Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD⁺ Concentration*

Abstract

Sirtuins are an evolutionarily conserved family of NAD⁺-dependent protein deacetylases that function in the regulation of gene transcription, cellular metabolism, and aging. Their activity requires the maintenance of an adequate intracellular NAD⁺ concentration through the combined action of NAD⁺ biosynthesis and salvage pathways. Nicotinamide (NAM) is a key NAD⁺ precursor that is also a byproduct and feedback inhibitor of the deacetylation reaction. In Saccharomyces cerevisiae, the nicotinamidase Pnc1 converts NAM to nicotinic acid (NA), which is then used as a substrate by the NAD⁺ salvage pathway enzyme NA phosphoribosyltransferase (Npt1). Isonicotinamide (INAM) is an isostere of NAM that stimulates yeast Sir2 deacetylase activity in vitro by alleviating the NAM inhibition. In this study, we determined that INAM stimulates Sir2 through an additional mechanism in vivo, which involves elevation of the intracellular NAD⁺ concentration. INAM enhanced normal silencing at the rDNA locus but only partially suppressed the silencing defects of an npt1Δ mutant. Yeast cells grown in media lacking NA had a short replicative life span, which was extended by INAM in a SIR2-dependent manner and correlated with increased NAD⁺. The INAM-induced increase in NAD⁺ was strongly dependent on Pnc1 and Npt1, suggesting that INAM increases flux through the NAD⁺ salvage pathway. Part of this effect was mediated by the NR salvage pathways, which generate NAM as a product and require Pnc1 to produce NAD⁺. We also provide evidence suggesting that INAM influences the expression of multiple NAD⁺ biosynthesis and salvage pathways to promote homeostasis during stationary phase.