Comparative proteomic analysis of foodborne Salmonella Enteritidis SE86 subjected to cold plasma treatment.

The increasing demand for high quality and safe food led to important technological innovations in food processing. Cold plasma appears as an emerging technology that has demonstrated efficiency in the removal of microbial contamination from fresh and minimally processed food. In this study, the proteomic profile of Salmonella Enteritidis SE86 subjected to cold plasma treatment was investigated. The number of viable S. Enteritidis SE86 cells was analyzed at different time intervals upon exposure to cold plasma and approximately 100 µg of S. Enteritidis SE86 protein extracts were analyzed by Multidimensional Protein Identification Technology (MudPIT). The results demonstrated that no significant changes in cell counts were detected for up to 20 min exposure to cold plasma, and 2 log reduction was achieved after 60 min. Overall, 1096 proteins were identified, with 249 detected only in plasma-treated samples, and 9 exclusive in non-treated control samples. The proteins uniquely detected in cold plasma-treated cells that showed higher abundance were glyoxalase I, ABC transporter substrate-binding protein and transcriptional activator OsmE, followed by some oxidoreductases. Proteins related with carbohydrate and nucleotide metabolism were mostly overexpressed in cold plasma treated cells, suggesting energy metabolism was increased.

Inhibition by active site directed covalent modification of human glyoxalase I.

The glyoxalase pathway is responsible for conversion of cytotoxic methylglyoxal (MG) to d-lactate. MG toxicity arises from its ability to form advanced glycation end products (AGEs) on proteins, lipids and DNA. Studies have shown that inhibitors of glyoxalase I (GLO1), the first enzyme of this pathway, have chemotherapeutic effects both in vitro and in vivo, presumably by increasing intracellular MG concentrations leading to apoptosis and cell death. Here, we present the first molecular inhibitor, 4-bromoacetoxy-1-(S-glutathionyl)-acetoxy butane (4BAB), able to covalently bind to the free sulfhydryl group of Cys60 in the hydrophobic binding pocket adjacent to the enzyme active site and partially inactivate the enzyme. Our data suggests that partial inactivation of homodimeric GLO1 is due to the modification at only one of the enzymatic active sites. Although this molecule may have limited use pharmacologically, it may serve as an important template for the development of new GLO1 inhibitors that may combine this strategy with ones already reported for high affinity GLO1 inhibitors, potentially improving potency and specificity.

Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant.

Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC-MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (-5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome.

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

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 P2(1)2(1)2, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 A and three dimers of the enzyme per asymmetric unit.

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

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.

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

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.

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

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.

Purification of glyoxalase I from onion bulbs and molecular cloning of its cDNA.

Glyoxalase I was highly purified from onion bulbs by DEAE-cellulose, hydroxyapatite, and S-hexylglutathione-agarose column chromatography. With 356 micromol min(-1) mg(-1) protein, the specific enzymatic activity of the purified enzyme is the highest reported to date in plants. The purified enzyme showed a single major band with a relative molecular mass of approximately 25,000 on SDS-PAGE. A cDNA encoding glyoxalase I was cloned and sequenced. Sequence comparison suggested that it is to be classified as a short-type glyoxalase I. The expression pattern of glyoxalase I in healthy onion plants and responses to various stresses were examined by Western blotting. Glyoxalase I exists at high concentration in roots, young bulbs, mature bulbs, and mature leaves, the highest concentration being in mature bulbs. Up-regulation of glyoxalase I and glyoxalase II enzyme activities were observed in response to various stresses, and an increase in Gly I protein was also seen by immunoblotting. Our results suggest an important role of the glyoxalase I gene in the plant abiotic stress response.

Polyproline-rod approach to isolating protein targets of bioactive small molecules: isolation of a new target of indomethacin.

Identification of protein targets of bioactive small molecules has been a technical hurdle of chemical genetics. Here we report a polyproline-rod approach to isolating protein targets of small molecules from cell lysates. The results indicate that insertion of a long, rigid polyproline helix between a small-molecule bait and a biotin tag boosts the capacity of affinity purification and thereby permits isolation of low-abundance or low-affinity proteins. In the course of the proof-of-concept experiments, we isolated glyoxalase 1 (GLO1) as a new target of indomethacin, a widely used antiinflammatory drug. Molecular biological experiments suggest that inhibition of GLO1 enzyme activity is related to the clinically recognized beneficial side effects of the indomethacin family of nonsteroidal antiinflammatory drugs.

Glyoxalase I from Leishmania donovani: a potential target for anti-parasite drug.

