This is particularly useful when analyzing clinical samples, which generate low amounts of poor-quality nucleic acids.

Most target-enrichment strategies require prior knowledge of both ends of the target region to be sequenced. Therefore, only gene fusions with known partners can be amplified for downstream NGS assays.

Archer’s Anchored Multiplex PCR (AMP™) technology overcomes this limitation, as it can enrich for novel fusions, while only requiring knowledge of one end of the fusion pair. At the heart of the AMP chemistry are unique Molecular Barcode (MBC) adapters, ligated to the 5′ ends of DNA fragments prior to amplification. The MBCs contain universal primer binding sites for PCR and a molecular barcode for identifying unique molecules. When combined with 3′ gene-specific primers, MBCs enable amplification of target regions with unknown 5′ ends.

“Tagging each molecule of input nucleic acid with a unique molecular barcode allows for de-duplication, error correction, and quantitative analysis, resulting in high sequencing consensus. With its low error rate and low limits of detection, AMP is revolutionizing the field of cancer genomics.”

In a proof-of-concept study, a single-tube 23-plex panel was designed to amplify the kinase domains of ALK, RET, ROS1, and MUSK genes by AMP. This enrichment strategy enabled identification of gene fusions with multiple partners and alternative splicing events in lung cancer, thyroid cancer, and glioblastoma specimens by NGS.

Over the last decade, the Biomarker/Translational Research Laboratory has focused on developing clinical genotyping and fluorescent in situ hybridization (FISH) assays for rapid personalized genomic testing.

“Initially, we analyzed the most prevalent hotspot mutations, about 160 in 25 cancer genes,” continued Dr. Borger. “However, this approach revealed mutations in only half of our patients. With the advent of NGS, we are able to sequence 190 exons in 39 cancer genes and obtain significantly richer genetic fingerprints, finding genetic aberrations in 92% of our cancer patients.”

Using multiplexed approaches, Dr. Borger’s team within the larger Center for Integrated Diagnostics (CID) program at MGH has established high-throughput genotyping service as an important component of routine care. While only a few susceptible molecular alterations may currently have a corresponding drug, the NGS-driven analysis may supply new information for inclusion of patients into ongoing clinical trials, or bank the result for future research and development.

“A significant impediment to discovery of clinically relevant genomic signatures is our current inability to interconnect the data,” explained Dr. Borger. “On the local level, we are striving to compile the data from clinical observations, including responses to therapy and genotyping. Globally, it is imperative that comprehensive public databases become available to the research community.”

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This image, from the Massachusetts General Hospital Cancer Center, shows multicolor fluorescence in situ hybridization (FISH) analysis of cells from a patient with esophagogastric cancer. Remarkably, the FISH analysis revealed that co-amplification of the MET gene (red signal) and the EGFR gene (green signal) existed simultaneously in the same tumor cells. A chromosome 7 control probe is shown in blue.

Tumor profiling at MGH have already yielded significant discoveries. Dr. Borger’s lab, in collaboration with oncologists at the MGH Cancer Center, found significant correlations between mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2) and certain types of cancers, such as cholangiocarcinoma and acute myelogenous leukemia (AML).

Historically, cancer signatures largely focus on signaling proteins. Discovery of a correlative metabolic enzyme offered a promise of diagnostics based on metabolic byproducts that may be easily identified in blood. Indeed, the metabolite 2-hydroxyglutarate accumulates to high levels in the tissues of patients carrying IDH1 and IDH2 mutations. They have reported that circulating 2-hydroxyglutarate as measured in the blood correlates with tumor burden, and could serve as an important surrogate marker of treatment response.  …..

Researchers Uncover How ‘Silent’ Genetic Changes Drive Cancer

Fri, 06/03/2016 – 8:41amby Rockeller University

http://www.dddmag.com/news/2016/06/researchers-uncover-how-silent-genetic-changes-drive-cancer

“Traditionally, it has been hard to use standard methods to quantify the amount of tRNA in the cell,” says Tavazoie. The lead authors of the article, Hani Goodarzi, formerly a postdoc in the lab and now a new assistant professor at UCSF, and research assistant Hoang Nguyen, devised and applied a new method that utilizes state-of-the-art genomic sequencing technology to measure the amount of tRNAs in different cell types.

