Glyoxalase II Research Articles

Discovery of a nanomolar glyoxalase-I inhibitor using integrated ligand-based pharmacophore modeling and molecular docking

The glyoxalase system, which is composed of two zinc metalloenzymes, glyoxalase I (GLO-I) and glyoxalase II (GLO-II) and a catalytic amount of GSH, has a pivotal role in the detoxification of cytotoxic methylglyoxal (MG), a glycolytic side product. Recent studies have revealed the overexpression of GLO-I in various cancer types such as breast carcinoma, invasive bladder, colon, prostate, and lung cancers. Consequently, GLO-I has become a validated target for the development of novel anticancer agents. In this study we were aiming to identify potent GLO-I inhibitors as potential candidates for the development of effective anticancer therapeutics using an integrated ligand- and structure-based drug design approach. A set of selective pharmacophore models was generated using an in-house tested set of flavonoids and used in virtual screening of Maybridge and Aldrich databases, collectively containing more than 64,000 compounds. Filtration of retained hits resulted in 362 compounds that were docked into the active site of the GLO-I enzyme. Then, the top 30% of docked compounds were visually inspected and 32 compounds were purchased and biologically evaluated. Five compounds showed good to excellent inhibitory activities with the most active one (compound 14, ID number ST018515) showing an IC50 of 336 nM. A high hit rate of actives implies the success of our approach. The five active compounds with considerable structural diversity were identified as novel leads that can be further optimized towards designing potent GLO-I inhibitors.

GLYI4 Plays A Role in Methylglyoxal Detoxification and Jasmonate-Mediated Stress Responses in Arabidopsis thaliana

Plant hormones play a central role in various physiological functions and in mediating defense responses against (a)biotic stresses. In response to primary metabolism alteration, plants can produce also small molecules such as methylglyoxal (MG), a cytotoxic aldehyde. MG is mostly detoxified by the combined actions of the enzymes glyoxalase I (GLYI) and glyoxalase II (GLYII) that make up the glyoxalase system. Recently, by a genome-wide association study performed in Arabidopsis, we identified GLYI4 as a novel player in the crosstalk between jasmonate (JA) and salicylic acid (SA) hormone pathways. Here, we investigated the impact of GLYI4 knock-down on MG scavenging and on JA pathway. In glyI4 mutant plants, we observed a general stress phenotype, characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness. Accumulation of MG in glyI4 plants led to lower efficiency of the JA pathway, as highlighted by the increased susceptibility of the plants to the pathogenic fungus Plectospherella cucumerina. Moreover, MG accumulation brought about a localization of GLYI4 to the plasma membrane, while MeJA stimulus induced a translocation of the protein into the cytoplasmic compartment. Collectively, the results are consistent with the hypothesis that GLYI4 is a hub in the MG and JA pathways.

Genome-wide analysis of glyoxalase-like gene families in grape (Vitis vinifera L.) and their expression profiling in response to downy mildew infection

BackgroundThe glyoxalase system usually comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII). This system converts cytotoxic methylglyoxal (MG) into non-toxic D-lactate in the presence of reduced glutathione (GSH) in two enzymatic steps. Recently, a novel type of glyoxalase III (GLYIII) activity has observed in Escherichia coli that can detoxify MG into D-lactate directly, in one step, without a cofactor. Investigation of the glyoxalase enzymes of a number of plant species shows the importance of their roles in response both to abiotic and to biotic stresses. Until now, glyoxalase gene families have been identified in the genomes of four plants, Arabidopsis, Oryza sativa, Glycine max and Medicago truncatula but no similar study has been done with the grapevine Vitis vinifera L.ResultsIn this study, four GLYI-like, two GLYII-like and three GLYIII-like genes are identified from the genome database of grape. All these genes were analysed in detail, including their chromosomal locations, phylogenetic relationships, exon-intron distributions, protein domain organisations and the presence of conserved binding sites. Using quantitative real-time PCR analysis (qRT-PCR), the expression profiles of these genes were analysed in different tissues of grape, and also when under infection stress from downy mildew (Plasmopara viticola). The study reveals that most VvGLY-like genes had higher expressions in stem, leaf, tendril and ovule but lower expressions in the flower. In addition, most of the VvGLY-like gene members were P. viticola responsive with high expressions 6-12 h and 96-120 h after inoculation. However, VvGLYI-like1 was highly expressed 48 h after inoculation, similar to VvPR1 and VvNPR1 which are involved in the defence response.ConclusionsThis study identified the GLYI-like, GLYII-like and GLYIII-like full gene families of the grapevine. Based on a phylogenetic analysis and the presence of conserved binding sites, we speculate that these glyoxalase-like genes in grape encode active glyoxalases. Moreover, our study provides a basis for discussing the roles of VvGLYI-like, VvGLYII-like and VvGLYIII-like genes in grape’s response to downy mildew infection. Our results shed light on the selection of candidate genes for downy mildew tolerance in grape and lay the foundation for further functional investigations of these glyoxalase genes.