Glyoxalases are involved in a ubiquitous detoxification pathway. In pursuit of a better understanding of the biological function of the enzyme, the recombinant glyoxalase I (LdGLOI) protein has been characterized from Leishmania donovani, the most important pathogenic Leishmania species that is responsible for visceral leishmaniasis. A 24kDa protein was heterologously expressed in Escherichia coli. LdGLOI showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOI protein could detect a band of anticipated size approximately 16kDa in promastigote extracts. Several inhibitors of human GLOI showed that they are weak inhibitors of L. donovani growth. Overexpression of GLOI gene in L. donovani using Leishmania expression vector pspalpha hygroalpha, we detected elevated expression of GLOI RNA and protein. Comparative modelling of the 3-D structure of LDGLOI shows that substrate-binding region of the model involves important differences compared to the homologues, such as E. coli, specific to glutathione. Most notably a substrate-binding loop of LDGLOI is characterized by a deletion of five residues compared to the E. coli homologue. Further, a critical Arg in the E. coli variant at the substrate-binding site is replaced by Tyr in LDGLOI. These major differences result in entirely different shapes of the substrate-binding loop and presence of very different chemical groups in the substrate-binding site of LDGLOI compared to E. coli homologue suggesting an explanation for the difference in the substrate specificity. Difference in the substrate specificity of the human and LDGLOI enzyme could be exploited for structure-based drug designing of selective inhibitors against the parasite.

Identification of glyoxalase-I as a protein marker in a mouse model of extremes in trait anxiety.

For >15 generations, CD1 mice have been selectively and bidirectionally bred for either high-anxiety-related behavior (HAB-M) or low-anxiety-related behavior (LAB-M) on the elevated plus-maze. Independent of gender, HAB-M were more anxious than LAB-M animals in a variety of additional tests, including those reflecting risk assessment behaviors and ultrasound vocalization, with unselected CD1 "normal" control (NAB-M) and cross-mated (CM-M) mice displaying intermediate behavioral scores in most cases. Furthermore, in both the forced-swim and tail-suspension tests, LAB-M animals showed lower scores of immobility than did HAB-M and NAB-M animals, indicative of a reduced depression-like behavior. Using proteomic and microarray analyses, glyoxalase-I was identified as a protein marker, which is consistently expressed to a higher extent in LAB-M than in HAB-M mice in several brain areas. The same phenotype-dependent difference was found in red blood cells with NAB-M and CM-M animals showing intermediate expression profiles of glyoxalase-I. Additional studies will examine whether glyoxalase-I has an impact beyond that of a biomarker to predict the genetic predisposition to anxiety- and depression-like behavior.

Identification of thermostable glyoxalase I in the fission yeast Schizosaccharomyces pombe.

Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I ( glo1(+)) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1(+) enhanced susceptibility to methylglyoxal, while expression of glo1(+) in a Delta glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1(+) gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34000 and the k(cat)/ K(m) value for methylglyoxal was 4.3 x 10(7) M(-1) x min(-1). Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn(2+), Co(2+), Ni(2+) and Mn(2+). The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60 degrees C for 4 h.

Purification and partial characterization of glyoxalase I from bovine brain.

Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.

A 33-kDa allergen from rice (Oryza sativa L. Japonica). cDNA cloning, expression, and identification as a novel glyoxalase I.

Cereal proteins are known to cause allergic reactions such as Baker's asthma and severe atopic dermatitis to certain populations. In rice allergy, proteins with molecular masses of 14-16, 26, 33, and 56 kDa have been demonstrated to be potentially allergenic. In this study, to identify and characterize the 33-kDa allergen, designated Glb33, this protein was first purified to homogeneity, and its cDNA clone was isolated. When expressed in Escherichia coli, the recombinant Glb33 was shown to be as reactive as the native Glb33 with mouse IgG and patients' IgE antibodies to Glb33. The Glb33 cDNA coded for a protein of 291 amino acids with two 120-amino acid residue repeats, and the amino acid sequence showed similarity to glyoxalase I from various organisms, including human, plant, yeast, and bacterium. As expected, both native Glb33 purified from rice seeds and the recombinant protein had glyoxalase I activity that catalyzes condensation of methylglyoxal and glutathione into S-lactoylglutathione. However, Glb33 had a higher sequence identity to the bacterial glyoxalase I rather than to known plant and yeast enzymes. Both the Glb33 transcript and the protein were detected not only in maturing seeds of rice but also in its stem and leaf. Taken all together, the rice allergen, Glb33, was identified to be a novel type of plant glyoxalase I that is expressed in various plant tissues, including maturing seeds.

Detection and characterisation of the genes encoding glyoxalase I and II from Neisseria meningitidis.

