Sirtuin regulation of lysine acylation targets diverse metabolic networks in hepatic mitochondria (981.3)

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Sirtuin 3 (SIRT3) and SIRT5 are NAD+‐dependent mitochondrial protein deacylases. While SIRT3 is the predominant mitochondrial deacetylase, SIRT5 has been identified as a lysine desuccinylase. Here, we use a label‐free quantitative proteomics called MS1 Filtering to characterize lysine acylation in liver mitochondria and its regulation by SIRT5 and SIRT3 using knockout mouse models. Using this approach we identified 1190 sites of lysine succinylation and 2187 sites of lysine acetylation from isolated mitochondria. Overall, we found 939 sites (~79%) were modified by both acetylation and succinylation, while 93 sites were targeted by both SIRT3 and SIRT5. Pathway analysis revealed that SIRT3 and SIRT5 are targeting similar mitochondrial pathways including β‐oxidation, branched‐chain amino acid catabolism, TCA cycle, and ketone body synthesis. Finally, we demonstrate that SIRT5 regulates succinylation of the rate‐limiting ketogenic enzyme 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 both in vivo and in vitro. Taken together, these findings establish SIRT5 and SIRT3 as global regulators of lysine acylation in mitochondria and suggest potential overlap in regulating metabolic networks.