The team chose to compare breast tissue from healthy individuals with tumor samples taken from breast cancer patients–including both primary tumors that had not spread from the breast to other body sites, and highly aggressive, metastatic tumors.

They found that the levels of two specific tRNAs were significantly higher in metastatic cells and metastatic tumors than in primary tumors that did not metastasize or healthy samples. “There are four different ways to encode for the protein building block arginine,” explains Tavazoie. “Yet only one of those–the tRNA that recognizes the codon CGG–was associated with increased metastasis.”

The tRNA that recognizes the codon GAA and encodes for a building block known as glutamic acid was also elevated in metastatic samples.

The team hypothesized that the elevated levels of these tRNAs may in fact drive metastasis. Working in mouse models of primary, non-metastatic tumors, the researchers increased the production of the tRNAs, and found that these cells became much more invasive and metastatic.

They also did the inverse experiment, with the anticipated results: reducing the levels of these tRNAs in metastatic cells decreased the incidence of metastases in the animals.

How do two tRNAs drive metastasis? The researchers teamed up with members of the Rockefeller University proteomics facility to see how protein expression changes in cells with elevated levels of these two tRNAs.

“We found global increases in many dozens of genes,” says Tavazoie, “so we analyzed their sequences and found that the majority of them had significantly increased numbers of these two specific codons.”

According to the researchers, two genes stood out among the list. Known as EXOSC2 and GRIPAP1, these genes were strongly and directly induced by elevated levels of the specific glutamic acid tRNA.

“When we mutated the GAA codons to GAG– a “silent” mutation because they both spell out the protein building block glutamic acid–we found that increasing the amount of tRNA no longer increased protein levels,” explains Tavazoie. These proteins were found to drive breast cancer metastasis.

The work challenges previous assumptions about how tRNAs function and suggests that tRNAs can modulate gene expression, according to the researchers. Tavazoie points out that “it is remarkable that within a single cell type, synonymous changes in genetic sequence can dramatically affect the levels of specific proteins, their transcripts, and the way a cell behaves.”

Testing Blood Metabolites Could Help Tailor Cancer Treatment

6/03/2016 1 Comment by Institute of Cancer Research
http://www.dddmag.com/news/2016/06/testing-blood-metabolites-could-help-tailor-cancer-treatment

Scientists have found that measuring how cancer treatment affects the levels of metabolites – the building blocks of fats and proteins – can be used to assess whether the drug is hitting its intended target.

This new way of monitoring cancer therapy could speed up the development of new targeted drugs – which exploit specific genetic weaknesses in cancer cells – and help in tailoring treatment for patients.

Scientists at The Institute of Cancer Research, London, measured the levels of 180 blood markers in 41 patients with advanced cancers in a phase I clinical trial conducted with The Royal Marsden NHS Foundation Trust.

They found that investigating the mix of metabolic markers could accurately assess how cancers were responding to the targeted drug pictilisib.

Their study was funded by the Wellcome Trust, Cancer Research UK and the pharmaceutical company Roche, and is published in the journal Molecular Cancer Therapeutics.

Pictilisib is designed to specifically target a molecular pathway in cancer cells, called PI3 kinase, which has key a role in cell metabolism and is defective in a range of cancer types.

As cancers with PI3K defects grow, they can cause a decrease in the levels of metabolites in the bloodstream.

The new study is the first to show that blood metabolites are testable indicators of whether or not a new cancer treatment is hitting the correct target, both in preclinical mouse models and also in a trial of patients.

Using a sensitive technique called mass spectrometry, scientists at The Institute of Cancer Research (ICR) initially analysed the metabolite levels in the blood of mice with cancers that had defects in the PI3K pathway.

They found that the blood levels of 26 different metabolites, which were low prior to therapy, had risen considerably following treatment with pictilisib. Their findings indicated that the drug was hitting its target, and reversing the effects of the cancer on mouse metabolites.

Similarly, in humans the ICR researchers found that almost all of the metabolites – 22 out of the initial 26 – once again rose in response to pictilisib treatment, as seen in the mice.

Blood levels of the metabolites began to increase after a single dose of pictilisib, and were seen to drop again when treatment was stopped, suggesting that the effect was directly related to the drug treatment.

