Abstract 18583: Posttranslational Modification of Sirt6 Activity by Reactive Nitrogen Species

Abstract

The mammalian sirtuin 6 (SIRT6) is a site-specific histone deacetylase that regulates chromatin structure and fundamental biological processes. It inhibits endothelial cell senescence and inflammation, prevents development of cardiac hypertrophy and heart failure, modulates glucose metabolism and represses tumor growth. The basic molecular mechanisms underlying regulation of Sirt6 enzymatic function remains unknown. Here we hypothesized that Sirt6 function can be regulated via posttranslational modification, focusing on the role of peroxynitrite. Peroxynitrite is one of the major reactive nitrogen species formed by excessive nitric oxide and superoxide generated during cardiovascular diseases. It induces nitration of protein tyrosine residue and alters the structure and function of protein.We found that incubation of purified recombinant Sirt6 protein with various doses (0.5, 1.0, 2.0, 5.0 mM ) of 3-morpholinosydnonimine (SIN-I, a peroxynitrite donor which generates NO and superoxide donor simultaneously) for 30 min at room temperature resulted in increase in Sirt6 tyrosine nitration by 6.6, 8.4, 11.1 and 14.3 fold, respectively. Correlated with the increase in Sirt6 nitration, the intrinsic catalytic activity of Sirt6 was reduced by 43.4%, 47.2%, 53.5% and 70.1% as determined with a Sirt6 deacetylase fluorometric assay kit. Nitration of endogenous Sirt6 were also observed in human umbilical vein endothelial cells and human retinal microvascular cells treated with SIN-1 (5 mM). To further test whether Sirt6 nitration occurs under pathological conditions, we monitored Sirt6 nitration using a model of endotoxin-induced retinal vascular inflammation. Measurement of 3-nitrotyrosine in immunoprecipitated Sirt6 showed that Sirt6 nitration was significantly increased in mice of inflammation. There are four tyrosine residues in Sirt6 which are conserved among human, mouse and rat. Analysis of protein nitration sites by mass spectrometry revealed that tyrosine 257 in Sirt6 was nitrated after SIN-1 treatment. These results demonstrate a novel regulatory mechanism of Sirt6 activity through reactive nitrogen species-mediated nitration under oxidative and nitrative stress.