Chemical Activation of Sir2-Dependent Silencing by Relief of Nicotinamide Inhibition

Abstract

Sir2 is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase involved in gene silencing and longevity. Cellular stresses affect Sir2 activity, but the mechanisms of Sir2 regulation are debated. Nicotinamide has been proposed as a physiological regulator that inhibits Sir2 deacetylase activity by chemical reversal of a covalent reaction intermediate. We demonstrate a chemical strategy to activate Sir2-dependent transcriptional silencing and present evidence that the endogenous level of nicotinamide limits Sir2 activity in wild-type (wt) yeast cells. Nicotinamide inhibition of Sir2 is antagonized in vitro by isonicotinamide, which causes an increase in Sir2 deacetylation activity. Isonicotinamide also substantially increases transcriptional silencing at Sir2-regulated loci in wt strains and in strains lacking key NAD+ salvage pathway enzymes (PNC1 and NPT1). Thus, a nicotinamide antagonist is a Sir2 agonist in vitro and in vivo.

Hormonal Control of Androgen Receptor Function through SIRT1

“…Evidence suggests that NAD ϩ -dependent protein deacetylases are also inhibited by nicotinamide in vivo (49). Addition of NAM (5 mM) to the prostate cancer cell line LNCaP induced accumulation of AR in the cells (Fig.…”

“…The sizes and numbers of GFP-positive colonies growing on soft-agar plates were scored on day 15. The thiazolyl blue (MTT) assay was performed as described previously (49).…”

“…The mass isolation window for the collision-induced dissociation mode was set at 3 mass units, and the relative collision energy was set arbitrarily at 30%. Reactions and analyses for yeast Sir2p and Archaeoglobus fulgidus Sir2af2 were performed similarly (49).…”

“…Nicotinamide is the first product from hydrolysis of the pyridinium-N-glycosidic bond of NAD, and it is an inhibitor of Sir2 enzymes from different organisms, including human, yeast, and Archaea enzymes (32,37,49). Evidence suggests that NAD ϩ -dependent protein deacetylases are also inhibited by nicotinamide in vivo (49).…”

Do Sirtuins Promote Mammalian Longevity?: A Critical Review on Its Relevance to the Longevity Effect Induced by Calorie Restriction

“…NAD + is hydrolyzed to NAM, which inhibits SIRT1 deacetylase activities (Bitterman et al, 2002;Fulco et al, 2008;Sauve et al, 2005) and O-acetyl-ADP-ribose (Borra et al, 2002;Tanner et al, 2000). Intracellular NAD + levels and SIRT1 function are regulated by nicotinamide phosphoribosyltransferase (NAMPT), which functions to resynthesize NAD + from NAM (Revollo et al, 2004).…”