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Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Diplomová práce se zaměřila na význam a funkci enzymu Enolázy a její vliv nejen při vzniku nádorových buněk, ale i v rámci boje imunitního systému proti nádorovým buňkám. V práci jsou popsány metody pro průkaz -Enolázy, stanovení její aktivity a také metody pro průkaz protilátek proti -Enoláze. V rámci experimentální části byla popsána a použita metoda sérologická imunoproteomová analýza, která byla použita k průkazu autoprotilátek anti ENO1 IgG v séru pacientů s mnohočetným myelomem. Součástí experimentální části bylo stanovení aktivity enzymu -Enoláza., The diploma thesis deals with the importance and function of the enzyme -Enolase and its influence not only in the formation of tumor cells, but also in the fight of the immune system against tumor cells. The work describes methods for the detection of -Enolase, determination of its activity, as well as methods for the detection of antibodies against -Enolase. As part of the experimental part, the method of serological immunoproteome analysis was described and used to detect anti ENO1 IgG autoantibodies in the serum of patients with multiple myeloma. Part of the experimental part was the determination of the activity of the enzyme -Enolase., Fakulta chemicko-technologická, 1. Prezentace výsledků diplomové práce. 2. Diskuze k posudkům vedoucího a oponenta diplomové práce. 3. Student/ka zodpověděl/la všechny dotazy a připomínky k DP., Dokončená práce s úspěšnou obhajobou

Redox cycling of thiol pairs on proximal cysteine residues in proteins, transitioning between dithiol and disulfide forms, is well established to underlie the catalytic activities of enzymes involved in redox homeostasis and protein folding but has not been widely studied as a means of redox-sensitive regulation of proteins. The Foley research group developed redox phenylarsine oxide-affinity chromatography to capture proteins from cells or tissues containing PAO-binding thiol pairs which can undergo reversible oxidations to presumed disulfide bonds. Prior work identified several metabolic enzymes among the disulfide bond-containing proteins, the so-called reducible disulfide proteome, from the brains of rats, used as a model organism and estimated the extents of disulfide bond formation in some of these. The present study sought to expand on earlier work by comparing, in extracts from rat brains, the extents of disulfide bond formation, in both total proteins and in metabolic enzymes of interest, under experimental conditions designed to prevent postmortem oxidations of thiols. Particularly unique to this study were (i) estimations of the extents of disulfide bond formation in the alpha and gamma isoforms of the glycolytic enzyme enolase and (ii) the effects of short-term delays to tissue freezing on protein thiol redox states. Among the enzymes compared, alpha-enolase was selectively enriched among the reducible disulfide proteome. Additionally, statistically-significant decreases in the extents of disulfide bond formation in both total proteins and in alpha-enolase following a 3 min, but not a 15 min delay to tissue freezing highlighted an apparent transient reductive stress occurring following animal euthanasia by decapitation, which induced global ischemia. These findings support the role of reversible oxidations of protein thiols to presumed disulfide bonds as potential regulatory switches in the healthy brain, in vivo, and suggest that ischemia, a risk factor

Teoretická část této práce je zaměřena na patogenezi onemocnění mnohočetný myelom. Práce popisuje fenomén tvorby autoprotilátek v rámci přirozené imunity s bližším zaměřením na problematiku určení vlastností a funkce protilátek v souvislostech s povahou onemocnění. V experimentální části je práce věnována metodice sérologické analýzy proteomu (SERPA) pro diagnostický screening autoprotilátek u pacientů s mnohočetným myelomem od doby stanovení diagnózy až po dosažení remise. Metodou SERPA je posuzována imunoreaktivita pacientských sér a význam nalézaných skvrn s bližším zaměřením na nálezy protilátek proti proteinu enoláza (ENO)., The theoretical part of this thesis is focused on the pathogenesis of multiple myeloma. It describes the phenomenon of autoantibody formation in innate immunity with a closer focus on the issue of determining the proberte and function of antibodies in relation to the nature of the disease. The experimental part is devoted to the methodology of serological analysis of proteome (SERPA) for diagnostic screening of autoantibodies in patients with multiple myeloma from the time of diagnosis to remission. Based od the used method SERPA is discussed revealed immunoreactivity of patient sera and the significance of the found spots with a closer focus on antibodies against protein enolase (ENO)., Fakulta chemicko-technologická, Prezentace výsledků diplomové práce. Diskuze k posudkům vedoucího a oponenta diplomové práce. Studentka zodpověděla všechny dotazy a připomínky k DP., Dokončená práce s úspěšnou obhajobou

