Showing papers on "Lactoylglutathione lyase published in 2010"

Hyperglycemia-Induced Reactive Oxygen Species Increase Expression of the Receptor for Advanced Glycation End Products (RAGE) and RAGE Ligands

Abstract: OBJECTIVE RAGE interacts with the endogenous ligands S100 calgranulins and high mobility group box 1 (HMGB1) to induce inflammation. Since hyperglycemia-induced reactive oxygen species (ROS) activate many pathways of diabetic tissue damage, the effect of these ROS on RAGE and RAGE ligand expression was evaluated. RESEARCH DESIGN AND METHODS Expression of RAGE, S100A8, S100A12, and HMGB1 was evaluated in human aortic endothelial cells (HAECs) incubated in normal glucose, high glucose, and high glucose after overexpression of either uncoupling protein 1 (UCP1), superoxide dismutase 2 (SOD2), or glyoxalase 1 (GLO1). Expression was also evaluated in normal glucose after knockdown of GLO1. Expression was next evaluated in high glucose after knockdown of nuclear factor (NF)-κB p65 (RAGE) and after knockdown of activated protein-1 (AP-1) (S100A8, S100A12, and HMGB1), and chromatin immunoprecipitation (ChIP) was performed ± GLO1 overexpression for NFκB p65 (RAGE promoter) and AP-1 (S100A8, S100A12, and HMGB1 promoters). Finally, endothelial cells from nondiabetic mice, STZ diabetic mice, and STZ diabetic mice treated with the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) were evaluated. RESULTS High glucose increased RAGE, S100A8, S100A12, and HMGB1 expression, which was normalized by overexpression of UCP1, SOD2, or GLO1. GLO1 knockdown mimicked the effect of high glucose, and in high glucose, overexpression of GLO1 normalized increased binding of NFκB p65 and AP-1. Diabetes increased RAGE, S100A8, and HMGB1 expression, and MnTBAP treatment normalized this. CONCLUSIONS These results show that hyperglycemia-induced ROS production increases expression of RAGE and RAGE ligands. This effect is mediated by ROS-induced methylglyoxal, the major substrate of glyoxalase 1.

Up-regulation of antioxidant and glyoxalase systems by exogenous glycinebetaine and proline in mung bean confer tolerance to cadmium stress

Abstract: The present study investigates the possible mediatory role of exogenously applied glycinebetaine (betaine) and proline on reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems in mung bean seedlings subjected to cadmium (Cd) stress (1 mM CdCl2, 48 h). Cadmium stress caused a significant increase in glutathione (GSH) and glutathione disulfide (GSSG) content, while the ascorbate (AsA) content decreased significantly with a sharp increase in hydrogen peroxide (H2O2) and lipid peroxidation level (MDA). Ascorbate peroxidase (APX), glutathione S-transferase (GST), glutathione peroxidase (GPX), and glyoxalase I (Gly I) activities were increased in response to Cd stress, while the activities of catalase (CAT), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and glyoxalase II (Gly II) were sharply decreased. Exogenous application of 5 mM betaine or 5 mM proline resulted in an increase in GSH and AsA content, maintenance of a high GSH/GSSG ratio and increased the activities of APX, DHAR, MDHAR, GR, GST, GPX, CAT, Gly I and Gly II involved in ROS and MG detoxification system as compared to the control and mostly also Cd-stressed plants, with a concomitant decrease in GSSG content, H2O2 and lipid peroxidation level. These findings together with our earlier findings suggest that both betaine and proline provide a protective action against Cd-induced oxidative stress by reducing H2O2 and lipid peroxidation levels and by increasing the antioxidant defense and MG detoxification systems.

Redox homeostasis, antioxidant defense, and methylglyoxal detoxification as markers for salt tolerance in Pokkali rice

