Brian K. Kennedy

Regulation of Yeast Replicative Life Span by TOR and Sch9 in Response to Nutrients

Calorie restriction increases life span in many organisms, including the budding yeast Saccharomyces cerevisiae. From a large-scale analysis of 564 single-gene–deletion strains of yeast, we identified 10 gene deletions that increase replicative life span. Six of these correspond to genes encoding components of the nutrient-responsive TOR and Sch9 pathways. Calorie restriction of tor1D or sch9D cells failed to further increase life span and, like calorie restriction, deletion of either SCH9 or TOR1 increased life span independent of the Sir2 histone deacetylase. We propose that the TOR and Sch9 kinases define a primary conduit through which excess nutrient intake limits longevity in yeast.

MECHANISM OF INHIBITION OF PROTEIN-TYROSINE PHOSPHATASES BY VANADATE AND PERVANADATE

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a Ki of 0.38 ± 0.02 μM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

Substrate-specific Activation of Sirtuins by Resveratrol

Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to act as a sirtuin activator, and this property has been proposed to account for its anti-aging effects. We show here that resveratrol is a substrate-specific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined.

Sirtuins in Aging and Age-Related Disease

Sirtuins have been the focus of intense scrutiny since the discovery of Sir2 as a yeast longevity factor. Functioning as either deacetylases or ADP ribosylases, Sirtuins are regulated by the cofactor NAD and thus may serve as sensors of the metabolic state of the cell and organism. Here we examine the roles of Sirtuins in diverse eukaryotic species, with special emphasis on their links to aging and age-related diseases including cancer, diabetes, and neurodegenerative disorders.

Mutation in the Silencing Gene SIR4 Can Delay Aging in S. cerevisiae

Aging in S. cerevisiae is exemplified by the fixed number of cell divisions that mother cells undergo (termed their life span). We have exploited a correlation between life span and stress resistance to identify mutations in four genes that extend life span. One of these, SIR4, encodes a component of the silencing apparatus at HM loci and telomeres. The sir4-42 mutation extends life span by more than 30% and is semidominant. Our findings suggest that sir4-42 extends life span by preventing recruitment of the SIR proteins to HM loci and telomeres, thereby increasing their concentration at other chromosomal regions. Maintaining silencing at these other regions may be critical in preventing aging. Consistent with this view, expression of only the carboxyl terminus of SIR4 interferes with silencing at HM loci and telomeres, which also extends life span. Possible links among silencing, telomere maintenance, and aging in other organisms are discussed.

Histone H4 lysine 16 acetylation regulates cellular lifespan.

Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD+-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.

LMNA mutations in atypical Werner's syndrome

BACKGROUND Werners syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werners syndrome (wild-type WRN). METHODS Of 129 index patients referred to our international registry for molecular diagnosis of Werners syndrome, 26 (20%) had wildtype WRN coding regions and were categorised as having atypical Werners syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RT-PCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS In four (15%) of 26 patients with atypical Werners syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werners syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION Our findings indicate that Werners syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy.

Redistribution of Silencing Proteins from Telomeres to the Nucleolus Is Associated with Extension of Life Span in S. cerevisiae

A prior genetic study indicated that activity of Sir silencing proteins at a hypothetical AGE locus is essential for long life span. In this model, the SIR4-42 mutation would direct the Sir protein complex to the AGE locus, giving rise to a long life span. We show by indirect immunofluorescence that Sir3p and Sir4p are redirected to the nucleolus in the SIR4-42 mutant. Furthermore, this relocalization is dependent on both UTH4 a novel yeast gene that extends life span, and its homologue YGL023. Strikingly, the Sir complex is relocalized from telomeres to the nucleolus in old wild-type cells. We propose that the rDNA is the AGE locus and that nucleolar function is compromised in old yeast cells in a way that may be mitigated by targeting of Sir proteins to the nucleolus.

Geroscience: Linking Aging to Chronic Disease

Mammalian aging can be delayed with genetic, dietary, and pharmacologic approaches. Given that the elderly population is dramatically increasing and that aging is the greatest risk factor for a majority of chronic diseases driving both morbidity and mortality, it is critical to expand geroscience research directed at extending human healthspan.

Yeast Life Span Extension by Depletion of 60S Ribosomal Subunits Is Mediated by Gcn4

In nearly every organism studied, reduced caloric intake extends life span. In yeast, span extension from dietary restriction is thought to be mediated by the highly conserved, nutrient-responsive target of rapamycin (TOR), protein kinase A (PKA), and Sch9 kinases. These kinases coordinately regulate various cellular processes including stress responses, protein turnover, cell growth, and ribosome biogenesis. Here we show that a specific reduction of 60S ribosomal subunit levels slows aging in yeast. Deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span. One mechanism by which reduced 60S subunit levels leads to life span extension is through induction of Gcn4, a nutrient-responsive transcription factor. Genetic epistasis analyses suggest that dietary restriction, reduced 60S subunit abundance, and Gcn4 activation extend yeast life span by similar mechanisms.