Práce se zabývá detekcí anti-ENOL protilátek v séru pacientů s mnohočetným myelomem v remisi. Jsou zde popsány základní charakteristiky nemoci, diagnostika a terapie onemocnění. Část práce je také věnovaná autoprotilátkám, protinádorové imunitě a také enoláze a jejímu dělení. Experimentální část je zaměřena na optimalizaci promývací stanice FW400, 2D-elektroforézu pacientských sér a jejich následnou imunoreaktivitu., The thesis deals with detection of anti-ENOL antibodies in the serum of patients with multiple myeloma in remission. The basic characteristics of the disease, diagnosis and therapy of the disease are described here. Part of the work is devoted to autoantibodies, anti-tumor immunity as well as enolase and important division. The experimental part is focused on the optimization of the FW400 washing station, 2D-electrophoresis of patient sera and their subsequent immunoreactivity., Fakulta chemicko-technologická, 1. Prezentace výsledků diplomové práce. 2. Diskuze k posudkům vedoucího a oponenta diplomové práce. 3. Student zodpověděl všechny dotazy a připomínky k DP., Dokončená práce s úspěšnou obhajobou

It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns.IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Informati

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Saw, J. H. W., Nunoura, T., Hirai, M., Takaki, Y., Parsons, R., Michelsen, M., Longnecker, K., Kujawinski, E. B., Stepanauskas, R., Landry, Z., Carlson, C. A., & Giovannoni, S. J. Pangenomics analysis reveals diversification of enzyme families and niche specialization in globally abundant SAR202 bacteria. Mbio, 11(1), (2020): e02975-19, doi:10.1128/mBio.02975-19., It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns. IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Informat, We thank the captain, crew, ROV and CTD operation teams, and science party of the JAMSTEC RV Kairei cruises KR11-11, KR12-19, and KR14-01. We thank the staff of the Bigelow Laboratory for Ocean Sciences’ Single Cell Genomics Center for the generation of single-cell genomic data. We thank Mark Dasenko from the Center for Genome Research and Biocomputing at Oregon State University for sequencing six of the Illumina SAG libraries. We thank the captain, crew and CTD operations team of the RV Atlantic Explorer (cruise AE1712). T.N. was supported in part by a Grant-in-Aid for Scientific Research (B) (30070015) from the Japan Society for the Promotion of Science (JSPS). The mass spectrometry samples were analyzed at the WHOI FT-MS Users’ Facility; funding for data collection and analysis came from the National Science Foundation (NSF Grant OCE-1154320 to E.B.K. and K.L.). This work was funded by Simons Foundation International as part of the BIOS-SCOPE initiative (S.J.G., C.A.C., and E.B.K.), and by NSF grants OCE-1335810 and DEB-1441717 to R.S. This work was funded by National Science Foundation grant OCE-1436865.