Combination of pharmacophore modeling and 3D-QSAR analysis of potential glyoxalase-I inhibitors as anticancer agents

Glyoxalase system is an ubiquitous system in human cells which has been examined thoroughly for its role in different diseases. It comprises two enzymes; Glyoxalase I (Glo-I) and Glyoxalase II (Glo-II) which perform detoxifying endogenous harmful metabolites, mainly methylglyoxal (MG) into non-toxic bystanders. In silico computer Aided Drug Design approaches were used and ninety two diverse pharmacophore models were generated from eighteen Glyoxalase I crystallographic complexes. Subsequent QSAR modeling followed by ROC evaluation identified a single pharmacophore model which was able to predict the expected Glyoxalase I inhibition. Screening of the National Cancer Institute (NCI) database using the optimal pharmacophore Hypo(3VW9) identified several promising hits. Thirty eight hits were successfully predicted then ordered and evaluated in vitro. Seven hits out of the thirty eight tested compounds showed more than 50% inhibition with low micromolar IC50.

GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance

Methylglyoxal(MG) is a potent cytotoxin that is produced as a byproduct of various metabolic reactions in the cell. The major enzymes for MG detoxification are Glyoxalase I(GLYI), Glyoxalase II(GLYII) and D-lactate dehydrogenase(D-LDH). These three enzymes work together and convert MG into D-pyruvate, which directly goes to TCA cycle. Here, a comparative study of the ability of MG detoxification of these three enzymes has been done in both E. coli and yeast. Ectopic expression of these three genes from Arabidopsis in E. coli in presence of different abiotic stress revealed the contribution of each of these genes in detoxifying MG. Yeast mutants of MG detoxification enzymes were also grown in different stress conditions to record the effect of each gene. These mutants were also used for complementation assays using the respective MG detoxifying genes from Arabidopsis in presence of various stress conditions. The MG content and the corresponding growth of cells was measured in all the bacterial as well as yeast strains. This study reveals differential contribution of MG detoxification enzymes in mitigating MG levels and alleviating stress in both prokaryotes as well as eukaryotes. GLYI and D-LDH were found to be key enzymes in MG detoxification under various abiotic stresses.

Genome-wide analysis and expression profiles of glyoxalase gene families in Chinese cabbage (Brassica rapa L).

The glyoxalase pathway is composed of glyoxalase I (GLYI) and glyoxalase II (GLYII) and is responsible for the detoxification of a cytotoxic metabolite methylglyoxal (MG) into the nontoxic S-D-lactoylglutathione. The two glyoxalase enzymes play a crucial role in stress tolerance in various plant species. Recently, the GLY gene families have well been analyzed in Arabidopsis, rice and soybean, however, little is known about them in Chinese cabbage (Brassica rapa). Here, 16 BrGLYI and 15 BrGLYII genes were identified in the B. rapa genome, and the BrGLYI and BrGLYII proteins were both clustered into five subfamilies. The classifications, chromosomal distributions, gene duplications, exon–intron structures, localizations, conserved motifs and promoter cis-elements were also predicted and analyzed. In addition, the expression pattern of these genes in different tissues and their response to biotic and abiotic stresses were analyzed using publicly available data and a quantitative real-time PCR analysis (RT-qPCR). The results indicated that the expression profiles of BrGLY genes varied among different tissues. Notably, a number of BrGLY genes showed responses to biotic and abiotic stress treatments, including Plasmodiophora brassicae infection and various heavy metal stresses. Taken together, this study identifies BrGLYI and BrGLYII gene families in B. rapa and offers insight into their roles in plant development and stress resistance, especially in heavy metal stress tolerance and pathogen resistance.

