Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases

Reversible post-translational modifications (PTMs) are
key to establishing
protein–protein and protein–nucleic acid interactions
that govern a majority of the signaling pathways in cells. Sequence-specific
PTMs are catalyzed by transferases, and their removal is carried out
by a class of reverse-acting enzymes termed “detransferases”.
Currently available chemoproteomic approaches have been valuable in
characterizing substrates of transferases. However, proteome-wide
cataloging of the substrates of detransferases is challenging, mostly
due to the loss of the epitope, rendering immunoprecipitation and
activity-based methods ineffective. Herein, we develop a general chemoproteomic
strategy called crosslinking-assisted substrate identification (CASI)
for systematic characterization of cellular targets of detransferases
and successfully apply it to lysine demethylases (KDMs) which catalyze
the removal of methyl groups from lysine sidechain in histones to
modulate gene transcription. By setting up a targeted azido-methylamino
photo-reaction deep inside the active site of KDM4, engineered to
carry p-azido phenylalanine, we reveal a novel “demethylome”
that has escaped the traditional methods. The proteomic survey led
to the identification of a battery of nonhistone substrates of KDM4,
extending the biological footprint of KDM4 beyond its canonical functions
in gene transcription. A notable finding of KDM4A-mediated demethylation
of an evolutionarily conserved lysine residue in eukaryotic translational
initiation factor argues for a much broader role of KDM4A in ribosomal
processes. CASI, representing a substantive departure from earlier
approaches by shifting focus from simple peptide-based probes to employing
full-length photo-activatable demethylases, is poised to be applied
to >400 human detransferases, many of which have remained poorly
understood
due to the lack of knowledge about their cellular targets.