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PubMed1998

Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells.

Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

Animals↗

PubMed1995

Free radicals generated during the glycation reaction of amino acids by methylglyoxal. A model study of protein-cross-linked free radicals.

The formation of alpha-dicarbonyl compounds seems to be an important step for cross-linking proteins in the glycation or Maillard reaction. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon alpha-dicarbonyl compound, methylglyoxal, and amino acids. Our results showed that this reaction generated yellow fluorescent products as formed in some glycated proteins. In addition, three types of free radical species were also produced, and their structures were determined by EPR spectroscopy. These free radicals are 1) the cross-linked radical cation, 2) the methylglyoxal radical anion as the counterion, and 3) the superoxide radical anion produced only in the presence of oxygen. The generation of the cross-linked radical cations and the methylglyoxal radical anions does not require metal ions or oxygens. These results indicate that dicarbonyl compounds cross-link free amino groups of protein by forming Schiff bases, which donate electrons directly to dicarbonyl compounds to form the cross-linked radical cations and the methylglyoxal radical anions. Oxygen can accept an electron from the radical anion to generate a superoxide radical anion, which can initiate damaging chain reactions. Time course studies suggest that the cross-linked radical cation is a precursor of yellow fluorescent glycation end products.

Aerobiosis↗

PubMed1996

Protein cross-linking by the Maillard reaction. Isolation, characterization, and in vivo detection of a lysine-lysine cross-link derived from methylglyoxal.

The Maillard reaction, initiated by nonenzymatic glycosylation of amino groups on proteins by reducing sugars, has been studied for its potential role in aging and the complications of diabetes. One of the major consequences of the advanced Maillard reaction in proteins is the formation of covalently cross-linked aggregates. The chemical nature of the cross-linking structures is largely unknown. Recently, methylglyoxal has been shown to be a potential glycating agent in vivo and suggested to be a common intermediate in the Maillard reaction involving glucose. Methylglyoxal can form enzymatically or nonenzymatically from glycolytic intermediates and by retro-aldol cleavage of sugars. Its elevation in tissues in diabetes and its high potency to glycate and cross-link proteins led us to investigate the chemical nature of its advanced Maillard products. Using an approach in which a synthetic model peptide was reacted with methylglyoxal, we isolated and purified a cross-linked peptide dimer. Characterization of this dimer revealed that the peptides are linked through epsilon amino groups of lysine residues. The actual cross-link was shown to be a methylimidazolium, formed from the reaction of two lysines and two methylglyoxal molecules. We have named this cross-link imidazolysine. Imidazolysine was detected in proteins by high performance liquid chromatography using a postcolumn derivatization method. Proteins incubated with methylglyoxal showed a time-dependent formation of imidazolysine. Quantification of imidazolysine in human serum proteins revealed a significant increase (p < 0.05) in diabetic samples (mean +/- S.D., 313.8 +/- 52.7 pmol/mg protein) when compared with normal samples (261.3 +/- 50.4). These values correlated with glycohemoglobin (p < 0.05). These results provide chemical evidence for protein cross-linking by dicarbonyl compounds in vivo.

Blood Proteins↗

PubMed1997

Selective induction of heparin-binding epidermal growth factor-like growth factor by methylglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes.

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2',7'-dichlorofluorescin diacetate. N-Acetyl-L-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC. DL-Buthionine-(S, R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

Acetylcysteine↗

PubMed1997

Identification and phenotypic analysis of two glyoxalase II encoding genes from Saccharomyces cerevisiae, GLO2 and GLO4, and intracellular localization of the corresponding proteins.

We have isolated and characterized two genes coding for the glyoxalase II enzyme from Saccharomyces cerevisiae. The coding region of the GLO2 gene corresponds to a protein with 274 amino acids and a molecular mass of 31,306 daltons. The open reading frame of the GLO4 gene could be translated into a protein with 285 amino acids and a molecular mass of 32,325 daltons. The amino acid sequences of the deduced proteins are 59.1% identical and show high similarities to the sequence of the human glyoxalase II. When grown on either glucose or glycerol as a carbon source, a glo2 glo4 double deletion strain contains no glyoxalase II activity at all and shows no obvious phenotype during vegetative growth. However, this strain showed a similar high sensitivity against exogenous methylglyoxal as compared with a glyoxalase I-deficient strain. Whereas the GLO2 gene is expressed on both glucose and glycerol, the GLO4 gene is only active on glycerol. The active Glo2p protein is localized in the cytoplasm and the active Glo4p in the mitochondrial matrix. Heterologous expression of the full-length GLO2 coding region in Escherichia coli resulted in an active protein. However, to get an active Glo4p protein in E. coli, the putative mitochondrial transit peptide at the N-terminal end had to be removed by shortening the 5' end of the GLO4 open reading frame.