The Role of Glyoxalase-I (Glo-I), Advanced Glycation Endproducts (AGEs), and Their Receptor (RAGE) in Chronic Liver Disease and Hepatocellular Carcinoma (HCC).

Glyoxalase-I (Glo-I) and glyoxalase-II (Glo-II) comprise the glyoxalase system and are responsible for the detoxification of methylglyoxal (MGO). MGO is formed non-enzymatically as a by-product, mainly in glycolysis, and leads to the formation of advanced glycation endproducts (AGEs). AGEs bind to their receptor, RAGE, and activate intracellular transcription factors, resulting in the production of pro-inflammatory cytokines, oxidative stress, and inflammation. This review will focus on the implication of the Glo-I/AGE/RAGE system in liver injury and hepatocellular carcinoma (HCC). AGEs and RAGE are upregulated in liver fibrosis, and the silencing of RAGE reduced collagen deposition and the tumor growth of HCC. Nevertheless, data relating to Glo-I in fibrosis and cirrhosis are preliminary. Glo-I expression was found to be reduced in early and advanced cirrhosis with a subsequent increase of MGO-levels. On the other hand, pharmacological modulation of Glo-I resulted in the reduced activation of hepatic stellate cells and therefore reduced fibrosis in the CCl4-model of cirrhosis. Thus, current research highlighted the Glo-I/AGE/RAGE system as an interesting therapeutic target in chronic liver diseases. These findings need further elucidation in preclinical and clinical studies.

Genome-Wide Identification of Glyoxalase Genes in Medicago truncatula and Their Expression Profiling in Response to Various Developmental and Environmental Stimuli

Glyoxalase is an evolutionary highly conserved pathway present in all organisms. Conventional glyoxalase pathway has two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII) that act sequentially to detoxify a highly cytotoxic compound methylglyoxal (MG) to D-lactate with the help of reduced glutathione. Recently, proteins with DJ-1/PfpI domain have been reported to perform the same conversion in a single step without the help of any cofactor and thus termed as “unique glyoxalase III” enzyme. Genome-wide analysis of glyoxalase genes have been previously conducted in Arabidopsis, rice and Soybean plants, but no such study was performed for one of the agricultural important model legume species, Medicago truncatula. A comprehensive genome-wide analysis of Medicago identified a total of putative 29 GLYI, 14 GLYII genes, and 5 glyoxalase III (DJ-1) genes. All these identified genes and their corresponding proteins were analyzed in detail including their chromosomal distribution, gene duplication, phylogenetic relationship, and the presence of conserved domain(s). Expression of all these genes was analyzed in different tissues as well as under two devastating abiotic stresses- salinity and drought using publicly available transcript data. This study revealed that MtGLYI-4, MtGLYII-6, and MtDJ-1A are the constitutive members with a high level of expression at all 17 analyzed tissues; while MtGLYI-1, MtGLYI-11, MtGLYI-5, MtGLYI-7, and MtGLYII-13 showed tissue-specific expression. Moreover, most of the genes displayed similar pattern of expression in response to both salinity and drought stress, irrespective of stress duration and tissue type. MtGLYI-8, MtGLYI-11, MtGLYI-6, MtGLYI-16, MtGLYI-21, and MtGLYII-9 showed up-regulation, while MtGLYI-17 and MtGLYI-7/9 showed down-regulation in response to both stresses. Interestingly, MtGLYI-14/15 showed completely opposite pattern of expression in these two stresses. This study provides an initial basis about the physiological significance of glyoxalase genes in plant development and stress response of Medicago that could be explored further.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes.

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni2+- and Zn2+-dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.

Genome-wide analysis and expression profiling of glyoxalase gene families in soybean (Glycine max) indicate their development and abiotic stress specific response.

