Showing papers on "Methylglyoxal published in 2003"

Glyoxalase I – structure, function and a critical role in the enzymatic defence against glycation

Abstract: Glyoxalase I is part of the glyoxalase system present in the cytosol of cells. The glyoxalase system catalyses the conversion of reactive, acyclic alpha-oxoaldehydes into the corresponding alpha-hydroxyacids. Glyoxalase I catalyses the isomerization of the hemithioacetal, formed spontaneously from alpha-oxoaldehyde and GSH, to S -2-hydroxyacylglutathione derivatives [RCOCH(OH)-SG-->RCH(OH)CO-SG], and in so doing decreases the steady-state concentrations of physiological alpha-oxoaldehydes and associated glycation reactions. Physiological substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclic alpha-oxoaldehydes. Human glyoxalase I is a dimeric Zn(2+) metalloenzyme of molecular mass 42 kDa. Glyoxalase I from Escherichia coli is a Ni(2+) metalloenzyme. The crystal structures of human and E. coli glyoxalase I have been determined to 1.7 and 1.5 A resolution. The Zn(2+) site comprises two structurally equivalent residues from each domain--Gln-33A, Glu-99A, His-126B, Glu-172B and two water molecules. The Ni(2+) binding site comprises His-5A, Glu-56A, His-74B, Glu-122B and two water molecules. The catalytic reaction involves base-catalysed shielded-proton transfer from C-1 to C-2 of the hemithioacetal to form an ene-diol intermediate and rapid ketonization to the thioester product. R - and S-enantiomers of the hemithioacetal are bound in the active site, displacing the water molecules in the metal ion primary co-ordination shell. It has been proposed that Glu-172 is the catalytic base for the S-substrate enantiomer and Glu-99 the catalytic base for the R-substrate enantiomer; Glu-172 then reprotonates the ene-diol stereospecifically to form the R-2-hydroxyacylglutathione product. By analogy with the human enzyme, Glu-56 and Glu-122 may be the bases involved in the catalytic mechanism of E. coli glyoxalase I. The suppression of alpha-oxoaldehyde-mediated glycation by glyoxalase I is particularly important in diabetes and uraemia, where alpha-oxoaldehyde concentrations are increased. Decreased glyoxalase I activity in situ due to the aging process and oxidative stress results in increased glycation and tissue damage. Inhibition of glyoxalase I pharmacologically with specific inhibitors leads to the accumulation of alpha-oxoaldehydes to cytotoxic levels; cell-permeable glyoxalase I inhibitors are antitumour and antimalarial agents. Glyoxalase I has a critical role in the prevention of glycation reactions mediated by methylglyoxal, glyoxal and other alpha-oxoaldehydes in vivo.

Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance

Abstract: The glyoxalase pathway involving glyoxalase I (gly I) and glyoxalase II (gly II) enzymes is required for glutathione-based detoxification of methylglyoxal. We had earlier indicated the potential of gly I as a probable candidate gene in conferring salinity tolerance. We report here that overexpression of gly I+II together confers improved salinity tolerance, thus offering another effective strategy for manipulating stress tolerance in crop plants. We have overexpressed the gly II gene either alone in untransformed plants or with gly I transgenic background. Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. Compared with nontransformants, several independent gly II transgenic lines showed improved capability for tolerating exposure to high methylglyoxal and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and WT plants under salinity stress. Ionic measurements revealed higher accumulation of Na+ and K+ in old leaves and negligible accumulation of Na+ in seeds of transgenic lines as compared with the WT plants. Comparison of various growth parameters and seed production demonstrated that there is hardly any yield penalty in the double transgenics under nonstress conditions and that these plants suffered only 5% loss in total productivity when grown in 200 mM NaCl. These findings establish the potential of manipulation of the glyoxalase pathway for increased salinity tolerance without affecting yield in crop plants.