Amino Acid Sequence↗

PubMed1998

Immunological evidence for methylglyoxal-derived modifications in vivo. Determination of antigenic epitopes.

The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino groups of proteins, contributes to the aging of proteins and to complications associated with diabetes. Methylglyoxal (MG) is a 2-oxoaldehyde derived from glycolytic intermediates and produced during the Maillard reaction. We reported previously the formation of a lysine-lysine protein cross-linking structure (imidazolysine) and a fluorescent arginine modification (argpyrimidine) from the Maillard reaction of MG. Here we show that rabbit antibodies to MG-modified ribonuclease A identify proteins modified by the Maillard reaction of glucose, fructose, ribose, glyceraldehyde, glyoxal, ascorbate, and ascorbate oxidation products (dehydroascorbate, 2,3-diketogulonate, L-xylosone, and L-threose) in addition to those modified by MG. The antibody recognized imidazolysine and argpyrimidine and a glyoxal-derived lysine-lysine cross-link. It did not react with Nepsilon-carboxymethyllysine. Incubations with amino acids revealed strongest reactivity with Nalpha-t-butoxycarbonylarginine and MG, and we identified argpyrimidine as one of the epitopes from this incubation mixture. Serum proteins from human diabetics reacted more strongly with the antibody than those from normal individuals, and the levels correlated with glycemic control. Collagen from human corneas contained MG-derived modifications, with those from older subjects containing higher levels of modified proteins than those from younger ones. An immunoaffinity-purified antibody showed higher reactivity with old corneas than with younger ones and localized the antigens primarily within the stromal region of the cornea. These results confirm reported MG-derived modifications in tissue proteins and show that dicarbonyl-mediated protein modification occurs during Maillard reactions in vivo.

Adult↗

PubMed1998

Role of the Maillard reaction in aging of tissue proteins. Advanced glycation end product-dependent increase in imidazolium cross-links in human lens proteins.

Dicarbonyl compounds such as glyoxal and methylglyoxal are reactive dicarbonyl intermediates in the nonenzymatic browning and cross-linking of proteins during the Maillard reaction. We describe here the quantification of glyoxal and methylglyoxal-derived imidazolium cross-links in tissue proteins. The imidazolium salt cross-links, glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), were measured by liquid chromatography/mass spectrometry and were present in lens protein at concentrations of 0. 02-0.2 and 0.1-0.8 mmol/mol of lysine, respectively. The lens concentrations of GOLD and MOLD correlated significantly with one another and also increased with lens age. GOLD and MOLD were present at significantly higher concentrations than the fluorescent cross-links pentosidine and dityrosine, identifying them as major Maillard reaction cross-links in lens proteins. Like the N-carboxy-alkyllysines Nepsilon-(carboxymethyl)lysine and Nepsilon-(carboxyethyl)lysine, these cross-links were also detected at lower concentrations in human skin collagen and increased with age in collagen. The presence of GOLD and MOLD in tissue proteins implicates methylglyoxal and glyoxal, either free or protein-bound, as important precursors of protein cross-links formed during Maillard reactions in vivo during aging and in disease.

Aging↗

PubMed1998

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation.

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with L-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995) J. Biol. Chem. 270, 28228-28233) or with Nalpha-acetyl-L-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochrome c in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free radicals.

Ascorbic Acid↗

PubMed1999

Different foci for the regulation of the activity of the KefB and KefC glutathione-gated K+ efflux systems.

KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization. The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles. By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins. A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC. Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB. Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity. The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB. We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations.

Amino Acid Sequence↗

PubMed1999

Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts.

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation of Nalpha-acetyl-L-arginine with MG at 37 degrees C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (Ndelta-(4-carboxy-4,6-dimethyl-5, 6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine). On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified as Ndelta-(5-methylimidazolon-2-yl)-L-ornithine (5-methylimidazolone), was found to be identical to Ndelta-(5-hydroxy-4, 6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak at m/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the 1H NMR spectrum of this product in d6-Me2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 degrees C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion.

Animals↗

PubMed2000

The role of alpha,beta -dicarbonyl compounds in the toxicity of short chain sugars.

