SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target.

Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD(+)-dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5(-/-) mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5(-/-) animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5(-/-) compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.

[1]  P. Sharp,et al.  Cre-lox-regulated conditional RNA interference from transgenes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[2]  小倉 雅仁.  Overexpression of SIRT5 confirms its involvement in deacetylation and activation of carbamoyl phosphate synthetase 1 , 2010 .

[3]  F. Alt,et al.  SIRT3 deacetylates mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 and regulates ketone body production. , 2010, Cell metabolism.

[4]  Alan J. Robinson,et al.  MitoMiner: a data warehouse for mitochondrial proteomics data , 2011, Nucleic Acids Res..

[5]  H. Lodish,et al.  Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells. , 1990, The American journal of physiology.

[6]  L. Guarente,et al.  SIRT 5 Deacetylates Carbamoyl Phosphate Synthetase 1 and Regulates the Urea Cycle Citation , 2022 .

[7]  Christopher T. Walsh,et al.  Protein Posttranslational Modifications: The Chemistry of Proteome Diversifications , 2006 .

[8]  Yingming Zhao,et al.  SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways. , 2013, Molecular cell.

[9]  Sean L Seymour,et al.  The Paragon Algorithm, a Next Generation Search Engine That Uses Sequence Temperature Values and Feature Probabilities to Identify Peptides from Tandem Mass Spectra*S , 2007, Molecular & Cellular Proteomics.

[10]  N. Grishin,et al.  Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. , 2006, Molecular cell.

[11]  David Saggerson,et al.  Malonyl-CoA, a key signaling molecule in mammalian cells. , 2008, Annual review of nutrition.

[12]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[13]  Matthew J. Rardin,et al.  Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site , 2013, The Journal of Biological Chemistry.

[14]  Robert V Farese,et al.  SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome. , 2011, Molecular cell.

[15]  Eric Verdin,et al.  Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2 , 2006, Proceedings of the National Academy of Sciences.

[16]  Peng Xue,et al.  Lysine Malonylation Is Elevated in Type 2 Diabetic Mouse Models and Enriched in Metabolic Associated Proteins* , 2014, Molecular & Cellular Proteomics.

[17]  Bing Li,et al.  Acetyl-CoA induces cell growth and proliferation by promoting the acetylation of histones at growth genes. , 2011, Molecular cell.

[18]  Xiang David Li,et al.  A chemical probe for lysine malonylation. , 2013, Angewandte Chemie.

[19]  Daniel Amador-Noguez,et al.  Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome*♦ , 2014, The Journal of Biological Chemistry.

[20]  Sebastian A. Wagner,et al.  Proteomic Investigations of Lysine Acetylation Identify Diverse Substrates of Mitochondrial Deacetylase Sirt3 , 2012, PloS one.

[21]  Matthew J. Rardin,et al.  SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. , 2013, Cell metabolism.

[22]  T. Kitamura,et al.  Plat-E: an efficient and stable system for transient packaging of retroviruses , 2000, Gene Therapy.

[23]  E. Verdin,et al.  The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide–dependent deacetylase , 2002, The Journal of cell biology.

[24]  G. R. Wagner,et al.  Widespread and Enzyme-independent Nϵ-Acetylation and Nϵ-Succinylation of Proteins in the Chemical Conditions of the Mitochondrial Matrix*♦ , 2013, The Journal of Biological Chemistry.

[25]  Johan Auwerx,et al.  Sirt5 Is a NAD-Dependent Protein Lysine Demalonylase and Desuccinylase , 2011, Science.

[26]  J. Boeke,et al.  Lysine Succinylation and Lysine Malonylation in Histones* , 2012, Molecular & Cellular Proteomics.

[27]  A. Emili,et al.  Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics , 2006, Nature Protocols.

[28]  Lloyd M. Smith,et al.  Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction. , 2011, Molecular cell.

[29]  Sean D. Mooney,et al.  Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways , 2013, Proceedings of the National Academy of Sciences.

[30]  P. Seglen.  Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver. , 1972, Experimental cell research.

[31]  L. Guarente,et al.  SIRT1 and other sirtuins in metabolism , 2014, Trends in Endocrinology & Metabolism.

[32]  D. Trono,et al.  A Third-Generation Lentivirus Vector with a Conditional Packaging System , 1998, Journal of Virology.

[33]  Yingming Zhao,et al.  Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. , 2014, Cell metabolism.

[34]  S. Yanagi,et al.  Distinct regulation of mitochondrial localization and stability of two human Sirt5 isoforms , 2011, Genes to cells : devoted to molecular & cellular mechanisms.

[35]  N. Ruderman,et al.  A malonyl-CoA fuel-sensing mechanism in muscle: effects of insulin, glucose, and denervation. , 1995, The American journal of physiology.

[36]  S. Snyder,et al.  H2S Signals Through Protein S-Sulfhydration , 2009, Science Signaling.

[37]  Yoshiharu Kawaguchi,et al.  MDM2–HDAC1‐mediated deacetylation of p53 is required for its degradation , 2002, The EMBO journal.

[38]  L. Cantley,et al.  Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation , 2009, Science.

[39]  Ronald J. Moore,et al.  A Method to Determine Lysine Acetylation Stoichiometries , 2014, International journal of proteomics.

[40]  J. McGarry,et al.  The role of malonyl-coa in the coordination of fatty acid synthesis and oxidation in isolated rat hepatocytes. , 1978, The Journal of biological chemistry.

[41]  Yi Zhang,et al.  The First Identification of Lysine Malonylation Substrates and Its Regulatory Enzyme* , 2011, Molecular & Cellular Proteomics.

[42]  E. Saggerson,et al.  Malonyl-CoA metabolism in cardiac myocytes. , 2000, The Biochemical journal.

[43]  N. Inagaki,et al.  Overexpression of SIRT5 confirms its involvement in deacetylation and activation of carbamoyl phosphate synthetase 1. , 2010, Biochemical and biophysical research communications.

[44]  Jimin Pei,et al.  AL2CO: calculation of positional conservation in a protein sequence alignment , 2001, Bioinform..

[45]  Michael J. MacCoss,et al.  Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline , 2012, Molecular & Cellular Proteomics.

[46]  R. Veech,et al.  The concentration of malonyl-coenzyme A and the control of fatty acid synthesis in vivo. , 1972, The Journal of biological chemistry.

[47]  S. Berger,et al.  Histone modifications in transcriptional regulation. , 2002, Current opinion in genetics & development.

[48]  Robert Burke,et al.  ProteoWizard: open source software for rapid proteomics tools development , 2008, Bioinform..

[49]  Yi Tang,et al.  Lysine Propionylation and Butyrylation Are Novel Post-translational Modifications in Histones*S , 2007, Molecular & Cellular Proteomics.