Widespread, Reversible Cysteine Modification by Methylglyoxal Regulates Metabolic Enzyme Function

Abstract

Methylglyoxal (MGO), a reactive metabolite byproduct of glucose metabolism, is known to form a variety of posttranslational modifications (PTMs) on nucleophilic amino acids. For example, cysteine, the most nucleophilic proteinogenic amino acid, forms reversible hemithioacetal and stable mercaptomethylimidazole adducts with MGO. The high reactivity of cysteine toward MGO and the rate of formation of such modifications provide the opportunity for mechanisms by which proteins and pathways might rapidly sense and respond to alterations in levels of MGO. This indirect measure of alterations in glycolytic flux would thereby allow disparate cellular processes to dynamically respond to changes in nutrient availability and utilization. Here we report the use of quantitative LC−MS/MS-based chemoproteomic profiling approaches with a cysteine-reactive probe to map the proteome-wide landscape of MGO modification of cysteine residues. This approach led to the identification of many sites of potential functional regulation by MGO. We further characterized the role that such modifications have in a catalytic cysteine residue in a key metabolic enzyme and the resulting effects on cellular metabolism.

Endogenous Cellular Metabolite Methylglyoxal Induces DNA–Protein Cross-Links in Living Cells

“…GAPDH is a moonlighting protein with many cellular functions including glycolysis and regulation of cell survival and apoptosis . In previous studies, GAPDH has been reported to be susceptible to modification by MGO at its catalytic Cys residue and additional Arg and Lys sites, which can inhibit its enzymatic function. − Our mass spectrometry results confirmed that MGO covalently modifies GAPDH at multiple Lys and Arg residues (Table S3 and Figures S8–14). Importantly, GAPDH has been reported to bind telomeric DNA sequences with a K d of 45 nM .…”

“…It should be noted that MGO is known to covalently modify GAPDH, leading to enzymatic inhibition and alteration in isoelectric point. 81,82,84 In neural precursor cells (NPCs), it was found that MGO modification of GAPDH impacted Notch signaling which affected NPC homeostasis. 104 MGOinduced GAPDH−DNA cross-linking may play an additional role in the association between MGO and aging processes linked to telomeres; however, more detailed studies are needed to clarify this hypothesis.…”

“…To confirm our results for cell culture treatments, in vitro experiments utilizing recombinant GAPDH, histone H3.1, and histone H4 were conducted in order to characterize the cross-linking mechanisms and to establish the kinetics of cross-linking. It should be noted that MGO is known to covalently modify GAPDH, leading to enzymatic inhibition and alteration in isoelectric point. ,, In neural precursor cells (NPCs), it was found that MGO modification of GAPDH impacted Notch signaling which affected NPC homeostasis . MGO-induced GAPDH–DNA cross-linking may play an additional role in the association between MGO and aging processes linked to telomeres; however, more detailed studies are needed to clarify this hypothesis. ,− MGO is also known to covalently modify histone proteins, leading to disruption of chromatin assembly and stability. , Breast cancer cells exhibit high levels of MGO-modified histone proteins .…”

Formaldehyde regulates S -adenosylmethionine biosynthesis and one-carbon metabolism

“…1B and table S1). In contrast to the conventional view of FA as an indiscriminate electrophile, the data revealed privileged FA-sensitive cysteine sites, similar to other reactive species such as hydrogen peroxide, nitric oxide, and methylglyoxal ( 27 , 28 ), and provides foundational information on FA-cysteine reactivity across the proteome in a systematic manner. Notably, modified cysteines were found enriched in protein families spanning FA-dependent pathways (e.g., carbon metabolism and glutathione) and one-carbon metabolism (e.g., one-carbon amino acids and folate) rather than demonstrating proteome-wide, indiscriminate reactivity (Fig.…”

PARK7 Catalyzes Stereospecific Detoxification of Methylglyoxal Consistent with Glyoxalase and Not Deglycase Function

“…Methylglyoxal (MGO) is a highly reactive metabolite produced by the degradation of the triose phosphates glyceraldehyde phosphate (GAP) and dihydroxyacetone phosphate (DHAP), which interconvert within glycolysis through the enzyme triosephosphate isomerase. MGO can chemically modify a variety of nucleophilic biomolecules including proteins, nucleic acids, and even metabolites. − The abundance of MGO-modified proteins has been associated with a number of diseases including diabetes, cancer, neurodegeneration, and aging, − with Parkinson’s disease-specific connections potentially being explained by a role for DJ-1 in protecting against accumulation of glycated α-synuclein. , MGO is principally detoxified by the glyoxalase system, which consists of the enzymes GLO1 and GLO2. − MGO reacts with reduced glutathione (GSH) to form a reversible hemithioacetal, which can be converted to lactoyl glutathione (Lac-GSH) by Zn 2+ metalloenzyme GLO1 . GLO2 then may hydrolyze Lac-GSH to recycle the GSH and produce d -lactate …”