The extent to which sugars serve as targets for superoxide was examined using glycolaldehyde as the simplest sugar and using superoxide dismutase (SOD)-replete and SOD-null strains growing under aerobic and anaerobic conditions. Glycolaldehyde was more toxic to the SOD-null strain than to its SOD-replete parent, and this differential effect was oxygen-dependent. The product, glyoxal, could be trapped in the medium by 1,2-diaminobenzene and assayed as quinoxaline. The SOD-null strain produced more glyoxal and eliminated it more slowly than the SOD-replete parent strain. Glyoxal was approximately 10 times more toxic than glycolaldehyde and was more toxic to the SOD-null strain than to the parental strain. 1,2-Diaminobenzene protected against the toxicity of glycolaldehyde. These Escherichia coli strains contained the glutathione-dependent glyoxalases I and II, as well as the glutathione-independent glyoxalase III. Of these enzymes, glyoxalase III was most abundant, and it was inactivated within the aerobic SOD-null strain and also in extracts when exposed to the flux of superoxide and hydrogen peroxide imposed by the xanthine oxidase reaction. Thus, it appears that short chain sugars are oxidized by superoxide yielding toxic dicarbonyls. Moreover, the defensive glyoxalase III is also inactivated by the oxidative stress imposed by the lack of SOD, thereby exacerbating the deleterious effect of sugar oxidation.

Acetaldehyde↗

PubMed2001

Methylglyoxal enhances cisplatin-induced cytotoxicity by activating protein kinase Cdelta.

The cytotoxic side effects of anti-neoplastic drugs are increased in patients with either type 1 or type 2 diabetes mellitus by a mechanism that is not clearly defined. We report that the circulating glucose metabolite, methylglyoxal (MGO), enhances cisplatin-induced apoptosis by activating protein kinase Cdelta (PKCdelta). We found that treatment of myeloma cells with the antioxidant N-acetylcysteine completely blocked cisplatin-dependent intracellular GSH oxidation, reactive oxygen species (ROS) generation, poly(ADP-ribose) polymerase cleavage, and apoptosis. Importantly, co-treatment of cells with the reactive carbonyl MGO and cisplatin increased apoptosis by 90% over the expected additive effect of combined MGO and cisplatin treatment. This same synergism was also observed when ROS generation was examined. MGO and cisplatin increased PKCdelta activity by 4-fold, and this effect was blocked by the PKCdelta inhibitor rottlerin but not by NAC. Furthermore, rottlerin blocked combined MGO and cisplatin-induced ROS generation and apoptosis. Finally, MGO and cisplatin induced c-Abl activation and c-Abl:PKCdelta association. Rottlerin blocked c-Abl activation, but the c-Abl inhibitor STI-571 increased MGO and cisplatin-induced apoptosis by 50%. Taken together these data indicate that MGO synergistically enhances cisplatin-induced apoptosis through activation of PKCdelta and that PKCdelta is critical to both cell death and cell survival pathways. These findings suggest that in the patient with diabetes mellitus heightened oxidative stress can enhance the cytotoxicity of agents that induce DNA damage.

Acetylcysteine↗

PubMed2002

Dihydroxyacetone kinases in Saccharomyces cerevisiae are involved in detoxification of dihydroxyacetone.

The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting K(m)((DHA)) of 22 and 5 microm and K(m)((ATP)) of 0.5 and 0.1 mm for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1 Delta dak2 Delta), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1 Delta dak2 Delta highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.

Base Sequence↗

PubMed2002

Modulation of heat-shock protein 27 (Hsp27) anti-apoptotic activity by methylglyoxal modification.

Methylglyoxal (MG) is one of the side-products in glycolysis, and it reacts with proteins under physiological conditions. Here, we identified heat-shock protein 27 (Hsp27) as a major MG-modified protein in cells. MG modification of Hsp27 selectively occurs at Arg-188 to form argpyrimidine, and mutation in the residue represses the formation of a large oligomer. This modification process is essential to its repressing activity for cytochrome c-mediated caspase activation. Inhibition of MG modification of Hsp27 causes sensitization of the cells to anti-tumor drug-induced apoptosis. Thus, MG is a novel modulator of cell survival by directly incorporating with the specific protein residue.