Abstract: To identify biochemical markers for salt tolerance, two contrasting cultivars of rice (Oryza sativa L.) differing in salt tolerance were analyzed for various parameters. Pokkali, a salt-tolerant cultivar, showed considerably lower level of H(2)O(2) as compared to IR64, a sensitive cultivar, and such a physiology may be ascribed to the higher activity of enzymes in Pokkali, which either directly or indirectly are involved in the detoxification of H(2)O(2). Enzyme activities and the isoenzyme pattern of antioxidant enzymes also showed higher activity of different types and forms in Pokkali as compared to IR64, suggesting that Pokkali possesses a more efficient antioxidant defense system to cope up with salt-induced oxidative stress. Further, Pokkali exhibited a higher GSH/GSSG ratio along with a higher ratio of reduced ascorbate/oxidized ascorbate as compared to IR64 under NaCl stress. In addition, the activity of methylglyoxal detoxification system (glyoxalase I and II) was significantly higher in Pokkali as compared to IR64. As reduced glutathione is involved in the ascorbate-glutathione pathway as well as in the methylglyoxal detoxification pathway, it may be a point of interaction between these two. Our results suggest that both ascorbate and glutathione homeostasis, modulated also via glyoxalase enzymes, can be considered as biomarkers for salt tolerance in Pokkali rice. In addition, status of reactive oxygen species and oxidative DNA damage can serve as a quick and sensitive biomarker for screening against salt and other abiotic stresses in crop plants.

Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress

Abstract: Aims/hypothesis Impaired nitric oxide (NO)-dependent vasorelaxation plays a key role in the development of diabetic vascular complications. We investigated the effect of hyperglycaemia on impaired vasoreactivity and a putative role therein of the AGE precursor methylglyoxal.

Hyperglycemia Impairs Proteasome Function by Methylglyoxal

Abstract: OBJECTIVE The ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo. RESEARCH DESIGN AND METHODS MGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2 Akita mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice. RESULTS In vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2 Akita , STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-β2 subunit was increased. CONCLUSIONS Hyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions.

Evidence for a role of exogenous glycinebetaine and proline in antioxidant defense and methylglyoxal detoxification systems in mung bean seedlings under salt stress

Abstract: In mung bean seedlings, salt stress (300 mM NaCl) caused a significant increase in reduced glutathione (GSH) content within 24 h of treatment as compared to control whereas a slight increase was observed after 48 h of treatment. Highest oxidized glutathione (GSSG) content was observed after 48 h to treatment with a concomitant decrease in glutathione redox state. Glutathione peroxidase, glutathione S-transferase, and glyoxalase II enzyme activities were significantly elevated up to 48 h, whereas glutathione reductase and glyoxalase I activities were increased only up to 24 h and then gradually decreased. Application of 15 mM proline or 15 mM glycinebetaine resulted in an increase in GSH content, maintenance of a high glutathione redox state and higher activities of glutathione peroxidase, glutathione S-transferase, glutathione reductase, glyoxalase I and glyoxalase II enzymes involved in the ROS and methylglyoxal (MG) detoxification system for up to 48 h, compared to those of the control and mostly also salt stressed plants, with a simultaneous decrease in GSSG content, H2O2 and lipid peroxidation level. The present study suggests that both proline and glycinebetaine provide a protective action against saltinduced oxidative damage by reducing H2O2 and lipid peroxidation level and by enhancing antioxidant defense and MG detoxification systems.

The Chaperone-Dependent Ubiquitin Ligase CHIP Targets HIF-1α for Degradation in the Presence of Methylglyoxal

Abstract: Hypoxia-inducible factor-1 (HIF-1) plays a key role in cell adaptation to low oxygen and stabilization of HIF-1 is vital to ensure cell survival under hypoxia. Diabetes has been associated with impairment of the cell response to hypoxia and downregulation of HIF-1 is most likely the event that transduces hyperglycemia into increased cell death in diabetes-associated hypoxia. In this study, we aimed at identifying the molecular mechanism implicated in destabilization of HIF-1 by high glucose. In this work, we identified a new molecular mechanism whereby methylglyoxal (MGO), which accumulates in high-glucose conditions, led to a rapid proteasome-dependent degradation of HIF-1α under hypoxia. Significantly, MGO-induced degradation of HIF-1α did not require the recruitment of the ubiquitin ligase pVHL nor did it require hydroxylation of the proline residues P402/P564 of HIF-1α. Moreover, we identified CHIP (Carboxy terminus of Hsp70-Interacting Protein) as the E3 ligase that ubiquitinated HIF-1α in the presence of MGO. Consistently, silencing of endogenous CHIP and overexpression of glyoxalase I both stabilized HIF-1α under hypoxia in the presence of MGO. Data shows that increased association of Hsp40/70 with HIF-1α led to recruitment of CHIP, which promoted polyubiquitination and degradation of HIF-1α. Moreover, MGO-induced destabilization of HIF-1α led to a dramatic decrease in HIF-1 transcriptional activity. Altogether, data is consistent with a new pathway for degradation of HIF-1α in response to intracellular accumulation of MGO. Moreover, we suggest that accumulation of MGO is likely to be the link between high glucose and the loss of cell response to hypoxia in diabetes.

