| Sirtuins are a conserved family of NAD+ dependent protein/histone deacetylases found in organisms ranging from bacteria to humans. A novel metabolite, O-Acetyl-ADP-ribose (OAADPr), was discovered as a result of acetyl group transfer from histones to the ADPr moiety of NAD+ catalyzed by Sirtuins.;We examined cellular metabolism of OAADPr by nudix hydrolase Ysa1. Ysa1 cleaves ADPr/OAADPr into ribose-phosphate/acetyl-ribose-phosphate and AMP. We developed a LC/MS-MS method to determine accurately cellular OAADPr concentration. In cells lacking Ysa1 (Deltaysa1 ), ADPr and OAADPr levels increased ∼50%. Strikingly, Deltaysa1 cells display higher resistance to exogenous reactive oxygen species (ROS), and 40% lower basal levels of endogenous ROS, compared to wild type. We investigated the biochemical basis for these differences and discovered that increased ADPr/OAADPr levels possibly protect cells via two pathways: (i) lowering ROS production through inhibition of complex I of the mitochondrial electron transport chain (ii) generation of higher levels of NADPH to suppress ROS damage, through diverting glucose into the pentose phosphate pathway and ADPr inhibition of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), a central enzyme of glycolysis.;Additionally, we developed methods utilizing photocrosslinking, isothermal titration calorimetry (ITC), Cibacron blue resin, and Biotin-OAADPr bound streptavidin conjugated agarose beads, to discover and characterize interactions between OAADPr and candidate proteins. Photocrosslinking of OAADPr to Sir3 protein, suggested that Sir2-4 complex interacted with OAADPr through Sir3, though Sir2 displayed direct binding to OAADPr in a separate binding assay. Dissociation constants for OAADPr binding to members of the sirtuin family were determined by ITC, yielding values between 5-10 muM. Cibacron blue resin was used to screen for OAADPr interacting proteins in the cell extracts yielding GAPDH, PGK and ADH, all of which are enzymes related to glycolysis.;While examining the cellular metabolism of OAADPr using S. cerevisiae as a model system, we discovered a specific nuclear metabolite derived from OAADPr, containing the acetyl group originating from OAADPr. The metabolite was purified via organic extraction, ion exchange, and reverse phase chromatography, and currently being analyzed by Mass Spectrometry and NMR. |