Glyoxal and Methylglyoxal Levels in Diabetic Patients: Quantitative Determination by a New GC/MS Method

Abstract: Determination of glyoxal and methylglyoxal levels in plasma is of great interest, since it allows us to evaluate oxidation processes occurring in glycated proteins. A method based on a simple derivatization procedure followed by gas chromatography/mass spectrometry (GC/MS) analysis has been developed. Ten diabetic patients were evaluated before and after improvement of glycemic control. Fasting plasma glucose, hemoglobin A1c (HbA1c), advanced glycation end products (AGE), pentosidine, glyoxal and methylglyoxal levels were measured. The percentage decreases of the levels of fasting plasma glucose, HbA1c and AGE were larger than those of pentosidine, glyoxal and methylglyoxal. These results may be explained by considering the different position of these compounds in the Maillard reaction pathways: these two sets of metabolic parameters give different pictures of patients' metabolic control. The measurement of glyoxal and methylglyoxal may be particularly important in the evaluation of the possible effect of oxidative stress. Other metabolic pathways can contribute to glyoxal production, and the observed minor decrease in these compounds can be, in principle, ascribed to such effect. However, a similar behavior of pentosidine indicates that these alternative pathways can be only partially responsible for glyoxal and methylglyoxal production.

Methylglyoxal-Derived Hydroimidazolone Advanced Glycation End-Products of Human Lens Proteins

Abstract: Purpose To determine the concentrations of methylglyoxal-derived advanced glycation end-products (AGEs), the hydroimidazolones MG-H1 and -H2, in soluble human lens proteins and compare them with the concentrations of other methylglyoxal-derived AGEs and pentosidine. Methods Lens protein samples were hydrolyzed enzymatically. AGEs were assayed without derivatization by HPLC with tandem mass spectrometry; the fluorescent AGEs argpyrimidine and pentosidine were assayed by fluorometric detection. MG-H1 and -H2 were resolved and assayed by fluorometric detection after derivatization with 6-aminoquinolyl-N-hydroxysuccimidylcarbamate (AQC). Results The methylglyoxal-derived hydroimidazolones MG-H1 and -H2 were detected and quantified in human lens proteins. AGE concentrations (mean +/- SEM) were: MG-H1 4609 +/- 411 pmol/mg protein, MG-H2 3085 +/- 328 pmol/mg protein, argpyrimidine 205 +/- 19 pmol/mg protein, and pentosidine 0.693 +/- 0.104 pmol/mg protein. The concentration of MG-H1 in human lens protein correlated positively with donor age (correlation coefficient = 0.28, P Conclusions Methylglyoxal hydroimidazolones are quantitatively major AGEs of human lens proteins. These substantial modifications of lens proteins may stimulate further glycation, oxidation, and protein aggregation leading to the formation of cataract.

Bacterial production of methylglyoxal: a survival strategy or death by misadventure?

Abstract: The production of MG (methylglyoxal) in bacterial cells must be maintained in balance with the capacity for detoxification and protection against this electrophile Excessive production of MG leads to cell death Survival of exposure to MG is best understood in the Gram-negative bacteria The major mechanism of protection is the spontaneous reaction of MG with GSH to form hemithiolacetal, followed by detoxification by the glyoxalase system leading to the production of D-lactate The KefB and KefC glutathione-gated K(+) efflux systems are integrated with the activity of the glyoxalase system to regulate the cytoplasmic pH in response to exposure to electrophiles Bacteria only produce MG when an imbalance occurs in metabolism Operation of the MG bypass enables cells to adapt, such that balance is restored to metabolism Excessive production of MG is an adaptive ploy, which, if it fails, has fatal consequences On this basis one might define MG-induced loss of life as "death by misadventure" rather than suicide!

Glutathione--functions and metabolism in the malarial parasite Plasmodium falciparum.

