ABSTRACT
Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.
Subject(s)
Glutathione/analogs & derivatives , Lysine/metabolism , Thiolester Hydrolases/metabolism , Alkynes/chemistry , Glutathione/chemistry , Glutathione/metabolism , Glycolysis/drug effects , Glycosylation , HEK293 Cells , Humans , Lactoylglutathione Lyase/deficiency , Lactoylglutathione Lyase/genetics , Lactoylglutathione Lyase/metabolism , Pyruvaldehyde/analogs & derivatives , Pyruvaldehyde/chemistry , Pyruvaldehyde/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Thiolester Hydrolases/deficiency , Thiolester Hydrolases/geneticsABSTRACT
Methylglyoxal (MG) is quantitatively the most important precursor to advanced glycation end-products (AGEs), and evidence is accumulating that it is also a causally linked to diabetes and aging related diseases. Living systems primarily reside on the glyoxalase system to detoxify MG into benign d-lactate. The flux to either glycation or detoxification, accordingly, is a key parameter for how well a system handles the ubiquitous glyoxal burden. Furthermore, insight into proteins and in particular their individual modification sites are central to understanding the involvement of MG and AGE in diabetes and aging related diseases. Here, we present a simple method to simultaneously monitor the flux of MG both to d-lactate and to protein AGE formation in a biological sample by employing an alkyne-labeled methylglyoxal probe. We apply the method to blood and plasma to demonstrate the impact of blood cell glyoxalase activity on plasma protein AGE formation. We move on to isolate proteins modified by the MG probe and accordingly can present the first general inventory of more than 100 proteins and 300 binding sites of the methylglyoxal probe on plasma as well as erythrocytic proteins. Some of the data could be validated against a number of in vivo and in vitro targets for advanced glycation previously known from the literature; the majority of proteins and specific sites however were previously unknown and may guide future research into MG and AGE to elucidate how these are functionally linked to diabetic disease and aging.
