SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks

Abstract

Summary Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

Mitochondrial Sirtuins and Their Relationships with Metabolic Disease and Cancer

“…Among the quantifiable sites identified by Park et al, more than 90% showed hypersuccinylation in Sirt5 KO cells, strongly suggesting that SIRT5 is a major regulator of lysine succinylation in mammals (116). Similarly, Rardin et al reported that SIRT5 deficiency resulted in hypersuccinylation of 32% sites in 56% of mitochondrial proteins overall (127). The preference of SIRT5 for negatively charged acyl groups was further corroborated by another recent study (156).…”

“…Via mass spectrometry approaches, two independent studies have identified multiple SIRT5 succinylated targets in mouse liver mitochondria (127), and globally in MEFs and liver tissues (116). Among the quantifiable sites identified by Park et al, more than 90% showed hypersuccinylation in Sirt5 KO cells, strongly suggesting that SIRT5 is a major regulator of lysine succinylation in mammals (116).…”

“…However, recently SIRT5 has been shown to preferentially deacylate negatively charged modifications: malonylation, succinylation, and glutarylation (34,116,121,127,156). Du et al showed that SIRT5 possesses minimal deacetylase activity compared with SIRT1 and SIRT3 (34); however, the catalytic efficiency of SIRT5 for demalonylation and desuccinylation was much higher than for deacetylation (34,121).…”

“…Consequently, SIRT5 KD resulted in elevated PDC E1a activity, along with a substantial increase in SDH activity and elevated cellular respiration (116). Rardin et al showed hypersuccinylation of HMGCS2 in the absence of SIRT5 (127). HMGCS2 is the rate-limiting enzyme of ketone body synthesis, and hypersuccinylation decreases its activity.…”

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis*

“…These succinylome studies have generated large data sets of lysine-succinylated proteins in both eukaryotes and prokaryotes and demonstrated the diverse cellular functions of this PTM. Notably, lysine succinylation is widespread among diverse mitochondrial metabolic enzymes that are involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (19,20). Thus, lysine succinylation is reported as a functional PTM with the potential to impact mitochondrial metabolism and coordinate different metabolic pathways in human cells and bacteria (14, 19 -22).…”