Abstract: When present as a trophozoite in human erythrocytes, the malarial parasite Plasmodium falciparum exhibits an intense glutathione metabolism. Glutathione plays a role not only in antioxidative defense and in maintaining the reducing environment of the cytosol. Many of the known glutathione-dependent processes are directly related to the specific lifestyle of the parasite. Reduced glutathione (GSH) supports rapid cell growth by providing electrons for deoxyribonucleotide synthesis and it takes part in detoxifying heme, a product of hemoglobin digestion. Free radicals generated in the parasite can be scavenged in reaction sequences involving the thiyl radical GS . as well as the thiolate GS-. As a substrate of glutathione S-transferase, glutathione is conjugated to nondegradable compounds including antimalarial drugs. Furthermore, it is the coenzyme of the glyoxalase system which detoxifies methylglyoxal, a byproduct of the intense glycolysis taking place in the trophozoite. Proteins involved in GSH-dependent processes include glutathione reductase, glutaredoxins, glyoxalase I and II, glutathione S-transferases, and thioredoxins. These proteins, as well as the ATP-dependent enzymes of glutathione synthesis, are studied as factors in the pathophysiology of malaria but also as potential drug targets. Methylene blue, an inhibitor of the structurally known P. falciparum glutathione reductase, appears to be a promising antimalarial medication when given in combination with chloroquine.

Protecting the genome: defence against nucleotide glycation and emerging role of glyoxalase I overexpression in multidrug resistance in cancer chemotherapy.

Abstract: Glycation of nucleotides in DNA forms AGEs (advanced glycation end-products). Nucleotide AGEs are: the imidazopurinone derivative dG-G [3-(2'-deoxyribosyl)-6,7-dihydro-6,7-dihydroxyimidazo[2,3-b]purin-9(8)one], CMdG ( N (2)-carboxymethyldeoxyguanosine) and gdC (5-glycolyldeoxycytidine) derived from glyoxal, dG-MG [6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one], dG-MG(2) [ N (2),7-bis-(1-hydroxy-2-oxopropyl)deoxyguanosine] and CEdG [ N (2)-(1-carboxyethyl)deoxyguanosine] derived from methylglyoxal, and dG-3DG [ N (2)-(1-oxo-2,4,5,6-tetrahydroxyhexyl)deoxyguanosine] derived from 3-deoxyglucosone and others. Glyoxal and methylglyoxal induce multi-base deletions, and base-pair substitutions - mostly occurring at G:C sites with G:C-->C:G and G:C-->T:A transversions. Suppression of nucleotide glycation by glyoxalase I and aldehyde reductases and dehydrogenases, and base excision repair, protects and recovers DNA from damaging glycation. The effects of DNA glycation may be most marked in diabetes and uraemia. Mutations arising from DNA glycation may explain the link of non-dietary carbohydrate intake to incidence of colorectal cancer. Overexpression of glyoxalase I was found in drug-resistant tumour cells and may be an example of an undesirable effect of the enzymatic protection against DNA glycation. Experimental overexpression of glyoxalase I conferred resistance to drug-induced apoptosis. Glyoxalase I-mediated drug resistance was found in human leukaemia and lung carcinoma cells. Methylglyoxal-mediated glycation of DNA may contribute to the cytotoxicity of some antitumour agents as a consequence of depletion of NAD(+) by poly(ADP-ribose) polymerase, marked increases in triosephosphate concentration and increased formation of methylglyoxal. S - p -Bromobenzylglutathione cyclopentyl diester is a cell-permeable glyoxalase I inhibitor. It countered drug resistance and was a potent antitumour agent against lung and prostate carcinoma. Glyoxalase I overexpression was also found in invasive ovarian cancer and breast cancer.

α-Oxoaldehyde metabolism and diabetic complications

Abstract: The factors responsible for variable susceptibility to diabetic nephropathy are not clear. According to the non-enzymatic glycation hypothesis, diabetes-related tissue damage occurs due to a complex mixture of toxic products, including alpha-oxoaldehydes, which are inherently toxic as well as serving as precursors for advanced glycation end-products. Protective mechanisms exist to control this unavoidable glycation, and these are determined by genetic or environmental factors that can regulate the concentrations of the reactive sugars or end-products. In diabetes these protective mechanisms become more important, since glycation stress increases, and less efficient defence systems against this stress could lead to diabetic complications. Some of these enzymatic control mechanisms, including those that regulate alpha-oxoaldehydes, have been identified. We have observed significant increases in production of the alpha-oxoaldehydes methylglyoxal and 3-deoxyglucosone in three human populations with biopsy-proven progression of nephropathy. The increase in methylglyoxal could be secondary to defects in downstream glycolytic enzymes (such as glyceraldehyde-3-phosphate dehydrogenase) that regulate its production, or in detoxification mechanisms such as glyoxalase. Other mechanisms, however, appear to be responsible for the observed increase in 3-deoxyglucosone levels. We present results of our studies on the mechanisms responsible for variable production of alpha-oxoaldehydes by measuring the activity and characteristics of these enzymes in cells from complication-prone and -resistant diabetic patients. New therapeutic interventions designed to control these endogenous mechanisms could potentially enhance protection against excessive glycation and prevent or reverse complications of long-term diabetes.

