Posttranslational modification of Sirt6 activity by peroxynitrite

The mammalian sirtuin 6 (Sirt6) is a site-specific histone deacetylase that regulates chromatin structure and many 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 are largely unknown. Here we hypothesized that Sirt6 function can be regulated via posttranslational modification, focusing on the role of peroxynitrite, one of the major reactive nitrogen species formed by excessive nitric oxide and superoxide generated during disease processes. We found that incubation of purified recombinant Sirt6 protein with 3-morpholinosydnonimine (SIN-1; a peroxynitrite donor that generates nitric oxide and superoxide simultaneously) increased Sirt6 tyrosine nitration and decreased its intrinsic catalytic activity. Similar results were observed in SIN-1-treated Sirt6, which was overexpressed in HEK293 cells, and in endogenous Sirt6 when human retinal microvascular endothelial cells were treated with SIN-1. To further investigate whether Sirt6 nitration occurs under pathological conditions, we determined Sirt6 nitration and activity in retina using a model of endotoxin-induced retinal inflammation. Our data showed that Sirt6 nitration was increased, whereas its activity was decreased, in this model. With mass spectrometry, we identified that tyrosine 257 in Sirt6 was nitrated after SIN-1 treatment. Mutation of tyrosine 257 to phenylalanine caused loss of Sirt6 activity and abolished SIN-1-induced nitration and decrease in its activity. Mass spectrometry analysis also revealed oxidation of methionine and tryptophan in Sirt6 after SIN-1 treatment. Our results demonstrate a novel regulatory mechanism controlling Sirt6 activity through reactive nitrogen species-mediated posttranslational modification under oxidative and nitrosative stress.