BackgroundGlyoxalase pathway consists of two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII) which detoxifies a highly cytotoxic metabolite methylglyoxal (MG) to its non-toxic form. MG may form advanced glycation end products with various cellular macro-molecules such as proteins, DNA and RNA; that ultimately lead to their inactivation. Role of glyoxalase enzymes has been extensively investigated in various plant species which showed their crucial role in salinity, drought and heavy metal stress tolerance. Previously genome-wide analysis of glyoxalase genes has been conducted in model plants Arabidopsis and rice, but no such study was performed in any legume species.ResultsIn the present study, a comprehensive genome database analysis of soybean was performed and identified a total of putative 41 GLYI and 23 GLYII proteins encoded by 24 and 12 genes, respectively. Detailed analysis of these identified members was conducted including their nomenclature and classification, chromosomal distribution and duplication, exon-intron organization, and protein domain(s) and motifs identification. Expression profiling of these genes has been performed in different tissues and developmental stages as well as under salinity and drought stresses using publicly available RNAseq and microarray data. The study revealed that GmGLYI-7 and GmGLYII-8 have been expressed intensively in all the developmental stages and tissues; while GmGLYI-6, GmGLYI-9, GmGLYI-20, GmGLYII-5 and GmGLYII-10 were highly abiotic stress responsive members.ConclusionsThe present study identifies the largest family of glyoxalase proteins to date with 41 GmGLYI and 23 GmGLYII members in soybean. Detailed analysis of GmGLYI and GmGLYII genes strongly indicates the genome-wide segmental and tandem duplication of the glyoxalase members. Moreover, this study provides a strong basis about the biological role and function of GmGLYI and GmGLYII members in soybean growth, development and stress physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0773-9) contains supplementary material, which is available to authorized users.

Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.

We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.

Promiscuous metallo-β-lactamases: MIM-1 and MIM-2 may play an essential role in quorum sensing networks

MIM-1 and MIM-2 are two recently identified metallo-β-lactamases (MBLs) from Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Since these organisms are non-pathogenic we speculated that the biological role(s) of MIM-1 and MIM-2 may not be related to their MBL activity. Although both sequence comparison and homology modeling indicate that these proteins are homologous to well-known MBLs such as AIM-1, the sequence analysis also indicated that MIM-1 and MIM-2 share similarities with N-acyl homoserine lactonases (AHLases) and glyoxalase II (GLX-II). Steady-state kinetic assays using a series of lactone substrates confirm that MIM-1 and MIM-2 are efficient lactonases, with catalytic efficiencies resembling those of well-known AHLases. Interestingly, unlike their MBL activity the AHLase activity of MIM-1 and MIM-2 is not dependent on the metal ion composition with Zn(II), Co(II), Cu(II), Mn(II) and Ca(II) all being able to reconstitute catalytic activity (with Co(II) being the most efficient). However, these enzymes do not turn over S-lactoylglutathione, a substrate characteristic for GLX-II activity. Since lactonase activity is linked to the process of quorum sensing the bifunctional activity of “non-pathogenic” MBLs such as MIM-1 and MIM-2 may provide insight into one possible evolutionary pathway for the emergence of antibiotic resistance.

Attractors in Sequence Space: Peptide Morphing by Directed Simulated Evolution.

Certain antimicrobial peptides (AMPs) and mitochondrial targeting peptides (mTPs) share common features such as a positive net charge, helical propensity, and the ability to interact with lipid membranes[1]. While a primary function of AMPs is to disrupt membrane integrity, many mTPs interact with the mitochondrial membranes and the translocator complex, leaving the membranes intact when proteolytically degraded by matrix-located protease(s)[2]. We present a computational approach that may help rationalize the delicate balance of these effects and other sequence-activity relationships. We implemented and applied a directed evolutionary strategy for stepwise peptide morphing of one peptide into another (MoPED, Morphing of Peptides by Evolutionary Design). For the prospective application, we chose cationic peptides as representatives of both peptide classes and converted an AMP (Protonectin, start sequence)[3] into an mTP (target or “attractor” sequence). Novel peptides were generated based on a chemical similarity index (Grantham substitution matrix).[4] The target sequence served as an attractor point for evolutionary exploration of sequence space. We synthesized and tested the individual peptides that were generated during the morphing process. The designed peptides showed systematic loss of membranolytic potential with increasing distance from the start sequence. The results of biophysical and bacterial growth experiments confirmed the applicability of MoPED to designing chemically motivated peptide derivatives. Receiver operating characteristic (ROC) analysis advocates the inducible peptide α-helicity as a semi-quantitative indicator of membranolytic antimicrobial activity.

