Chemoproteomic Profiling of Signaling Metabolite Fructose-1,6-Bisphosphate Interacting Proteins in Living Cells

Fructose-1,6-bisphosphate (FBP), a cellular endogenous
sugar metabolite
in the glycolytic pathway, has recently been reported to act as a
signaling molecule to regulate various cellular events through the
engagement of important proteins. Though tremendous progress has been
made in identifying specific FBP–protein interactions, the
comprehensive identification of FBP-interacting proteins and their
regulatory mechanisms remains largely unexplored. Here, we describe
a concise synthetic approach for the scalable preparation of a photoaffinity
FBP probe that enables the quantitative chemoproteomic profiling of
FBP–protein interactions based on photoaffinity labeling (PAL)
directly in living cells. Using such a protocol, we captured known
FBP targets including PKM2 and MDH2. Furthermore, among unknown FBP-interacting
proteins, we identified a mitochondrial metabolic enzyme aldehyde
dehydrogenase 2 (ALDH2), against which FBP showed inhibitory activity
and resulted in cellular ROS upregulation accompanied by mitochondrial
fragmentation. Our findings disclosed a new mode of glucose signaling
mediating by the FBP-ALDH2-ROS axis.