Anti-SIRT5 (C-terminal) polyclonal antibody

The ability to probe for catalytic activities of enzymes and to detect their abundance in complex biochemical contexts has traditionally relied on a combination of kinetic assays and techniques such as western blots that use expensive reagents such as antibodies. The ability to simultaneously detect activity and isolate a protein catalyst from a mixture is even more difficult and currently impossible in most cases. In this manuscriptwe describe a chemical approach that achieves this goal for a unique family of enzymes called sirtuins using novel chemical tools, enabling rapid detection of activity and isolation of these protein catalysts. Sirtuin deacetylases are implicated in the regulation of many physiological functions including energy metabolism, DNA-damage response, and cellular stress resistance. We synthesized an aminooxy-derivatized NAD(+) and a pan-sirtuin inhibitor that reacts on sirtuin active sites to form a chemically stable complex that can subsequently be crosslinked to an aldehyde-substituted biotin. Subsequent retrieval of the biotinylated sirtuin complexes on streptavidin beads followed by gel electrophoresis enabled simultaneous detection of active sirtuins, isolation and molecular weight determination. We show that these tools are cross reactive against a variety of human sirtuin isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and can react with microbial derived sirtuins as well. Finally, we demonstrate the ability to simultaneously detect multiple sirtuin isoforms in reaction mixtures with this methodology, establishing proof of concept tools for chemical studies of sirtuins in complex biological samples.

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 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including beta-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium-and long-chain acylcarnitines and decreased beta-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.