Weak association of glyoxalase 1 (GLO1) variants with autism spectrum disorder

The prevalence of the autism spectrum disorder (ASD) was recently estimated to 1 in 88 children by the CDC MMWR. In up to 25 % of the cases, the genetic cause can be identified. Past studies identified increased level of advanced glycation end products (AGE) in the brain samples of ASD patients. The methylglyoxal (MG) is one of the main precursors for AGE formation. Humans developed effective mechanism of the MG metabolism involving two enzymes glyoxalase 1 (GLO1) and hydroxyacylglutathione hydrolase (HAGH). Our aim was to analyse genetic variants of GLO1 and HAGH in population of 143 paediatric participants with ASD. We detected 7 genetic variants in GLO1 and 16 variants in HAGH using high-resolution melting (HRM) analysis. A novel association between variant rs1049346 and ASD [OR (allele C)] = 1.5; 95 % CI = 1.1-2.2 and p < 0.05) was identified, and weak association between ASD and variant rs2736654 [OR (allele A)] = 2.2; 95 % CI = 0.99-4.9; p = 0.045) was confirmed. Additionally, a novel genetic variant (GLO1 c.484G > A, p.Ala161Thr) with predicted potentially damaging effect on the activity of the glyoxalase 1 that may contribute to the aetiology of ASD was identified in one participant with ASD. No association between genetic variants of the HAGH gene and ASD was found. Increased level of MG and, consequently, AGEs can induce oxidative stress, mitochondrial dysfunction and inflammation all of which have been implicated to act in the aetiology of the ASD. Our results indicate potential importance of MG metabolism in ASD. However, these results must be interpreted with caution until a causative relation is demonstrated.

Activity, regulation, copy number and function in the glyoxalase system

Molecular, catalytic and structural properties of glyoxalase pathway enzymes of many species are now known. Current research has focused on the regulation of activity and expression of Glo1 (glyoxalase I) and Glo2 (glyoxalase II) and their role in health and disease. Human GLO1 has MRE (metal-response element), IRE (insulin-response element), E2F4 (early gene 2 factor isoform 4), AP-2α (activating enhancer-binding protein 2α) and ARE (antioxidant response-element) regulatory elements and is a hotspot for copy number variation. The human Glo2 gene, HAGH (hydroxyacylglutathione hydrolase), has a regulatory p53-response element. Glo1 is linked to healthy aging, obesity, diabetes and diabetic complications, chronic renal disease, cardiovascular disease, other disorders and multidrug resistance in cancer chemotherapy. Mathematical modelling of the glyoxalase pathway predicts that pharmacological levels of increased Glo1 activity markedly decrease cellular methylglyoxal and related glycation, and pharmacological Glo1 inhibition markedly increases cellular methylglyoxal and related glycation. Glo1 inducers are in development to sustain healthy aging and for treatment of vascular complications of diabetes and other disorders, and cell-permeant Glo1 inhibitors are in development for treatment of multidrug-resistant tumours, malaria and potentially pathogenic bacteria and fungi.

Expression of abiotic stress inducible ETHE1-like protein from rice is higher in roots and is regulated by calcium

ETHYLMALONIC ENCEPHALOPATHY PROTEIN 1 (ETHE1) encodes sulfur dioxygenase (SDO) activity regulating sulfide levels in living organisms. It is an essential gene and mutations in ETHE1 leads to ethylmalonic encephalopathy (EE) in humans and embryo lethality in Arabidopsis. At present, very little is known regarding the role of ETHE1 beyond the context of EE and almost nothing is known about factors affecting its regulation in plant systems. In this study, we have identified, cloned and characterized OsETHE1, a gene encoding ETHE1-like protein from Oryza sativa. ETHE1 proteins in general are most similar to glyoxalase II (GLYII) and hence OsETHE1 has been earlier annotated as OsGLYII1, a putative GLYII gene. Here we show that OsETHE1 lacks GLYII activity and is instead an ETHE1 homolog being localized in mitochondria like its human and Arabidopsis counterparts. We have isolated and analyzed 1618 bp OsETHE1 promoter (pOsETHE1) to examine the factors affecting OsETHE1 expression. For this, transcriptional promoter pOsETHE1: 5-bromo-5-chloro-3-indolyl-β-D-glucuronide (GUS) fusion construct was made and stably transformed into rice. GUS expression pattern of transgenic pOsETHE1:GUS plants reveal a high root-specific expression of OsETHE1. The pOsETHE1 activity was stimulated by Ca(II) and required light for induction. Moreover, pOsETHE1 activity was induced under various abiotic stresses such as heat, salinity and oxidative stress, suggesting a potential role of OsETHE1 in stress response.

