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

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.

Fig. 1

Experimental approach for studying interactions and substrates of SIRT4. (a) Timeline for the generation of MRC5 fibroblasts (bottom row) stably expressing SIRT4-EGFP (orange cells) by transduction of retrovirus generated from Phoenix cells (top row). Two rounds of transduction are performed, followed by G418 selection of EGFP-tagged SIRT4 expressing cell. MRC5 cells that survive the selection are expanded to 5 × 15 cm plates in normal growth medium. (b) Scheme for isolation of mitochondria from EGFP-tagged SIRT4 expressing MRC5 cells. A crude organelle fraction is first obtained by 3× passage of MRC5 cells through a pressure lysis apparatus containing two polycarbonate filter disks. Organelles are then resolved by ultracentrifugation through a self-prepared OptiPrep density gradient. Western blot analysis should be used to confirm mitochondria are enriched in fractions 3 and 4, and also to check purity. (c) Graphic workflow for proteomic analysis of SIRT4-containing complexes isolated from purified mitochondria. Using optimized lysis buffer, SIRT4-EGFP and associated proteins are extracted from mitochondria and mixed with EGFP-conjugated magnetic beads. Affinity purified proteins are then eluted from the beads, resolved by SDS-PAGE, and digested in-gel with trypsin. Tryptic peptide mixtures are then analyzed by reverse-phase nanoliquid chromatography (RP-nLC) coupled to nanoelectrospray (nESI) tandem mass spectrometry. The total number of spectra collected and assigned to each protein (spectral counts) between control and SIRT4 isolations are used to determine specific interactions, which are subsequently analyzed by bioinformatics