Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes.

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.

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cells in vitro.

Glyoxalase 1 (Glo1) is a cytoplasmic enzyme with a cytoprotective function linked to metabolism of the cytotoxic side product of glycolysis, methylglyoxal (MG). It prevents dicarbonyl stress - the abnormal accumulation of reactive dicarbonyl metabolites, increasing protein and DNA damage. Increased Glo1 expression delays ageing and suppresses carcinogenesis, insulin resistance, cardiovascular disease and vascular complications of diabetes and renal failure. Surprisingly, gene trapping by the International Mouse Knockout Consortium (IMKC) to generate putative Glo1 knockout mice produced a mouse line with the phenotype characterised as normal and healthy. Here, we show that gene trapping mutation was successful, but the presence of Glo1 gene duplication, probably in the embryonic stem cells (ESCs) before gene trapping, maintained wild-type levels of Glo1 expression and activity and sustained the healthy phenotype. In further investigation of the consequences of dicarbonyl stress in ESCs, we found that prolonged exposure of mouse ESCs in culture to high concentrations of MG and/or hypoxia led to low-level increase in Glo1 copy number. In clinical translation, we found a high prevalence of low-level GLO1 copy number increase in renal failure where there is severe dicarbonyl stress. In conclusion, the IMKC Glo1 mutant mouse is not deficient in Glo1 expression through duplication of the Glo1 wild-type allele. Dicarbonyl stress and/or hypoxia induces low-level copy number alternation in ESCs. Similar processes may drive rare GLO1 duplication in health and disease.

A Glyoxalase-1 Knockdown Does Not Have Major Short Term Effects on Energy Expenditure and Atherosclerosis in Mice.

Objective. Glyoxalase-1 is an enzyme detoxifying methylglyoxal (MG). MG is a potent precursor of advanced glycation endproducts which are regarded to be a key player in micro- and macrovascular damage. Yet, the role of Glo1 in atherosclerosis remains unclear. In this study, the effect of Glo1 on mouse metabolism and atherosclerosis is evaluated. Methods. Glo1 knockdown mice were fed a high fat or a standard diet for 10 weeks. Body weight and composition were investigated by Echo MRI. The PhenoMaster system was used to measure the energy expenditure. To evaluate the impact of Glo1 on atherosclerosis, Glo1(KD) mice were crossed with ApoE-knockout mice and fed a high fat diet for 14 weeks. Results. Glo1 activity was significantly reduced in heart, liver, and kidney lysates derived from Glo1(KD) mice. Yet, there was no increase in methylglyoxal-derived AGEs in all organs analyzed. The Glo1 knockdown did not affect body weight or body composition. Metabolic studies via indirect calorimetry did not show significant effects on energy expenditure. Glo1(KD) mice crossed to ApoE(-/-) mice did not show enhanced formation of atherosclerosis. Conclusion. A Glo1 knockdown does not have major short term effects on the energy expenditure or the formation of atherosclerotic plaques.

A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells.

Methylglyoxal is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Methylglyoxal cytotoxicity appears to occur through cell-cycle arrest but, more often, through induction of apoptosis. In this study we examined whether, and through which molecular mechanism, methylglyoxal affects the growth of poorly aggressive LNCaP and invasive PC3 human prostate cancer cells, where its role has not been exhaustively investigated yet. We demonstrated that methylglyoxal is cytotoxic on LNCaP and PC3 and that such cytotoxicity occurs not via cell proliferation but apoptosis control. Moreover, we demonstrated that methylglyoxal cytotoxicity, potentiated by the silencing of its major scavenging enzyme Glyoxalase I, occurred via different apoptotic responses in LNCaP and PC3 cells that also showed a different susceptibility to this metabolite. Finally, we showed that the observed methylglyoxal apoptogenic role involved different molecular pathways, specifically mediated by methylglyoxal or methylglyoxal-derived argpyrimidine intracellular accumulation and NF-kB signaling-pathway. In particular, in LNCaP cells, methylglyoxal, through the accumulation of argpyrimidine, desensitized the key cell survival NF-kB signaling pathway, which was consistent with the modulation of NF-kB-regulated genes, triggering a mitochondrial apoptotic pathway. The results suggest that this physiological compound merits investigation as a potential chemo-preventive/-therapeutic agent, in differently aggressive prostate cancers.

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans.

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C. elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan.

Involvement of the detoxifying enzyme lactoylglutathione lyase in Streptococcus mutans aciduricity.

Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Its main virulence factors are acidogenicity and aciduricity, the abilities to produce acid and to survive and grow at low pH, respectively. Metabolic processes are finely regulated following acid exposure in S. mutans. Proteome analysis of S. mutans demonstrated that lactoylglutathione lyase (LGL) was up-regulated during acid challenge. The LGL enzyme catalyzes the conversion of toxic methylglyoxal, derived from glycolysis, to S-D-lactoylglutathione. Methylglyoxal inhibits the growth of cells in all types of organisms. The current study aimed to investigate the relationship between LGL and aciduricity and acidogenicity in S. mutans. An S. mutans isogenic mutant defective in lgl (LGLKO) was created, and its growth kinetics were characterized. Insertional inactivation of lgl resulted in an acid-sensitive phenotype. However, the glycolytic rate at pH 5.0 was greater for LGLKO than for S. mutans UA159 wild-type cells. LGL was involved in the detoxification of methylglyoxal, illustrated by the absence of enzyme activity in LGLKO and the hypersensitivity of LGLKO to methylglyoxal, compared with UA159 (MIC of 3.9 and 15.6 mM, respectively). Transcriptional analysis of lgl conducted by quantitative real-time PCR revealed that lgl was up-regulated (approximately sevenfold) during the exponential growth phase compared with that in the stationary growth phase. Gene expression studies conducted at low pH demonstrated that lgl was induced during acidic growth (approximately 3.5-fold) and following acid adaptation (approximately 2-fold). This study demonstrates that in S. mutans, LGL functions in the detoxification of methylglyoxal, resulting in increased aciduricity.

