Chemical Probes Reveal Sirt2's New Function as a Robust "Eraser" of Lysine Lipoylation.
Lysine lipoylation, a highly conserved lysine PTM, plays a critical role in regulating cell metabolism. The catalytic activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH) depends on lysine lipoylation. Despite its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we designed a powerful affinity based probe KPlip to interrogate the interactions of lipoylated peptide/proteins under native cellular environment. Large scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including Sirt2, an NAD+ dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed a single step fluorogenic probe KTlip, which reports delipoylation activity in a continuous manner. The results showed that Sirt2 led to significant delipoylation of KTlip, displaying up to 60 fold fluorescence increase in the assay. Further kinetic experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT PDH, K259) with a remarkable catalytic efficiency (kcat/Km) of 3.26 x 103 s 1M 1. The activity is about 400 fold higher than that of Sirt4, the only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demonstrated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lipoylation in biology and diseases.
Journal of the American Chemical Society