Conserved Enzymatic Production and Biological Effect of O-Acetyl-ADP-ribose by Silent Information Regulator 2-like NAD+-dependent Deacetylases

Abstract

Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD ؉ -dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD ؉ and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose.

Sir2 Protein Deacetylases: Evidence for Chemical Intermediates and Functions of a Conserved Histidine

“…Synthetic 11-mer H3 peptide and acetylated H3 peptide (AcH3), corresponding to the 11 amino acids surrounding and including lysine-14 of the histone H3 N-terminal tail; H 2 N-KSTGGK (Ac)APRKQ-CONH 2 , were purchased from the University of Wisconsin-Madison peptide synthesis center, 3 H-labeled Acetyl H3-peptide ( 3 H-AcH3) was synthesized enzymatically from the 11-mer H3 peptide as previously reported (33). 18 O-labelled water was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA).…”

“…This unusual consumption of NAD + in sirtuins (class III HDACs) with linked generation of the novel product OAADPr, has led to the idea that OAADPr possesses unique biological properties and whose cellular levels may be tightly regulated (32). Indeed, OAADPr was shown to be metabolized by a variety of cell extracts (33,34 (39,40). In addition, the 2'-hydroxyl of NAD + does not play a significant role in the first chemical step since replacement with fluorine results in only a slight decrease in the nicotinamide exchange rate (39).…”

“…This unusual consumption of NAD + in sirtuins (class III HDACs) with linked generation of the novel product OAADPr, has led to the idea that OAADPr possesses unique biological properties and whose cellular levels may be tightly regulated (32). Indeed, OAADPr was shown to be metabolized by a variety of cell extracts (33,34). In more recent evidence, OAADPr was shown to bind specifically to the histone splice variant macro H2A1.1 (35) and to induce formation of the yeast Sir2/Sir3/Sir4 complex in vitro (36).…”

Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation

“…To this end, we transfected HEK293T cells with Flag-tagged FOXO3, together with SIRT1, SIRT1-H363Y deacetylase mutant (Wang and Chen, 2010), SIRT2 or SIRT2-H187A deacetylase mutant (Finnin et al, 2001;Borra et al, 2002). Flag-tagged FOXO3 protein was immunoprecipitated with anti-Flag agarose beads and its ubiquitination level was detected using anti-ubiquitin antibody.…”

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).…”