Glyoxalase enzymes I and II are involved in a detoxification process consisting of conversion of reactive dicarbonyl compounds (e.g., methylglyoxal) to less reactive hydroxy acids. The structural gene for meningococcal glyoxalase I (gloA) was identified by screening an expression library with a rabbit antiserum. The meningococcal gloA gene consisted of 138 deduced amino acids, with a calculated mol. wt of 15.7 kDa. The DNA and deduced protein sequence of gloA was compared to known sequences of glyoxalase I enzymes and showed high homology with gloA of several eukaryotic and prokaryotic species. Insertion of a gloA-containing plasmid in Escherichia coli increased the host organism's tolerance to methylglyoxal from <2 mM to >4 mM, thus demonstrating its functional identity. A databank search also revealed the presence of a putative gloB gene, encoding glyoxalase II (GlxII), in the recently released genomic sequences of Neisseria meningitidis and N. gonorrhoeae.

Glyoxalase I from Brassica juncea is a calmodulin stimulated protein.

Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1. 5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg2+) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca2+/CaM). In the presence of Ca2+ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca2+ dependent mobility shift on denaturing gels. Its Ca2+ binding was confirmed by Chelex-100 assay and gel overlays using 45CaCl2. Glyoxalase I was activated by over 7-fold in the presence of Ca2+ (25 microM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 microM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in the presence of Mg2+ and Ca2+; however, magnesium alone also showed glyoxalase I activation by CaM.

Isolation and sequencing of a gene coding for glyoxalase I activity from Salmonella typhimurium and comparison with other glyoxalase I sequences.

The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn2+ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.

Mutagenesis of residue 157 in the active site of human glyoxalase I.

Met-157 in the active site of human glyoxalase I was changed by site-directed mutagenesis into alanine, glutamine or histidine in order to evaluate its possible role in catalysis. The glyoxalase I mutants were expressed in Escherichia coli and purified on an S-hexylglutathione affinity gel. The physicochemical properties of the mutant proteins were similar to those of the wild-type enzyme. The glutamine mutant exhibited the same high specific activity as wild-type glyoxalase I, whereas the alanine and histidine mutants had approx. 20% of wild-type activity. The kcat/Km values of the mutant glyoxalase I determined with the hemithioacetal adduct of glutathione and methylglyoxal were reduced to between 10 and 40% of the wild-type value. This reduction was due to lower kcat values for the alanine and histidine mutants and a twofold increase in the Km value for the glutamine mutant. With the hemithioacetal of glutathione and phenylglyoxal, the kinetic parameters of the mutants were also of the same magnitude as those of wild-type glyoxalase I. Studies with the competitive inhibitors S-hexyl- and S-benzyl-glutathione revealed that the affinity was reduced to 7-11% of the wild-type affinity for the glutamine and alanine mutants and to 30-40% for the histidine mutant, as measured by a comparison of Ki values. The results show that Met-157 has no direct role in catalysis, but is rather involved in forming the substrate-binding site of human glyoxalase I. The high activity of the glutamine mutant suggests that a structurally equivalent glutamine residue in the N-terminal half of Saccharomyces cerevisiae glyoxalase I may be part of a catalytically competent active site.

Interaction of aldehydes with glyoxalase I and the status of several aldehyde metabolizing enzymes of Ehrlich ascites carcinoma cells.

The possible effect of several physiologically important aldehydes has been tested on partially purified glyoxalase I of Ehrlich ascites carcinoma (EAC) cells. The results indicate that D, and L-lactaldehyde are strong non-competitive inhibitors of glyoxalase I and the effect with the D-isomer is more pronounced, whereas both D,L-glyceraldehyde and acetaldehyde are moderately inhibitory and the nature of inhibition is strictly competitive. Moreover, D,L-glyceraldehyde strongly inhibits the utilization of methylglyoxal by intact EAC cells. A search for the presence of several aldehyde metabolizing enzymes in EAC cells indicates that non-specific aldehyde reductase, methylglyoxal reductase, aldehyde dehydrogenase and alcohol dehydrogenase are apparently absent in this rapidly growing, highly de-differentiated malignant cell.

Analysis of glyoxalase-I from normal and tumor tissue from human colon.

Glyoxalase-I (Gly-I) is part of the glyoxalase system which converts methylglyoxal to D-lactic acid via an S-D-lactoylglutathione intermediate. This glutathione (GSH)-binding protein was purified from human colon tumors and corresponding normal tissue. The GSH-affinity purified fraction from normal human colon tissue showed enzyme activity of 30.6 +/- 11.5 mumol/min per mg protein, with methylglyoxal as substrate. Corresponding fractions from carcinomas showed significantly elevated Gly-I activity of 54.5 +/- 15 mumol/min per mg protein. Polyclonal antibodies made against human Gly-I cross-reacted weakly with mouse liver Gly-I but not with yeast Gly-I. Isoelectric points of Gly-I from human, mouse and yeast were determined to be 4.6, 4.9 and 7.0, respectively, by horizontal IEF. Immunohistochemical analysis confirmed the increase of Gly-I in human colon carcinoma in 16 out of 21 samples when compared to corresponding normal tissue. The elevated levels of Gly-I in colon tumors may be an indicator of the enhanced proliferative status of the neoplastic condition.