Metabolites vary naturally depending on the time of day or how much food a patient has eaten. But the researchers were able to provide the first strong evidence that despite this variation metabolites can be used to test if a drug is working, and could help guide decisions about treatment.

New Metabolic Pathway Reveals Aspirin-Like Compound’s Anti-Cancer Properties

http://www.genengnews.com/gen-news-highlights/new-metabolic-pathway-reveals-aspirin-like-compound-s-anti-cancer-properties/81252777/

Researchers at the Gladstone Institutes say they have found a new pathway by which salicylic acid, a key compound in the nonsteroidal anti-inflammatory drugs aspirin and diflunisal, stops inflammation and cancer.

In a study (“Salicylate, Diflunisal and Their Metabolites Inhibit CBP/p300 and Exhibit Anticancer Activity”) published in eLife, the investigators discovered that both salicylic acid and diflunisal suppress two key proteins that help control gene expression throughout the body. These sister proteins, p300 and CREB-binding protein (CBP), are epigenetic regulators that control the levels of proteins that cause inflammation or are involved in cell growth.

By inhibiting p300 and CBP, salicylic acid and diflunisal block the activation of these proteins and prevent cellular damage caused by inflammation. This study provides the first concrete demonstration that both p300 and CBP can be targeted by drugs and may have important clinical implications, according to Eric Verdin, M.D., associate director of the Gladstone Institute of Virology and Immunology .

“Salicylic acid is one of the oldest drugs on the planet, dating back to the Egyptians and the Greeks, but we’re still discovering new things about it,” he said. “Uncovering this pathway of inflammation that salicylic acid acts upon opens up a host of new clinical possibilities for these drugs.”

Earlier research conducted in the laboratory of co-author Stephen D. Nimer, M.D., director of Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, and a collaborator of Verdin’s, established a link between p300 and the leukemia-promoting protein AML1-ETO. In the current study, scientists at Gladstone and Sylvester worked together to test whether suppressing p300 with diflunisal would suppress leukemia growth in mice. As predicted, diflunisal stopped cancer progression and shrunk the tumors in the mouse model of leukemia. ……

Novel Protein Agent Targets Cancer and Host of Other Diseases

http://www.genengnews.com/gen-news-highlights/new-protein-agent-targets-cancer-and-host-of-other-diseases/81252780/

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Researchers at Georgia State University have designed a new protein compound that can effectively target the cell surface receptor integrin v3, mutations in which have been linked to a number of diseases. Initial results using this new molecule show its potential as a therapeutic treatment for an array of illnesses, including cancer.

The novel protein molecule targets integrin v3 at a novel site that has not been targeted by other scientists. The researchers found that the molecule induces apoptosis, or programmed cell death, of cells that express integrin v3. This integrin has been a focus for drug development because abnormal expression of v3 is linked to the development and progression of various diseases.

“This integrin pair, v3, is not expressed in high levels in normal tissue,” explained senior study author Zhi-Ren Liu, Ph.D., professor in the department of biology at Georgia State. “In most cases, it’s associated with a number of different pathological conditions. Therefore, it constitutes a very good target for multiple disease treatment.”

“Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, which binds to integrin α_vβ₃ outside the classical ligand-binding site,” the authors wrote. “We show ProAgio induces apoptosis of integrin α_vβ₃-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin α_vβ₃.”

The findings from this study were published recently in Nature Communications in an article entitled “Rational Design of a Protein That Binds Integrin α_vβ₃ Outside the Ligand Binding Site.”   …..

“We took a unique angle,” Dr. Lui noted. “We designed a protein that binds to a different site. Once the protein binds to the site, it directly triggers cell death. When we’re able to kill pathological cells, then we’re able to kill the disease.”

The investigators performed extensive cell and molecular testing that confirmed ProAgio interacts and binds well with integrin v3. Interestingly, they found that ProAgio induces apoptosis by recruiting caspase 8—an enzyme that plays an essential role in programmed cell death—to the cytoplasmic area of integrin v3. ProAgio was much more effective in inducing cell death than other agents tested.

Noncoding RNAs Not So Noncoding

Bits of the transcriptome once believed to function as RNA molecules are in fact translated into small proteins.

