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

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

Eri Maria Sol  1 , Sebastian A Wagner, Brian T Weinert, Amit Kumar, Hyun-Seok Kim, Chu-Xia Deng, Chunaram Choudhary
Affiliations

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

Eri Maria Sol et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Experimental strategy for mapping of Sirt3-regulated acetylation sites.
(A) SILAC-based proteomics strategy for quantification of Sirt3 regulated acetylation sites. Wild type mouse embryonic fibroblasts (MEFs) were labeled with light amino acids, whereas Sirt3 knockout MEFs were labeled with heavy amino acids. Lysine acetylation was analyzed as described previously . (B) Overlap between the acetylation sites quantified in two independent experimental replicates. (C) Correlation of SILAC ratios determined in two biological replicates. Correlation was calculated using Pearson's correlation coefficient.
Figure 2
Figure 2. Cellular and functional annotations of Sirt3 regulated proteins.
(A) Cellular distribution of identified acetylation sites. (B) Significantly elevated acetylation of mitochondrial sites in Sirt3 knockout cells. Logarithmized SILAC ratios of acetylated peptides from mitochondrial and non-mitochondrial proteins were plotted. The data shows the upwardly shifted distribution of SILAC ratios of mitochondrial acetylation sites in Sirt3 deficient cells. Statistical significance was calculated using Wilcoxon rank sum test. (C) Gene Ontology and KEGG pathway enrichment analysis of Sirt3-regulated proteins. Proteins with increased acetylation in Sirt3 knockout cells showed significant enrichment of several mitochondrial associated GOCC or KEGG terms.
Figure 3
Figure 3. Identification of Sirt3-regulated acetylation sites in human cells.
(A) Generation of Sirt3 knockdown and overexpression U2OS cells. U2OS cells were either transfected with a human Sirt3 encoding cDNA or with a Sirt3-specific shRNA. To induce expression of Sirt3-shRNA, cells were treated with doxycycline for the indicated time periods. Expression of Sirt3 was analyzed by immunostaining using anti-Sirt3 antibody. (B) Distribution of identified acetylation sites between mitochondrial or non-mitochondrial cellular compartments. (C) Increased acetylation of mitochondrial acetylation in Sirt3 knockdown cells. Logarithmized SILAC ratios of acetylated peptides from mitochondrial and non-mitochondrial proteins were plotted. These data shows the upwardly shifted distribution of SILAC ratios of mitochondrial acetylation sites in Sirt3 knockdown cells. (D) Gene Ontology enrichment analysis of Sirt3-regulated proteins. Proteins with increased acetylation in Sirt3 knockdown cells showed significant enrichment of mitochondrial associated GO terms.
Figure 4
Figure 4. Sirt3 increases acetylation of enzymes involved in fatty acid metabolism and the TCA cycle.
(A and B) The panel A shows enzymes involved in the fatty acid metabolism, and the panel B shows enzymes involved in the TCA cycle. Enzymes in these pathways are color coded (indicated at the top of the figure) based on their identification in our experiments, and their increase in acetylation in Sirt3 knockout or knockdown cells. (C) Acetylation of PDHX is increased in Sirt3 knockout cells. Acetylated proteins were immunoprecipitated using an anti-acetyllysine antibody and immunoblotted with anti-PDHX or anti-acetyltubulin antibodies. The lower panel shows protein levels of PDHX in whole cell lysates used as input for immunoprecipitations, and vinculin was used as loading control.
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Grants and funding

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.

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