Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

PLoS One. 2012;7(12):e50545. doi: 10.1371/journal.pone.0050545. Epub 2012 Dec 7.

Abstract

Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site-specific acetylation in wild-type murine embryonic fibroblasts to Sirt3 knockout cells. We confirm Sirt3-regulated acetylation of several mitochondrial proteins in human cells by comparing acetylation in U2OS cells overexpressing Sirt3 to U2OS cells in which Sirt3 expression was reduced by shRNA. Our data demonstrate that ablation of Sirt3 significantly increases acetylation at dozens of sites on mitochondrial proteins. Substrates of Sirt3 are implicated in various metabolic pathways, including fatty acid metabolism and the tricarboxylic acid cycle. These results imply broader regulatory roles of Sirt3 in the mitochondria by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Animals
  • Cell Line
  • Fibroblasts / metabolism*
  • Humans
  • Lysine / genetics
  • Lysine / metabolism*
  • Mice
  • Mice, Knockout
  • Mitochondria / genetics
  • Mitochondria / metabolism
  • Proteomics
  • Sirtuin 3 / genetics
  • Sirtuin 3 / metabolism*

Substances

  • Sirt3 protein, mouse
  • Sirtuin 3
  • Lysine

Grant support

This work is supported in part by the European Commission's 7th Framework Programme grant Proteomics Research Infrastructure Maximising knowledge EXchange and access (XS) (INFRASTRUCTURES-F7-2010-262067/PRIME-XS), and the Lundbeck Foundation (R48-A4649). The Center for Protein Research is funded by a generous grant from the Novo Nordisk Foundation. EMS is supported by a postdoctoral fellowship from Swedish Research Council (2009-7387). SAW is supported by a postdoctoral grant from Danish Council for Independent Research (FSS: 10-085134). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.