Imidazopurinones are markers of physiological genomic damage linked to DNA instability and glyoxalase 1-associated tumour multidrug resistance

Abstract: Glyoxal and methylglyoxal are reactive dicarbonyl metabolites formed and metabolized in physiological systems. Increased exposure to these dicarbonyls is linked to mutagenesis and cytotoxicity and enhanced dicarbonyl metabolism by overexpression of glyoxalase 1 is linked to tumour multidrug resistance in cancer chemotherapy. We report herein that glycation of DNA by glyoxal and methylglyoxal produces a quantitatively important class of nucleotide adduct in physiological systems—imidazopurinones. The adduct derived from methylglyoxal-3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one isomers—was the major quantitative adduct detected in mononuclear leukocytes in vivo and tumour cell lines in vitro. It was linked to frequency of DNA strand breaks and increased markedly during apoptosis induced by a cell permeable glyoxalase 1 inhibitor. Unexpectedly, the DNA content of methylglyoxal-derived imidazopurinone and oxidative marker 7,8-dihydro-8-oxo-2′-deoxyguanosine were increased moderately in glyoxalase 1-linked multidrug resistant tumour cell lines. Together these findings suggest that imidazopurinones are a major type of endogenous DNA damage and glyoxalase 1 overexpression in tumour cells strives to counter increased imidazopurinone formation in tumour cells likely linked to their high glycolytic activity.

Posttranslational modification of human glyoxalase 1 indicates redox-dependent regulation.

Abstract: Background Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic α-oxo-aldehydes into the corresponding α-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown.

GLO1 overexpression in human malignant melanoma.

Abstract: Glyoxalase I [lactoylglutathione lyase (EC 4.4.1.5) encoded by GLO1] is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. Accumulative evidence suggests an important role of GLO1 expression in protection against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. Based on the hypothesis that GLO1 upregulation may play a functional role in glycolytic adaptations of cancer cells, we examined GLO1 expression status in human melanoma tissue. Quantitative RT-PCR analysis of a cDNA tissue array containing 40 human melanoma tissues (stages III and IV) and 13 healthy controls revealed pronounced upregulation of GLO1 expression at the mRNA level. Immunohistochemical analysis of a melanoma tissue microarray confirmed upregulation of glyoxalase 1 protein levels in malignant melanoma tissue versus healthy human skin. Consistent with an essential role of GLO1 in melanoma cell defense against methylglyoxal cytotoxicity, siRNA interference targeting GLO1-expression (siGLO1) sensitized A375 and G361 human metastatic melanoma cells towards the antiproliferative, apoptogenic, and oxidative stress-inducing activity of exogenous methylglyoxal. Protein adduction by methylglyoxal was increased in siGLO1-transfected cells as revealed by immunodetection using a monoclonal antibody directed against the major methylglyoxal-derived epitope argpyrimidine that detected a single band of methylglyoxal-adducted protein in human LOX, G361, and A375 total cell lysates. Using 2D-proteomics followed by mass spectrometry the methylglyoxal-adducted protein was identified as heat shock protein 27 (Hsp27; HSPB1). Taken together, our data suggest a function of GLO1 in the regulation of detoxification and target-adduction by the glycolytic byproduct methylglyoxal in malignant melanoma.

Molecular cloning and characterization of a novel glyoxalase I gene TaGly I in wheat (Triticum aestivum L.)

Abstract: Methylglyoxal is a kind of poisonous metabolite that can react with RNA, DNA and protein, which generally results in a number of side advert effects to cell. Glyoxalase I is a member of glyoxalase system that can detoxify methylglyoxal. An EST encoding a glyoxalase I was isolated from a SSH (suppression subtractive hybridization)-cDNA library of wheat spike inoculated by Fusarium graminearum. The corresponding full length gene, named TaGly I, was cloned, sequenced and characterized. Its genomic sequence consists of 2,719 bp, including seven exons and six introns, and its coding sequence is 929 bp with an open reading frame encoding 291 amino acids. Sequence alignment showed that there were two glyoxalase I domains in the deduced protein sequence. By using specific primers, TaGly I was mapped to chromosome 7D of wheat via a set of durum wheat ‘Langdon’ D-genome disomic-substitution lines. The result of Real-time quantitative polymerase chain reaction demonstrated that TaGly I was induced by the inoculation of Fusarium graminearum in wheat spikes. Additionally, it was also induced by high concentration of NaCl and ZnCl2. When TaGly I was overexpressed in tobacco leaves via Agrobacterium tumefaciens infection, the transgenic tobacco showed stronger tolerance to ZnCl2 stress relative to transgenic control with GFP. The above facts indicated that TaGly I might play a role in response to diverse stresses in plants.

