Chemical Probes Reveal Sirt2’s New Function as a Robust “Eraser” of Lysine Lipoylation

Lysine
lipoylation, a highly conserved lysine post-translational
modification, 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 sirtuin 2 (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 a 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 × 103 s–1 M–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.