The role of glyoxalases for sugar stress and aging, with relevance for dyskinesia, anxiety, dementia and Parkinson's disease

Carbohydrates are the primordial source of energy and carbon for certain unicellular organisms as well as for the complex mammalian brain and its synaptic functions [1]. However, for all these cells the degradation of carbohydrates poses several problems, e.g. the formation of toxic by-products such as the glycating electrophile methylglyoxal (MG, also named 2-oxo-propanal) or the excessive generation of lactic acid with ensuing pH change [2]. Under conditions of high carbon flux and under limited availability of NAD+, e.g. during anaerobic glycolysis [3], the triose phosphates dihydroxyacetone phosphate and glyceraldehyde-3-phosphate spontaneously decompose to MG, a compound known to contribute to the generation of advanced glycation endproducts (AGEs) [4] – with possibly irreversible damage to lipids, nucleic acids and proteins, in particular of mitochondria [5] – and to the induction of ubiquitin conjugates [6]. In a pathway with very high conservation during evolution, all these cells use the enzyme glyoxalase I (lactoylglutathione lyase) in the presence of glutathione (GSH) to convert MG into S-lactoyl-glutathione (SLG) and then use glyoxalase II (hydroxyacylglutathione hydrolase) to liberate D-lactate and glutathione. In spite of this toxicity of MG, some bacteria use the enzyme methylglyoxal synthase to generate MG, apparently to regulate carbon flux and growth rate [7]. Furthermore, the ratio between GSH and SLG in such bacteria modulates potassium efflux and intracellular acidification [8]. Acting as a signal initiator, MG activates the osmosensor Sln1, the HOG-MAP kinase cascade and the calcium(2+) signalling pathway in yeast [9]. In human cells, MG has a well established anti-proliferative effect in cancerous cells with enhanced glycolysis (Warburg-effect) [10, 11]. MG was recently reported to play a physiological role in the modulation of hypoxia-induced-factor-1α (HIF-1alpha) levels and thus in the modulation of the balance between anaerobic and aerobic bioenergetics [12]. Interestingly, an anxiety-suppressing effect of MG infusions into the brain was observed in mice [13]. And in patients with ditary dyskinesias, episodic disorders of spontaneous movement, it is now known that either defects in the cerebral glucose transport or defects in the putative neuronal SLG sensor protein MR-1 can be responsible for these symptoms [14,15]. These observations suggest that MG and SLG are not simply toxic by-products to be eliminated, but might play an important physiological function in bioenergetic signaling.

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

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 modelPodospora 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.

Metal-dependent inhibition of glyoxalase II: A possible mechanism to regulate the enzyme activity

Glyoxalase II (GLX2, EC 3.1.2.6., hydroxyacylglutathione hydrolase) is a metalloenzyme involved in crucial detoxification pathways. Different studies have failed in identifying the native metal ion of this enzyme, which is expressed with iron, zinc and/or manganese. Here we report that GloB, the GLX2 from Salmonella typhimurium, is differentially inhibited by glutathione (a reaction product) depending on the bound metal ion, and we provide a structural model for this inhibition mode. This metal-dependent inhibition was shown to occur in metal-enriched forms of the enzyme, complementing the spectroscopic data. Based on the high levels of free glutathione in the cell, we suggest that the expression of the different metal forms of GLX2 during Salmonella infection could be exploited as a mechanism to regulate the enzyme activity.

The effect of electroaucpuncture for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced proteomic changes in the mouse striatum

Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinson's disease mouse model, we investigated protein expression changes associated with the action of electroacupuncture (EA) in the mouse striatum. Twelve-week-old male C57BL/6 mice were injected intraperitoneally with 30 mg/kg of MPTP at 24-h intervals for 5 days, and the 100-Hz EA stimulation was performed at GB34 and GB39 once a day for 12 days consecutively from the first injection. With the EA, the MPTP-induced dopaminergic neuronal destruction was reduced. Of the 13 proteins that were differentially expressed between control and MPTP treated mice, cytosolic malate dehydrogenase, munc18-1, and hydroxyacylglutathione hydrolase, which were increased by MPTP, and cytochrome c oxidase subunit Vb, which was decreased by MPTP, were restored to the level of the saline group after EA treatment. These proteins are likely related to cellular metabolism. Altogether, we propose that the EA may exert neuroprotective effects in mice striatum through reducing MPTP-induced toxicity such as oxidative stress.