Abstract
Sirtuin enzymes depend on NAD+ to catalyze protein deacetylation. Therefore, the lowering of NAD+ during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD+-depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog,SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD+-limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms.
Highlights
Discovered for its role in yeast genomic silencing, Sir2 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that mediates gene silencing and aging (Imai et al, 2000)
We identified two mutations in SIR2 that extend replicative lifespan (RLS) and increase enzymatic activity in low NAD+
Strains Used to Select Sir2 Mutants The main pathways involved in NAD+ biosynthesis in yeast and other organisms are de novo synthesis of NAD+ from tryptophan and the salvage pathway generating NAD+ from nicotinamide and nicotinic acid
Summary
Discovered for its role in yeast genomic silencing, Sir is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that mediates gene silencing and aging (Imai et al, 2000). Sir2-related proteins, or sirtuins—especially the mammalian Sir isoform SIRT1—have been shown to modulate numerous diseases in mouse models, including metabolic disorders, neurodegeneration, cardiovascular disease, cancers, and aging (Imai and Guarente, 2014; Guarente, 2013). Acetylation of specific lysine (Lys) residues in the amino terminus of histone proteins is recognized as a fundamental epigenetic mark that affects transcriptional regulation (Rothbart and Strahl, 2014). Histone acetylation is generally associated with transcriptional activation, as Lys residues in histone proteins neutralize negative charges along the DNA backbone. Deacetylation, results in chromatin condensation, which is associated with transcriptional repression and gene silencing. Post-translational modifications, including deacetylation, recruit regulatory molecules to chromatin and determine the transcriptional status of genes (Allis and Jenuwein, 2016). Sirtuins deacetylate many nonhistone proteins, such as transcription factors and metabolic enzymes, and can exert a profound effect on cellular physiology
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