Posttranslational modification of Sirt6 activity by peroxynitrite

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Sirtuin 6 (Sirt6) is a crucial histone deacetylase regulating diverse biological processes.
• The molecular mechanisms governing Sirt6 enzymatic activity remain largely uncharacterized.
• Reactive nitrogen species (RNS) are implicated in various disease processes.

Purpose of the Study:

• To investigate the role of peroxynitrite, a major RNS, in regulating Sirt6 enzymatic function.
• To identify potential posttranslational modifications of Sirt6 induced by RNS.
• To explore the pathological relevance of Sirt6 modification by RNS.

Main Methods:

• Incubation of purified recombinant Sirt6 with a peroxynitrite donor (SIN-1).
• Overexpression of Sirt6 in HEK293 cells and treatment with SIN-1.
• Analysis of endogenous Sirt6 in human retinal microvascular endothelial cells treated with SIN-1.
• Assessment of Sirt6 nitration and activity in an endotoxin-induced retinal inflammation model.
• Mass spectrometry to identify nitrated residues and other modifications.
• Site-directed mutagenesis of identified tyrosine residues.

Main Results:

• Peroxynitrite donor treatment increased Sirt6 tyrosine nitration and decreased its catalytic activity.
• Similar effects were observed in cellular models, including endogenous Sirt6.
• Sirt6 nitration was elevated, and activity reduced in a model of endotoxin-induced retinal inflammation.
• Tyrosine 257 was identified as the primary site of nitration, and its mutation abolished activity and RNS-mediated inhibition.
• Oxidation of methionine and tryptophan residues was also observed.

Conclusions:

• Sirt6 activity is regulated by RNS-mediated posttranslational modification, specifically tyrosine nitration at Y257.
• This regulatory mechanism is relevant under conditions of oxidative and nitrosative stress.
• The findings reveal a novel pathway controlling Sirt6 function in disease pathogenesis.