Antiglycation effects of carnosine and other compounds on the long-term survival of Escherichia coli.

Abstract: Glycation, or nonenzymatic glycosylation, is a chemical reaction between reactive carbonyl-containing compounds and biomolecules containing free amino groups. Carbonyl-containing compounds include reducing sugars such as glucose or fructose, carbohydrate-derived compounds such as methylglyoxal and glyoxal, and nonsugars such as polyunsaturated fatty acids. The latter group includes molecules such as proteins, DNA, and amino lipids. Glycation-induced damage to these biomolecules has been shown to be a contributing factor in human disorders such as Alzheimer's disease, atherosclerosis, and cataracts and in diabetic complications. Glycation also affects Escherichia coli under standard laboratory conditions, leading to a decline in bacterial population density and long-term survival. Here we have shown that as E. coli aged in batch culture, the amount of carboxymethyl lysine, an advanced glycation end product, accumulated over time and that this accumulation was affected by the addition of glucose to the culture medium. The addition of excess glucose or methylglyoxal to the culture medium resulted in a dose-dependent loss of cell viability. We have also demonstrated that glyoxylase enzyme GloA plays a role in cell survival during glycation stress. In addition, we have provided evidence that carnosine, folic acid, and aminoguanidine inhibit glycation in prokaryotes. These agents may also prove to be beneficial to eukaryotes since the chemical processes of glycation are similar in these two domains of life.

Metabolic engineering of glyoxalase pathway for enhancing stress tolerance in plants.

Abstract: Glyoxalase system consists of two enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I detoxifies methylglyoxal (MG), a cytotoxic byproduct of glycolysis, to S-lactoylglutathione (SLG) where it uses one molecule of reduced glutathione. Subsequently, SLG is converted to lactate by Gly II and one molecule of reduced glutathione is recycled back into the system. The level of MG, which is produced ubiquitously in all living organisms, is enhanced upon exposure to different abiotic stresses in plants. Overexpression of glyoxalase pathway genes in transgenic plants has been found to keep a check on the MG level under stress conditions, regulate glutathione homeostasis, and the transgenic plants are able to survive and grow under various abiotic stresses.

The critical role of S-lactoylglutathione formation during methylglyoxal detoxification in Escherichia coli

Abstract: Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.

Distinct subcellular localization in the cytosol and apicoplast, unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases

Abstract: Summary The ubiquitous glyoxalase system removes methylglyoxal as a harmful by-product of glycolysis Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways Using a variety of methods, cGloII was found to exist in a monomer–dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance The compounds methyl-gerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P falciparum GloI Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P falciparum

Modulation of the glyoxalase system in the aging model Podospora anserina: effects on growth and lifespan.

Abstract: The eukaryotic glyoxalase system consists of two enzymatic components, glyoxalase I (lactoylglutathione lyase) and glyoxalase II (hydroxyacylglutathione hydrolase). These enzymes are dedicated to the removal of toxic α-oxoaldehydes like methylglyoxal (MG). MG is formed as a by-product of glycolysis and MG toxicity results from its damaging capability leading to modifications of proteins, lipids and nucleic acids. An efficient removal of MG appears to be essential to ensure cellular functionality and viability. Here we study the effects of the genetic modulation of genes encoding the components of the glyoxalase system in the filamentous ascomycete and aging model Podospora anserina. Overexpression of PaGlo1 leads to a lifespan reduction on glucose rich medium, probably due to depletion of reduced glutathione. Deletion of PaGlo1 leads to hypersensitivity against MG added to the growth medium. A beneficial effect on lifespan is observed when both PaGlo1 and PaGlo2 are overexpressed and the corresponding strains are grown on media containing increased glucose concentrations. Notably, the double mutant has a ‘healthy’ phenotype without physiological impairments. Moreover, PaGlo1/PaGlo2_OEx strains are not long-lived on media containing standard glucose concentrations suggesting a tight correlation between the efficiency and capacity to remove MG within the cell, the level of available glucose and lifespan. Overall, our results identify the up-regulation of both components of the glyoxalase system as an effective intervention to increase lifespan in P. anserina.

