Abstract
Recent studies have highlighted the three mitochondrial human sirtuins (SIRT3, SIRT4, and SIRT5) as critical regulators of a wide range of cellular metabolic pathways. A key factor to understanding their impact on metabolism has been the discovery that, in addition to their ability to deacetylate substrates, mitochondrial sirtuins can have other prominent enzymatic activities. SIRT4, one of the least characterized mitochondrial sirtuins, was shown to be the first known cellular lipoamidase, removing lipoyl modifications from lysine residues of substrates. Specifically, SIRT4 was found to delipoylate and modulate the activity of the pyruvate dehydrogenase complex (PDH), a protein complex critical for the production of acetyl-CoA. Furthermore, SIRT4 is well known to have ADP-ribosyltransferase activity and to regulate the activity of the glutamate dehydrogenase complex (GDH). Adding to its impressive range of enzymatic activities are its ability to deacetylate malonyl-CoA decarboxylase (MCD) to regulate lipid catabolism, and its newly recognized ability to remove biotinyl groups from substrates that remain to be defined. Given the wide range of enzymatic activities and the still limited knowledge of its substrates, further studies are needed to characterize its protein interactions and its impact on metabolic pathways. Here, we present several proven protocols for identifying SIRT4 protein interaction networks within the mitochondria. Specifically, we describe methods for generating human cell lines expressing SIRT4, purifying mitochondria from crude organelles, and effectively capturing SIRT4 with its interactions and substrates.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Guarente L (2000) Sir2 links chromatin silencing, metabolism, and aging. Genes Dev 14(9):1021–1026
Imai S, Armstrong CM, Kaeberlein M et al (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403(6771):795–800
Donmez G (2012) The neurobiology of sirtuins and their role in neurodegeneration. Trends Pharmacol Sci 33(9):494–501
Min SW, Sohn PD, Cho SH et al (2013) Sirtuins in neurodegenerative diseases: an update on potential mechanisms. Front Aging Neurosci 5:53
Roth M, Chen WY (2014) Sorting out functions of sirtuins in cancer. Oncogene 33(13):1609–1620
Sebastian C, Satterstrom FK, Haigis MC et al (2012) From sirtuin biology to human diseases: an update. J Biol Chem 287(51):42444–42452
Winnik S, Auwerx J, Sinclair DA et al (2015) Protective effects of sirtuins in cardiovascular diseases: from bench to bedside. Eur Heart J. doi:10.1093/eurheartj/ehv290
Yuan H, Su L, Chen WY (2013) The emerging and diverse roles of sirtuins in cancer: a clinical perspective. Onco Targets Ther 6:1399–1416
Haigis MC, Mostoslavsky R, Haigis KM et al (2006) SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell 126(5):941–954
Michishita E, Park JY, Burneskis JM et al (2005) Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol Biol Cell 16(10):4623–4635
Lombard DB, Alt FW, Cheng HL et al (2007) Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation. Mol Cell Biol 27(24):8807–8814
Rardin MJ, Newman JC, Held JM et al (2013) Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways. Proc Natl Acad Sci U S A 110(16):6601–6606
Brautigam CA, Wynn RM, Chuang JL et al (2006) Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3 binding protein of human pyruvate dehydrogenase complex. Structure 14(3):611–621
Laurent G, German NJ, Saha AK et al (2013) SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase. Mol Cell 50(5):686–698
Mathias RA, Greco TM, Oberstein A et al (2014) Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity. Cell 159(7):1615–1625
Du J, Zhou Y, Su X et al (2011) Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 334(6057):806–809
Peng C, Lu Z, Xie Z, et al. (2011) The first identification of lysine malonylation substrates and its regulatory enzyme. Mol Cell Proteomics 10(12), DOI:10.1074/mcp.M111.012658
Tan M, Peng C, Anderson KA et al (2014) Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. Cell Metab 19(4):605–617
Feldman JL, Baeza J, Denu JM (2013) Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins. J Biol Chem 288(43):31350–31356
Miteva YV, Cristea IM (2014) A proteomic perspective of Sirtuin 6 (SIRT6) phosphorylation and interactions and their dependence on its catalytic activity. Mol Cell Proteomics 13(1):168–183
Tsai YC, Greco TM, Boonmee A et al (2012) Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription. Mol Cell Proteomics 11(5):60–76
Joshi P, Greco TM, Guise AJ et al (2013) The functional interactome landscape of the human histone deacetylase family. Mol Syst Biol 9(1):672
Diner BA, Li T, Greco TM et al (2015) The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA. Mol Syst Biol 11(1):787
Choi H, Larsen B, Lin ZY et al (2011) SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods 8(1):70–73
Cristea IM, Williams R, Chait BT et al (2005) Fluorescent proteins as proteomic probes. Mol Cell Proteomics 4(12):1933–1941
Greco TM, Miteva Y, Conlon FL et al (2012) Complementary proteomic analysis of protein complexes. Methods Mol Biol 917:391–407
Greco TM, Diner BA, Cristea IM (2014) The impact of mass spectrometry-based proteomics on fundamental discoveries in virology. Annu Rev Virol 1(1):581–604
Choi H, Liu G, Mellacheruvu D et al (2012) Analyzing protein-protein interactions from affinity purification-mass spectrometry data with SAINT. Curr Protoc Bioinformatics 39(8.15):1–23
Mellacheruvu D, Wright Z, Couzens AL et al (2013) The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10(8):730–736
Acknowledgements
We are grateful for funding from NIH grants R01HL127640 and R21AI102187 (I.M.C.), an NHMRC of Australia Early Career CJ Martin Fellowship #APP1037043 (R.A.M.), and an NJCCR postdoctoral fellowship (T.M.G.).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Mathias, R.A., Greco, T.M., Cristea, I.M. (2016). Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators. In: Sarkar, S. (eds) Histone Deacetylases. Methods in Molecular Biology, vol 1436. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3667-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3667-0_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3665-6
Online ISBN: 978-1-4939-3667-0
eBook Packages: Springer Protocols