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Methods Mol Biol. 2016;1436:213-39. doi: 10.1007/978-1-4939-3667-0_15.

Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators.

Author information

1
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Room 423 LIMS1, Melbourne, VIC, 3086, Australia. r.mathias@latrobe.edu.au.
2
Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Washington Road, Princeton, NJ, 08544, USA.
3
Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Washington Road, Princeton, NJ, 08544, USA. icristea@princeton.edu.

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.

KEYWORDS:

Lipoamide; Lipoic acid; Lipoyl; PDH; Protein–protein interactions; Sirtuin 4

PMID:
27246218
PMCID:
PMC4919573
DOI:
10.1007/978-1-4939-3667-0_15
[Indexed for MEDLINE]
Free PMC Article

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