By Ruth Williams | June 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/46150/title/Noncoding-RNAs-Not-So-Noncoding

In 2002, a group of plant researchers studying legumes at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, discovered that a 679-nucleotide RNA believed to function in a noncoding capacity was in fact a protein-coding messenger RNA (mRNA).¹ It had been classified as a long (or large) noncoding RNA (lncRNA) by virtue of being more than 200 nucleotides in length. The RNA, transcribed from a gene called early nodulin 40 (ENOD40), contained short open reading frames (ORFs)—putative protein-coding sequences bookended by start and stop codons—but the ORFs were so short that they had previously been overlooked. When the Cologne collaborators examined the RNA more closely, however, they found that two of the ORFs did indeed encode tiny peptides: one of 12 and one of 24 amino acids. Sampling the legumes confirmed that these micropeptides were made in the plant, where they interacted with a sucrose-synthesizing enzyme.

Five years later, another ORF-containing mRNA that had been posing as a lncRNA was discovered inDrosophila.^2,3 After performing a screen of fly embryos to find lncRNAs, Yuji Kageyama, then of the National Institute for Basic Biology in Okazaki, Japan, suppressed each transcript’s expression. “Only one showed a clear phenotype,” says Kageyama, now at Kobe University. Because embryos missing this particular RNA lacked certain cuticle features, giving them the appearance of smooth rice grains, the researchers named the RNA “polished rice” (pri).

Turning his attention to how the RNA functioned, Kageyama thought he should first rule out the possibility that it encoded proteins. But he couldn’t. “We actually found it was a protein-coding gene,” he says. “It was an accident—we are RNA people!” The pri gene turned out to encode four tiny peptides—three of 11 amino acids and one of 32—that Kageyama and colleagues showed are important for activating a key developmental transcription factor.⁴

Since then, a handful of other lncRNAs have switched to the mRNA ranks after being found to harbor micropeptide-encoding short ORFs (sORFs)—those less than 300 nucleotides in length. And given the vast number of documented lncRNAs—most of which have no known function—the chance of finding others that contain micropeptide codes seems high.

Overlooked ORFs

From the late 1990s into the 21st century, as species after species had their genomes sequenced and deposited in databases, the search for novel genes and their associated mRNAs duly followed. With millions or even billions of nucleotides to sift through, researchers devised computational shortcuts to hunt for canonical gene and mRNA features, such as promoter regions, exon/intron splice sites, and, of course, ORFs.

ORFs can exist in practically any stretch of RNA sequence by chance, but many do not encode actual proteins. Because the chance that an ORF encodes a protein increases with its length, most ORF-finding algorithms had a size cut-off of 300 nucleotides—translating to 100 amino acids. This allowed researchers to “filter out garbage—that is, meaningless ORFs that exist randomly in RNAs,” says Eric Olsonof the University of Texas Southwestern Medical Center in Dallas.

Of course, by excluding all ORFs less than 300 nucleotides in length, such algorithms inevitably missed those encoding genuine small peptides. “I’m sure that the people who came up with [the cut-off] understood that this rule would have to miss anything that was shorter than 100 amino acids,” saysNicholas Ingolia of the University of California, Berkeley. “As people applied this rule more and more, they sort of lost track of that caveat.” Essentially, sORFs were thrown out with the computational trash and forgotten.

Aside from statistical practicality and human oversight, there were also technical reasons that contributed to sORFs and their encoded micropeptides being missed. Because of their small size, sORFs in model organisms such as mice, flies, and fish are less likely to be hit in random mutagenesis screens than larger ORFs, meaning their functions are less likely to be revealed. Also, many important proteins are identified based on their conservation across species, says Andrea Pauli of the Research Institute of Molecular Pathology in Vienna, but “the shorter [the ORF], the harder it gets to find and align this region to other genomes and to know that this is actually conserved.”

As for the proteins themselves, the standard practice of using electrophoresis to separate peptides by size often meant micropeptides would be lost, notes Doug Anderson, a postdoc in Olson’s lab. “A lot of times we run the smaller things off the bottom of our gels,” he says. Standard protein mass spectrometry was also problematic for identifying small peptides, says Gerben Menschaert of Ghent University in Belgium, because “there is a washout step in the protocol so that only larger proteins are retained.”

