title: Posttranslational modification of Sirt6 activity by peroxynitrite.
abstract: 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.
authors.affiliation: Institute of Emergency Rescue Medicine, Xuzhou Medical College, Xuzhou, Jiangsu, China; Department of Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA.
authors.forename: Shuqun
authors.initials: S
authors.lastname: Hu
authors.affiliation: Center for Biomedical Engineering, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA.
authors.forename: Yonju
authors.initials: Y
authors.lastname: Ha
authors.affiliation: Biomolecular Resource Facility, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Luo
authors.affiliation: Department of Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA; Center for Biomedical Engineering, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA.
authors.forename: Massoud
authors.initials: M
authors.lastname: Motamedi
authors.affiliation: Department of Surgery, Committee on Molecular and Cellular Physiology, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Mahesh P
authors.initials: MP
authors.lastname: Gupta
authors.affiliation: Department of Physiology, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 731 04, USA.
authors.forename: Jian-Xing
authors.initials: JX
authors.lastname: Ma
authors.affiliation: Department of Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA; Internal Medicine, Division of Endocrinology and Stark Diabetes Center, and The University of Texas Medical Branch, Galveston, TX 77555-0144, USA.
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Tilton
authors.affiliation: Department of Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA; Center for Biomedical Engineering, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA; Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, Galveston, TX 77555-0144, USA. Electronic address: we2zhang@utmb.edu.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D012160:Retina / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017386:Sequence Homology, Amino Acid; D037761:Sirtuins / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D008070:Lipopolysaccharides; D030421:Peroxynitrous Acid; C516592:SIRT6 protein, human; D037761:Sirtuins
keywords: Catalytic activity; Free radicals; Inflammation; Nitration; Nitrosative stress; Oxidation; Oxidative stress; Reactive nitrogen species; Sirt6; Sirtuin