Glyoxalase I deficiency is associated with an unusual level of advanced glycation end products in a hemodialysis patient.

BACKGROUND: Advanced glycation of proteins and their attendant advanced glycation end products (AGEs) contribute to the complications associated with diabetes mellitus or uremia. Regulatory mechanisms of AGE formation in vivo remain an issue of particular interest. We investigated a role of the glyoxalase detoxification system of precursor reactive carbonyl compounds (RCOs) in the in vivo AGE formation. METHODS: Plasma levels of AGEs [pentosidine and Nepsilon-carboxymethyllysine (CML)], their RCO precursors, d-lactate (the final product resulting from the glyoxalase detoxification pathway), as well as of various compounds known to generate AGE precursors and surrogate markers for oxidative stress (antioxidant enzymes and glutathione), were measured in both hemodialysis (HD) patients and normal subjects. The activity and protein expression of glyoxalase I, an enzyme essential for the detoxification of alpha-oxoaldehydes, in red blood cells (RBC) were also examined. RESULTS: In one 69-year-old lady who had been on hemodialysis (HD) for three years and had suffered from recurrent cardiovascular complications despite the absence of significant risk factors, plasma levels of pentosidine (77.3 +/- 2.4 pmol/mg protein) and CML (330.8 +/- 8.2 pmol/mg protein) were markedly elevated as compared to other HD patients (N = 20: 26.6 +/- 11.8 pmol/mg protein for pentosidine and 224.4 +/- 51.7 pmol/mg protein for CML). The plasma level of RCO precursors for pentosidine and CML was also higher in this patient than in other HD patients. Further investigation disclosed a very low activity in RBC of glyoxalase I (1.5 +/- 0.4 mU/106 RBC), as compared to other HD patients (3.9 +/- 0.6 mU/106 RBC) or normal subjects (4.0 +/- 0.6 mU/106 RBC). The glyoxalase I protein level, assessed in RBC by immunoblot analysis with a specific antibody, was markedly lower than that observed in HD patients and normal subjects. The causes of this deficiency remain unknown. Nucleotide sequencing of the products of reverse transcription-polymerase chain reaction from the patient's mononuclear cells revealed no genetic mutation within the coding region of the glyoxalase I gene. Plasma d-lactate level was also in the lower range (0.18 +/- 0.03 mg/dL) of the values measured in the other HD patients (0.27 +/- 0.09 mg/dL) and normal subjects (0.35 +/- 0.12 mg/dL). The plasma levels of various compounds known to generate AGE precursors (glucose, lipids and ascorbic acid) were either normal or low. The surrogate markers for oxidative stress such as antioxidant enzymes (glutathione peroxidases and superoxide dismutase) and glutathione were all within the range observed in the other HD patients. CONCLUSION: The unusually high levels of AGEs in this patient implicate a deficient glyoxalase detoxification of RCO precursors. The present clinical observation implicates, to our knowledge for the first time, the glyoxalase detoxification system and, in particular, glyoxalase in the actual level of AGEs in a uremic patient.

Alteration of glucose tolerance in mice fed low levels of aflatoxins and with depressed glyoxalase-I activity.

Glyoxalase-I activity plays an important role in glucose metabolism and has been reported to be depressed in mice fed low levels of aflatoxin B1. In the present study examination of glyoxalase-I activity, glucose tolerance and pancreatic beta cell sensitivity was made in mice fed 0.045 ng aflatoxin B1 + 0.450 ng aflatoxin G1/g feed prenatally and for 6 mo after birth. After glucose challenge the ratios between 0-h and 2-h serum glucose levels were significantly higher than controls, indicating an increase in tolerance of glucose in the aflatoxin-fed mice with lower glyoxalase-I activity. Pancreatic beta cell sensitivity to stimulation by tolbutamide was similar in both groups. However, liver malonic dialdehyde was significantly higher in the aflatoxin-fed mice, suggesting that the altered tolerance for glucose in the aflatoxin-fed mice might be a consequence of aflatoxin mediated peroxidative actions in the liver.

Glyoxalase 2 deficiency in the erythrocytes of a horse: 1H NMR studies of enzyme kinetics and transport of S-lactoylglutathione.

In mammalian red blood cells the metabolism of methylglyoxal, and some alpha-ketoaldehydes, takes place via two, generally, highly active enzymes, glyoxalase 1 and 2. The 1H NMR spin-echo spectra of horse erythrocytes, and the various reactants in the glyoxalase system, were characterized as a prelude to obtaining series of spectra in time courses of methylglyoxal metabolism. We characterized the kinetics of the enzyme system in red cells from a normal horse and also from one which had very low activity of glyoxylase 2. The kinetics of the reaction scheme, with methylglyoxal as the starting substrate, were obtained from 1H NMR spectra and analyzed with a computer model of the scheme. The most salient feature of the normal system was the very high feed-forward inhibition (KiHTA = 0.1 microM) of glyoxalase 2 by the hemithioacetal which is the substrate of glyoxalase 1. The glyoxalase-2-deficient red cells were used to test whether S-lactoylglutathione is transported from red cells via the glutathione-S-conjugate transporter; this transport appeared not to occur. Because methylglyoxal is extremely rapidly removed (half-life, approximately 5 min) from normal red cells, it is difficult to assess the effect of this compound on glycolysis but the slow decline evident in the deficient cells allowed a study of the effects on L-lactate production; no effects were apparent.