But as researchers take a deeper dive into the function of the thousands of lncRNAs believed to exist in genomes, they continue to uncover surprise micropeptides. In February 2014, for example, Pauli, then a postdoc in Alex Schier’s lab at Harvard University, discovered a hidden code in a zebrafish lncRNA. She had been hunting for lncRNAs involved in zebrafish development because “we hadn’t really anticipated that there would be any coding regions out there that had not been discovered—at least not something that is essential,” she says. But one lncRNA she identified actually encoded a 58-amino-acid micropeptide, which she called Toddler, that functioned as a signaling protein necessary for cell movements that shape the early embryo.⁵

Then, last year, Anderson and his colleagues reported another. Since joining Olson’s lab in 2010, Anderson had been searching for lncRNAs expressed in the heart and skeletal muscles of mouse embryos. He discovered a number of candidates, but one stood out for its high level of sequence conservation—suggesting to Anderson that it might have an important function. He was right, the RNA was important, but for a reason that neither Anderson nor Olson had considered: it was in fact an mRNA encoding a 46-amino-acid-long micropeptide.⁶

“When we zeroed in on the conserved region [of the gene], Doug found that it began with an ATG [start] codon and it terminated with a stop codon,” Olson says. “That’s when he looked at whether it might encode a peptide and found that indeed it did.” The researchers dubbed the peptide myoregulin, and found that it functioned as a critical calcium pump regulator for muscle relaxation.

With more and more overlooked peptides now being revealed, the big question is how many are left to be discovered. “Were there going to be dozens of [micropeptides]? Were there going to be hundreds, like there are hundreds of microRNAs?” says Ingolia. “We just didn’t know.”

see more at  http://www.the-scientist.com/?articles.view/articleNo/46150/title/Noncoding-RNAs-Not-So-Noncoding

Research at Micro- and Nanoscales

From whole cells to genes, closer examination continues to surprise.

By Mary Beth Aberlin | June 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/46129/title/Research-at-Micro–and-Nanoscales

Little things mean a lot. To any biologist, this time-worn maxim is old news. But it’s worth revisiting. As several articles in this issue of The Scientist illustrate, how researchers define and examine the “little things” does mean a lot.

Consider this month’s cover story, “Noncoding RNAs Not So Noncoding,” by TS correspondent Ruth Williams. Combing the human genome for open reading frames (ORFs), sequences bracketed by start and stop codons, yielded a protein-coding count somewhere in the neighborhood of 24,000. That left a lot of the genome relegated to the category of junk—or, later, to the tens of thousands of mostly mysterious long noncoding RNAs (lncRNAs). But because they had only been looking for ORFs that were 300 nucleotides or longer (i.e., coding for proteins at least 100 amino acids long), genome probers missed so-called short ORFs (sORFs), which encode small peptides. “Their diminutive size may have caused these peptides to be overlooked, their sORFs to be buried in statistical noise, and their RNAs to be miscategorized, but it does not prevent them from serving important, often essential functions, as the micropeptides characterized to date demonstrate,” writes Williams.

How little things work definitely informs another field of life science research: synthetic biology. As the functions of genes and gene networks are sussed out, bioengineers are using the information to design small, synthetic gene circuits that enable them to better understand natural networks. In “Synthetic Biology Comes into Its Own,” Richard Muscat summarizes the strides made by synthetic biologists over the last 15 years and offers an optimistic view of how such networks may be put to use in the future. And to prove him right, just as we go to press, a collaborative group led by one of syn bio’s founding fathers, MIT’s James Collins, has devised a paper-based test for Zika virus exposure that relies on a freeze-dried synthetic gene circuit that changes color upon detection of RNAs in the viral genome. The results are ready in a matter of hours, not the days or weeks current testing takes, and the test can distinguish Zika from dengue virus. “What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” commented one researcher about the test.

Moving around little things is the name of the game when it comes to delivering a package of drugs to a specific target or to operating on minuscule individual cells. Mini-scale delivery of biocompatible drug payloads often needs some kind of boost to overcome fluid forces or size restrictions that interfere with fine-scale manipulation. To that end, ingenious solutions that motorize delivery by harnessing osmotic changes, magnets, ultrasound, and even bacterial flagella are reviewed in “Making Micromotors Biocompatible.”

….  http://www.the-scientist.com/?articles.view/articleNo/46129/title/Research-at-Micro–and-Nanoscales

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