Proteomics


(previous session)

Craig Skinner (Lin Lab, UC Davis): Identification of potential calorie restriction mimics in yeast using a nitric oxide-based screen. Yeast are an important model system in biogerontology, useful not only for genetic studies of longevity control but also for discovery of bioactive compounds. Calorie restriction (CR) in yeast causes increased levels of nitric oxide (NO) — somewhat surprising in that yeast cells lack a homolog of nitric oxide synthase — and elevated NO is sufficient to extend yeast lifespan. These observations led Skinner to screen a yeast deletion library for elevated NO levels, yielding several genes that extend lifespan.

Mark Lucanic (Lithgow Lab, Buck): Endocannabinoid signaling mediates the effect of diet on lifespan in C. elegans. Mutants in the dauer pathway in C. elegans often influence longevity; the daf-2 mutation, which causes constitutive dauer formation at elevated temperatures, extends lifespan by several fold. Lucanic discovered that endocannabinoids are involved in the regulation of the dauer pathway — and therefore, of longevity — either independently of or far downstream of daf-2 and daf-16. Endocannabinoids are upregulated under well-fed conditions, and shorten lifespan.

Delia David (Kenyon Lab, UCSF): Widespread protein aggregation is an inherent part of aging in C. elegans. Protein aggregates are a hallmark of many age-related neurodegenerative diseases, leading to the hypotheses that the cellular mileu changes with age in a manner that causes native, aggregation-prone proteins to form aggregates. David used mass spectrometry to identify a subset of normal worm proteins aggregate as a function of age. As with the proteins associated with neurodegeneration, specific proteins aggregate in specific cell types. Mutations that extend lifespan (such as daf-2) decrease aggregation, and tend to downregulate the expression of genes encoding aggregation-prone proteins. Curiously, regulators of protein homeostasis tend to aggregate themselves, leading to a destructive positive feedback loop in which the very factors that protect the cell from proteotoxicity disappear into aggregates, leading to further aggregation.

Cherry Tang (Zhong Lab, Berkeley): The Clearance of Ubiquitinated Protein Aggregates Via Autophagy. Autophagic protein degradation has been implicated in control of lifespan: autophagy slows cell and tissue aging. Tang has identified a protein that participates in degradation of ubiquitinated proteins and co-localizes with autophagosomes; when the protein is knocked down, protein aggregates become more toxic.

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Tinkering with insulin signaling can dramatically slow the aging process and extend survival. The details of this effect have best been studied in C. elegans. What distinguishes long-lived insulin signaling mutants from normal animals? Many studies have tacked the transcriptional side of this question, comparing gene expression profiles by microarray analysis. A new study addresses the post-transcriptional side of this story. Continuing a recent streak of high profile papers, workers from the labs of Andy Dillin and John Yates examined protein abundance in worms made to live longer by RNAi knockdown of the insulin receptor DAF-2. Dong et al. (“Quantitative Mass Spectrometry Identifies Insulin Signaling Targets in C. elegans,” Science, August 3, 2007) differentially labeled worms with two different nitrogen isotopes and compared relative protein abundance by mass spectrometry. Of 1685 identifiable proteins, a total of 86 varied markedly between wild type and mutant animals — 47 were more abundant in daf-2-depleted animals and 39 were less abundant. These proteins were mostly from the usual laundry list of metabolic processes, but 51 of them had not been previously come to light in microarray studies.

Lowering daf-2 activity changes all kinds of random processes, only some of which are relevant to longevity. To figure out which of these proteins were involved in aging, Dong et al. knocked each of them down with RNAi and looked for an effect on diapause or lifespan. Not surprisingly, these two phenomena could be separated— depletion of a number of these proteins promoted diapause but did not affect lifespan. The surprise came from the characterization of a group of proteins involved in carbohydrate metabolism and translation initiation. These proteins, including ACO-2 (aconitase / iron response element binding protein), FBP-1 (fructose bisphosphatase) and other ostensibly boring enzymes, are up in daf-2-deficient worms, yet when directly depleted by RNAi cause daf-2 mutant animals to live yet longer. Ditto for TAX-6, the worm’s calcineurin A homolog, which these workers showed to undergo nuclear localization upon daf-2 knockdown. However, depleting each of these proteins in wild type animals didn’t affect survival one way or another.

At first, these findings seem counterintuitive. Genetically, the knockdown experiments say that these proteins ordinarily act to shorten lifespan, but only in daf-2 mutants. The authors invoke a model of compensatory regulation, wherein daf-2 loss of function promotes the accumulation of “pro-aging” proteins, tempering the beneficial effect of low insulin activity. Thus, for further increase our longevity, we should not only aim to inhibit our insulin receptors but also to shut down glycolysis. The Atkins Diet is looking better and better.

As cells age, their protein composition changes. Two recent papers address these changes: the first addresses the proteome of colon epithelia, and the second focuses on a specific chemical modification in the proteins of the cerebellum.

Li et al. perform a comprehensive analysis of the colon epithelial proteome over the course of aging. The approach is intellectually straightforward with few surprises, but it’s technically sound, and I think it’s important for biogerontologists to appreciate how far proteomic technology has come — the approach used by these authors could be applied to any system of interest, comparing the old and young states and allowing us to learn about the mechanisms of age-related change in our favorite tissues:

In order to screen the aging related proteins in human normal colon epithelia, the comparative proteomics analysis was applied to get the two-dimensional electrophoresis (2-DE) profiles with high resolution and reproducibility from normal colon epithelial tissues of young and aged people. Differential proteins between the colon epithelia of two age groups were found with PDQuest software. The thirty five differential protein-spots were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and database searching. … The identified differential proteins appear to be involved in metabolism, energy generation, chaperone, antioxidation, signal transduction, protein folding and apoptosis. The data will help to understand the molecular mechanisms of human colon epithelial aging.

The second paper, also a good example of the best technology and technique currently available, focuses not on differential protein expression but rather on differences in the chemical alteration of cellular protein. The specific alteration of interest here is nitration, a marker of oxidative stress. Gokulrangan et al.:

3-Nitrotyrosine (3-NT) is a useful biomarker of increasing oxidative stress and protein nitration during biological aging. The proteomic analysis of cerebellar homogenate from Fisher 344/Brown Norway (BN/F1) rats shows an age-dependent increase in protein nitration … When proteins were separated by solution isoelectrofocusing and analyzed by NSI MS/MS, we obtained MS/MS spectra of 3-NT containing peptides of four proteins – similar to ryanodine receptor 3, low density lipoprotein related receptor 2, similar to nebulin-related anchoring protein isoform C and 2,3 cyclic nucleotide 3-phosphodiesterase. Although the functional consequences of protein nitration for these targets are not yet known, our proteomic experiments serve as a first screen for the more targeted analysis of nitrated proteins from aging cerebellum for functional characterization.

Studies of this latter kind are an important complement to the sort represented by the former kind: Covalent modifications such as nitration (and oxidation, and glycation, and ubiquitination, and sumoylation, and…) can dramatically alter protein function, but they are not encoded in the genome; hence this information is lost in a simple enumeration of the identities and abundances of every protein in the cell. In order to understand the peculiarities of aging cells’ behavior, we must learn not only about their gene expression and proteome but also the condition in which the macromolecules find themselves at any given point in time.