DNA damage by carbonyl stress in human skin cells.

Abstract: Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N e -(carboxymethyl)- l -lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger d -penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA–protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

Role of methylglyoxal adducts in the development of vascular complications in diabetes mellitus

Abstract: Various theories have been proposed to explain the hyperglycaemia-induced pathogenesis of vascular complications of diabetes, including detrimental effects of AGEs (advanced glycation end products) on vascular tissues. Increased formation of the very reactive dicarbonyl compound MGO (methylglyoxal), one of the side-products of glycolysis, and MGO-derived AGEs seem to be implicated in the development of diabetic vascular complications. Although the exact role of MGO and MGO adducts in the development of vascular complications is unknown, receptor-mediated activation of vascular cells by the MGO–arginine adduct hydroimidazolone, as well as intracellular modifications of protein by MGO, seem to be involved. The aim of this mini-review is to assess to what extent MGO is related to vascular complications in diabetes.

Enhancement of chaperone function of alpha-crystallin by methylglyoxal modification.

Abstract: The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.

Identification of the binding site of methylglyoxal on glutathione peroxidase: methylglyoxal inhibits glutathione peroxidase activity via binding to glutathione binding sites Arg 184 and 185.

Abstract: Methylglyoxal (MG), a physiological alpha-dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272, 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.

The enzymatic defence against glycation in health, disease and therapeutics: a symposium to examine the concept.

Abstract: Glycation of proteins, nucleotides and basic phospholipids by glucose, glyoxal, methylglyoxal, 3-deoxyglucosone and other saccharide derivatives is potentially damaging to the proteome and mutagenic. It is now recognized that there is an enzymatic defence against glycation – a group of enzymes that suppress the physiological levels of potent glycating agents and repair glycated proteins: glyoxalase I, aldehyde reductases and dehydrogenases, amadoriase and fructosamine 3-phosphokinase. The enzymatic defence against glycation influences morbidity and the efficiency of drug therapy in certain diseases. Improved understanding of the balance between glycation and the enzymatic anti-glycation defence will advance disease diagnosis and therapy.

Modification and inactivation of human Cu,Zn-superoxide dismutase by methylglyoxal.

Abstract: Methylglyoxal (MG) has been identified as an intermediate in non-enzymatic glycation, and increased levels have been reported in patients with diabetes. In this study, the effect of MG on the structure and function of human Cu,Zn-superoxide dismutase (SOD) was investigated. MG modifies Cu,Zn-SOD, as indicated by the formation of fluorescent products. When Cu, Zn-SOD was incubated with MG, covalent crosslinking of the protein increased progressively. MG-mediated modification of Cu,Zn-SOD led to loss of enzymatic activity and release of copper ions from the protein. Radical scavengers inhibited the crosslinking of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to MG was analyzed, glycine, histidine, lysine, and valine residues were found to be particularly sensitive. It is suggested that oxidative damage to Cu,Zn-SOD by MG may perturb cellular antioxidant defense systems and damage cells. This effect may account, in part, for organ deterioration in diabetes.

