An Azidoribose Probe to Track Ketoamine Adducts in Histone Ribose Glycation

Reactive
cellular metabolites can modify macromolecules and form
adducts known as nonenzymatic covalent modifications (NECMs). The
dissection of the mechanisms, regulation, and consequences of NECMs,
such as glycation, has been challenging due to the complex and often
ambiguous nature of the adducts formed. Specific chemical tools are
required to directly track the formation of these modifications on
key targets in order to uncover their underlying physiological importance.
Here, we present the novel chemoenzymatic synthesis of an active azido-modified
ribose analog, 5-azidoribose (5-AR), as well as the synthesis
of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new
probes we found that, similar to methylglyoxal (MGO) glycation, ribose
glycation specifically accumulates on histones. In addition to fluorescent
labeling, we demonstrate the utility of the probe in enriching modified
targets, which were identified by label-free quantitative proteomics
and high-resolution MS/MS workflows. Finally, we establish that the
known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K),
recognizes and facilitates the removal of 5-AR glycation
adducts in live cells, supporting the dynamic regulation of ribose
glycation as well as validating the probe as a new platform to monitor
FN3K activity. Altogether, we demonstrate this probe’s utilities
to uncover ribose-glycation and deglycation events as well as track
FN3K activity toward establishing its potential as a new cancer vulnerability.