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. 2020 Feb 20;27(2):206-213.e6.
doi: 10.1016/j.chembiol.2019.11.005. Epub 2019 Nov 22.

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes

Affiliations

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes

Dominique O Gaffney et al. Cell Chem Biol. .

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.

Keywords: GLO2; HAGH; glyoxalase; hydroxyacylglutathione hydrolase; lactoyllysine; lactyllysine; methylglyoxal; post-translational modification.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. LactoylLys is generated through a non-enzymatic acyl transfer from LGSH to Lys.
(A) Mechanism of LactoylLys formation. (B) Synthetic standards for CEL-d4 and LactoylLys demonstrate chromatographic separation, allowing for quantitation via MRM-MS. (C) Recombinant histone H4 (2 μg) was treated with either MGO or LGSH (1mM) for 24 h at 37°C. (D-G) QuARKMod was performed to quantify MG-H1, CEA, CEL, and LactoylLys modifications following incubation with either MGO or LGSH, as described in (C). (H) MS/Ms of a LactoylLys modification detected on H4K31 (5.72 ppm mass error).
Figure 2.
Figure 2.. GLO2 regulates GSH and LGSH in cells.
(A) The glyoxalase cycle. (B) Generation of GLO1−/−, GLO2−/−, and GLO1/2−/− HEK293 cells. (C) MGO detoxification is achieved through GLO1. (D) Significant increases in LGSH are observed in GLO2−/− cells, resulting in a concomitant decrease in (E) GSH. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3.
Figure 3.. Reactivity-based protein profiling demonstrates marked elevations in modified proteins in GLO2−/− cells.
(A) Overview of the reactivity-based protein profiling approach performed. (B) Modified proteins are observed in each knockout cell line treated with 50 μM alkMGO for 6 h. (C-F) QuARKMod was performed to determine the composition of MGO-derived PTMs in each cell line in vehicle and 50 μM MGO-treated cells. The absence of GLO1 results in a significant increase in Arg-derived MGO modifications, while ablation of GLO2 leads to a robust increase in LactoylLys modifications. N = 6, *P < 0.05, **P < 0.01, ***P < 0.001. (G-J) Basal levels of MGO-derived PTMs were quantified in tissues collected from WT C57Bl6/J mice (N = 3).
Figure 4.
Figure 4.. LactoylLys modifications are enriched on glycolytic proteins.
(A) Enrichment strategy to identify and quantify protein targets for LactoylLys modifications in cells. (B) 350 proteins were identified using this approach; DAVID analysis was performed on identified proteins to reveal carbon metabolism and glycolysis as heavily enriched pathways in this data set. (C) Proteins identified (red) are mapped to glycolysis, TCA, and pentose phosphate pathways demonstrating clear enrichment for glycolysis. (D) Targeted metabolomics reveals a global reduction in glycolytic metabolites in GLO2−/− cells, which is exacerbated in the presence of MGO (50 μM, 6 h). Data are presented as a Log2 fold-change of an N = 6.

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