PROJECTS
Project 1: Expanding our understanding of methylglyoxal metabolism.
Methylglyoxal (MGO) is generated as a by-product of glycolysis (~0.1% - 0.4% of the total glycolytic flux). Under physiologic conditions, MGO is detoxified by the glyoxalase system, which is comprised of two enzymes (glyoxalase 1, GLO1, and GLO2). When this system is compromised (e.g. diabetes), concentrations of MGO are elevated and it can adduct proteins and DNA, yielding stable, long-lived adducts on Lys, Arg, and deoxyguanosine. As a result, systems are in place to remove these potentially damaging modifications. We have shown that the deglycase, DJ-1 , is capable of removing the intermediate aminocarbinol adduct, yielding the unmodified Arg and lactate. We are interested in fully understanding these enzymatic reactions and elucidating the consequences of protein adduction when these detoxification mechanisms are compromised. Enzymatic reactions are shown in green.
Project 2: Deciphering the impact of methylglyoxal-modified histones on gene expression.
Histones are modified by MGO. (A) Western blotting demonstrates complete knockout of GLO1. (B) GLO1-/- cells lack any measurable GLO1 activity. (C) Western blotting of chromatin fractions using isoform-specific MG-H antibodies (courtesy of Dr. David Spiegel) reveals histones as targets for modification with markedly increased levels observed in GLO1-/- cells treated with electrophile. Due to a lack of measurable MG-H2 protein adduction, chromatin was treated with 5 mM MGO for 6 h (‘Control’) to serve as a positive control for MGO modification. (D,E) RNA-Seq reveals transcripts altered by MGO. Venn diagrams displaying significantly decreased or increased protein coding transcripts in GLO1-/- cells treated with either vehicle, 50 µM MGO, or 500 µM MGO compared with WT vehicle control. WT cells were also analyzed, revealing minimal alterations in gene expression, likely a result of the rapid metabolism of MGO by GLO1. Collectively, these data demonstrate that MGO mediates alterations in gene expression, which may be attributed, in part, to the covalent modification of histones.