Amino Acid Sequence↗

PubMed2004

Methylglyoxal, a metabolite derived from glycolysis, functions as a signal initiator of the high osmolarity glycerol-mitogen-activated protein kinase cascade and calcineurin/Crz1-mediated pathway in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis, although it inhibits the growth of cells in all types of organism. Hence, it has been questioned why such a toxic metabolite is synthesized via the ubiquitous energy-generating pathway. We have previously reported that expression of GLO1, coding for the major enzyme detoxifying MG, was induced by osmotic stress in a high osmolarity glycerol (HOG)-mitogen-activated protein (MAP) kinase-dependent manner in Saccharomyces cerevisiae. Here we show that MG activates the HOG-MAP kinase cascade. Two osmosensors, Sln1 and Sho1, have been identified to function upstream of the HOG-MAP kinase cascade, and we reveal that MG initiates the signal transduction to this MAP kinase cascade through the Sln1 branch. We also demonstrate that MG activates the Msn2 transcription factor. Moreover, MG activated the uptake of Ca(2+) in yeast cells, thereby stimulating the calcineurin/Crz1-mediated Ca(2+) signaling pathway. We propose that MG functions as a signal initiator in yeast.

Glycolysis↗

PubMed2004

Peptide mapping identifies hotspot site of modification in human serum albumin by methylglyoxal involved in ligand binding and esterase activity.

Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone N(delta)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (92% of total modification) with a minor formation of argpyrimidine, N(epsilon)-(1-carboxyethyl)lysine, and methylglyoxal lysine dimer. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-218, and Arg-428. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pK(a) = 7.9 +/- 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pK(a) to 8.8 +/- 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of arginine-directed hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation.

Amino Acid Sequence↗

PubMed2005

The glycolytic metabolite methylglyoxal activates Pap1 and Sty1 stress responses in Schizosaccharomyces pombe.

Methylglyoxal, a toxic metabolite synthesized in vivo during glycolysis, inhibits cell growth. One of the mechanisms protecting eukaryotic cells against its toxicity is the glyoxalase system, composed of glyoxalase I and II (glo1 and glo2), which converts methylglyoxal into d-lactic acid in the presence of glutathione. Here we have shown that the two principal oxidative stress response pathways of Schizosaccharomyces pombe, Sty1 and Pap1, are involved in the response to methylglyoxal toxicity. The mitogen-activated protein kinase Sty1 is phosphorylated and accumulates in the nucleus following methylglyoxal treatment. Moreover, glo2 expression is induced by methylglyoxal and environmental stresses in a Sty1-dependent manner. The transcription factor Pap1 also accumulates in the nucleus, activating the expression of its target genes following methylglyoxal treatment. Our studies showed that the C-terminal cysteine-rich domain of Pap1 is sufficient for methylglyoxal sensing. Furthermore, the redox status of Pap1 is not changed by methylglyoxal. We propose that methylglyoxal treatment triggers Pap1 and Sty1 nuclear accumulation, and we describe the molecular basis of such activation mechanisms. In addition, we discuss the potential physiological significance of these responses to a natural toxic metabolite.

Basic-Leucine Zipper Transcription Factors↗

PubMed2006

Methylglyoxal as a signal initiator for activation of the stress-activated protein kinase cascade in the fission yeast Schizosaccharomyces pombe.

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis. We have recently found that MG activates transcription factors such as Yap1 and Msn2, and triggers a Hog1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae. Regarding the activation of Hog1 by MG, we found that Sln1, an osmosensor possessing histidine kinase activity, functions as a sensor of MG (Maeta, K., Izawa, S., and Inoue, Y. (2005) J. Biol. Chem. 280, 253-260). To gain further insight into the role of MG as a signal initiator, here we analyze the response of Schizosaccharomyces pombe to extracellular MG. Spc1, a stress-activated protein kinase (SAPK), was phosphorylated following the treatment with MG. No phosphorylation was observed in a wis1Delta mutant. The His-to-Asp phosphorelay system consisting of three histidine kinases (Phk1, Phk2, and Phk3), a phosphorelay protein (Spy1), and a response regulator (Mcs4) exists upstream of the Spc1-SAPK pathway. The phosphorylation of Spc1 following MG treatment was observed in phk1Deltaphk2Deltaphk3Delta and spy1Delta cells, but not in mcs4Delta cells. These results suggest that S. pombe has an alternative module(s) that directs the MG signal to the SAPK pathway via Mcs4. Additionally, we found that the transcription factor Pap1 is concentrated in the nucleus in response to MG, independent of the Spc1-SAPK pathway.

Basic-Leucine Zipper Transcription Factors↗