Glycolaldehyde induces oxidative stress in the heart: a clue to diabetic cardiomyopathy?

Abstract: Cardiovascular complications account for 80% of the mortality related to diabetes mellitus. Hyperglycemia is believed to be the major culprit of angiopathy and cardiomyopathy. High glucose levels and oxidative stress cause elevation of Advanced Glycation End-products that are known to contribute to diabetic complications and correlate with many diseases. However, there are few reports describing the effects of glycating agents other than glucose. Here, we aimed to evaluate the effects of glycolaldehyde (GA) on oxidative stress parameters in the heart of Wistar rats. Male Wistar rats received a single injection of GA (10, 50 or 100 mg/Kg) and were sacrificed 6, 12 or 24 h after injection. As indexes of oxidative stress, we quantified protein carbonylation, lipid peroxidation and total reduced thiols. The activities of superoxide dismutase, catalase and glyoxalase I were assayed. Also, the content of N ɛ-(carboxymethyl)lysine (CML) was quantified. Glycolaldehyde induced an imbalance in the redox status, with increased protein carbonylation and lipoperoxidation. Catalase and glyoxalase I had a decrease in their activities. Despite the oxidative stress, we observed no increase in CML content. These results suggest that short-chain aldehydes such as GA might have a significant role in the development of diabetic cardiomyopathy.

The role of glyoxalase system in renal hypoxia.

Abstract: Methylglyoxal (MG), a highly reactive α-oxoaldehyde generated by oxidation of carbohydrate and glycolysis, binds to proteins and forms advanced glycation end products (AGE). MG and MG adducts have been implicated in oxidative stress-related diseases, therefore, MG detoxifying system such as the glyoxalase system (glyoxalase I) also contributes to progression of these diseases. Recent papers have emphasized the pathophysiological effects of MG and the glyoxalase system in acute hypoxic injury, which is associated with acute oxidative stress. We investigated the kinetics of MG level and glyoxalase I activity in renal acute hypoxic injury induced by ischemia-reperfusion (I/R). I/R induced tubulointerstitial injury and the histological changes were associated with a significant decrease in renal glyoxalase I activity and an increase in MG level in the damaged tubular cells. Of note, rats over expressing human glyoxalase I showed amelioration of I/R-induced histological and functional damages and it was associated with a decrease in MG level in the lesion resulting in reduction of oxidative stress and tubular cell apoptosis. In conclusion, glyoxalase I has renoprotective effects in renal hypoxia such as I/R injury via a reduction in cytotoxic MG level in tubular cells.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyoxalase I from Leishmania infantum.

Abstract: Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-d-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized. Two crystal forms were obtained: a cube-shaped form and a rod-shaped form. While the cube-shaped form did not diffract X-rays at all, the rod-­shaped form exhibited diffraction to about 2.0 A resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 A and three dimers of the enzyme per asymmetric unit.

Converting GLX2-1 into an active glyoxalase II

Abstract: Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level S-lactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited a 1.5−2-fold increase in kcat and a decrease in Km as compared to the values exhibited by the double mutant. In contrast, the R246H mutant of GLX2-1 did not exhibit glyoxalase 2 activity. Zn(II)-loaded R246H GLX2-1 enzyme bound 2 equiv of Zn(II), and 1H NMR spectra of the Co(II)-subst...

Troglitazone Reduces Glyoxalase I Protein Expression in Glioma and Potentiates the Effects of Chemotherapeutic Agents

Abstract: Despite resistance of most gliomas to chemotherapy, approximately 2/3 of oligodendrogliomas show sensitivity to such agents. This sensitivity has been associated with deletions on chromosome 1p alone or in combination with 19q. Higher expression of the enzyme glyoxalase I has been found in oligodendrogliomas with chromosome 1p intact compared to those with a deletion. Higher expression of this enzyme is also associated with tumor chemoresistance in other cancers. The present study tested whether the drug troglitazone would make a glioma cell line more sensitive to chemotherapeutic agents. This drug was chosen because it has been shown to decrease glyoxalase I enzyme activity in cells. Treatment with troglitazone decreased expression of glyoxalase I, and potentiated cell death when used in combination with chemotherapeutic agents. This decrease in glyoxalase I protein may be one mechanism by which this potentiation occurs, and troglitazone may be a candidate for use in glioma therapy.