Glycation and glycoxidation of low‐density lipoproteins by glucose and low‐molecular mass aldehydes

Abstract: Patients with diabetes mellitus suffer from an increased incidence of complications including cardiovascular disease and cataracts; the mechanisms responsible for this are not fully understood. One characteristic of such complications is an accumulation of advanced glycation end-products formed by the adduction of glucose or species derived from glucose, such as low-molecular mass aldehydes, to proteins. These reactions can be nonoxidative (glycation) or oxidative (glycoxidation) and result in the conversion of low-density lipoproteins (LDL) to a form that is recognized by the scavenger receptors of macrophages. This results in the accumulation of cholesterol and cholesteryl esters within macrophages and the formation of foam cells, a hallmark of atherosclerosis. The nature of the LDL modifications required for cellular recognition and unregulated uptake are poorly understood. We have therefore examined the nature, time course, and extent of LDL modifications induced by glucose and two aldehydes, methylglyoxal and glycolaldehyde. It has been shown that these agents modify Arg, Lys and Trp residues of the apoB protein of LDL, with the extent of modification induced by the two aldehydes being more rapid than with glucose. These processes are rapid and unaffected by low concentrations of copper ions. In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions. No evidence was obtained for the stimulation of lipid or protein oxidation by glucose or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation or direct oxidation of lipid or proteins) only occur to any significant extent at later time points. This ‘carbonyl-stress’ may facilitate the formation of foam cells and the vascular complications of diabetes.

The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli

Abstract: In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh(-) strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh(-) mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal.

Glyoxalase I inhibitors in cancer chemotherapy.

Abstract: Several recent developments suggest that the GSH-dependent glyoxalase enzyme system deserves renewed interest as a potential target for antitumour drug development. This summary focuses on the design and development of new classes of tumoricidal agents that specifically target this elementary detoxification pathway in order to induce elevated concentrations of cytotoxic methylglyoxal in tumour cells. Special emphasis is placed on structure- and mechanism-based inhibitors of GlxI (glyoxalase I), the first enzyme in the pathway. A new class of bivalent transition-state analogues is described that simultaneously bind the active site on each subunit of the homodimeric human GlxI, resulting in K (i) values as low as 1 nM. Also described is a new family of bromoacyl esters of GSH that function as active-site-directed irreversible inhibitors of GlxI. Newer prodrugs for delivering the GSH-based inhibitors into tumour cells include reactive sulphoxide esters that undergo acyl exchange with endogenous GSH to give the inhibitors, and polymethacrylamide esters of the inhibitors that are potentially tumour-selective on the basis of the "enhanced permeability and retention effect". Finally, a preliminary evaluation of the efficacy of selected GlxI inhibitors in tumour-bearing mice is given.

Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast Saccharomyces cerevisiae

Abstract: Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH-dependent) is encoded by GRE2 (YOL151w). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI-TOF the proteins in candidate bands on SDS-PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione-S-transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae, by our estimate).

Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes. Formation of modified and oxidized particles.

Abstract: Patients with diabetes mellitus suffer from an increased incidence of complications including cardiovascular disease and cataracts; the mechanisms responsible for this are not fully understood. One characteristic of such complications is an accumulation of advanced glycation end-products formed by the adduction of glucose or species derived from glucose, such as low-molecular mass aldehydes, to proteins. These reactions can be nonoxidative (glycation) or oxidative (glycoxidation) and result in the conversion of low-density lipoproteins (LDL) to a form that is recognized by the scavenger receptors of macrophages. This results in the accumulation of cholesterol and cholesteryl esters within macrophages and the formation of foam cells, a hallmark of atherosclerosis. The nature of the LDL modifications required for cellular recognition and unregulated uptake are poorly understood. We have therefore examined the nature, time course, and extent of LDL modifications induced by glucose and two aldehydes, methylglyoxal and glycolaldehyde. It has been shown that these agents modify Arg, Lys and Trp residues of the apoB protein of LDL, with the extent of modification induced by the two aldehydes being more rapid than with glucose. These processes are rapid and unaffected by low concentrations of copper ions. In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions. No evidence was obtained for the stimulation of lipid or protein oxidation by glucose or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation or direct oxidation of lipid or proteins) only occur to any significant extent at later time points. This 'carbonyl-stress' may facilitate the formation of foam cells and the vascular complications of diabetes.

Enhancement of Chaperone Function of Alpha-crystallin by Methylglyoxal

Abstract: The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.

A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity.

Abstract: A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel β-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The Km for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.