In yeast cells, Pnc1 (nicotinamidase) is an evolutionarily conserved enzyme in the NAD+ salvage pathway and a master regulator of aging, as (1) expression is induced by environmental stresses and (2) is necessary and sufficient for lifespan extension by caloric restriction and stress. Yeast Pnc1 is thought to control the sirtuin family of NAD+-dependent deacetylases, enzymes previously implicated in aging and stress. Given this work, we hypothesized that PNC-1 in C. elegans may play a role in longevity and stress protection, and that specific tissues may be important for these biological effects. To study the enzymatic activity of PNC-1 in vitro, recombinant protein was expressed in bacterial cells, purified, assayed, and shown biochemically to have robust nicotinamidase activity. Moreover, site-directed mutagenesis of a conserved residue in the active site of PNC-1, as predicted by yeast Pnc1 crystallographic data, abolishes nicotinamidase enzymatic function. PNC-1 over-expressing transgenic animals were generated, employing a ubiquitous driver, to study the biological effects of over-expression in vivo. Using previously described dietary assays, PNC-1 over-expressing animals were tested for longevity and found to outlive controls. To assess a conserved role in stress responses, PNC-1 over-expressing animals were subjected to an established salt stress paradigm, to which they were remarkably resistant, suggesting that PNC-1 may play an important role in integrating nutritional signals and environmental conditions to determine lifespan. Lastly, to pinpoint sites of endogenous expression, a
pnc-1 promoter fragment was cloned into a green fluorescent protein reporter system; green expression was observed in the pharynx and specific neurons. Also, epistasis studies are in progress to address how
pnc-1 acts in relation to known regulators of aging. Collectively, all of these approaches will better define conserved pro-survival pathways across different organisms, which will contribute significantly to our basic understanding of the biology of aging.