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Saw, J. H. W., Nunoura, T., Hirai, M., Takaki, Y., Parsons, R., Michelsen, M., Longnecker, K., Kujawinski, E. B., Stepanauskas, R., Landry, Z., Carlson, C. A., & Giovannoni, S. J. Pangenomics analysis reveals diversification of enzyme families and niche specialization in globally abundant SAR202 bacteria. Mbio, 11(1), (2020): e02975-19, doi:10.1128/mBio.02975-19., It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns. IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Informat, We thank the captain, crew, ROV and CTD operation teams, and science party of the JAMSTEC RV Kairei cruises KR11-11, KR12-19, and KR14-01. We thank the staff of the Bigelow Laboratory for Ocean Sciences’ Single Cell Genomics Center for the generation of single-cell genomic data. We thank Mark Dasenko from the Center for Genome Research and Biocomputing at Oregon State University for sequencing six of the Illumina SAG libraries. We thank the captain, crew and CTD operations team of the RV Atlantic Explorer (cruise AE1712). T.N. was supported in part by a Grant-in-Aid for Scientific Research (B) (30070015) from the Japan Society for the Promotion of Science (JSPS). The mass spectrometry samples were analyzed at the WHOI FT-MS Users’ Facility; funding for data collection and analysis came from the National Science Foundation (NSF Grant OCE-1154320 to E.B.K. and K.L.). This work was funded by Simons Foundation International as part of the BIOS-SCOPE initiative (S.J.G., C.A.C., and E.B.K.), and by NSF grants OCE-1335810 and DEB-1441717 to R.S. This work was funded by National Science Foundation grant OCE-1436865.

Background Thermodynamic methods are finding more and more applications in systems biology, which attempts to understand cell functions mechanistically. Unfortunately, the state variables used (reaction enthalpy and Gibbs energy) do not take sufficient account of the conditions inside of cells, especially the crowding with macromolecules. Methods For this reason, the influence of crowding agents and various other parameters such as salt concentrations, pH and temperature on equilibrium position and reaction enthalpy of the glycolytic example reaction 9 (2-Phospoglycerate - > Phosphoenolpyruvate + H2O) was investigated. The conditions were chosen to be as close as possible to the cytosolic conditions. Poly(ethylene glycol) MW = 20,000 g mol−1 (PEG 20,000) was used to analyze the influence of crowding with macromolecules. The equation of state electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) was applied to consider the influence of crowding agents on the reaction equilibria. Results and conclusions For the reaction enthalpies and for the equilibria, it was found that the influence of salts and temperature is not pronounced while that of pH and PEG 20,000 concentration is considerable. Furthermore, it could be shown that under identical measurement conditions there are no differences between the van ‘t Hoff and the calorimetrically determined reaction enthalpy. General significance The results show how important it is to consider the special cytosolic conditions when applying thermodynamic data in systems biology.

Melatonin is a bioactive compound that is present in fermented beverages and synthesized by yeast during alcoholic fermentation. Many studies have shown that melatonin interacts with some mammalian proteins, such as sirtuins or orphan receptor family proteins. The aim of this study was to determine the intracellular synthesis profile of melatonin in Saccharomyces cerevisiae and to identify the proteins that may interact with this molecule in yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced intracellularly in the lag phase of yeast growth and was exported to the extracellular media during the stationary phase. During this period, melatonin was bound to six proteins with molecular weights from 55 to 35 kDa. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes. An RNA-binding protein was also identified, the elongation alpha factor, which is related to mitochondria. This study reports for the first time the interaction of melatonin and proteins inside yeast cells. These results highlight the possible role of melatonin as a signal molecule and provide a new perspective for understanding its role in yeast.

QC 20220301

In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.

Tomato spotted wilt virus (TSWV) is mainly vectored by Frankliniella occidentalis Pergande, and it potentially activates the vector's immune response. However, molecular background of the altered immune response is not clearly understood. Therefore, using a proteomic approach, we investigated the immune pathways that are activated in F. occidentalis larvae after 24 h exposure to TSWV. Two-dimensional isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-IEF/SDS/PAGE) combined with mass spectrometry (MS), were used to identify proteins that were differentially expressed upon viral infection. High numbers of proteins were abundantly expressed in F. occidentalis exposed to TSWV (73%) compared to the non-exposed (27%), with the majority functionally linked to the innate immune system such as: signaling, stress response, defense response, translation, cellular lipids and nucleotide metabolism. Key proteins included: 70 kDa heat shock proteins, Ubiquitin and Dermcidin, among others, indicative of a responsive pattern of the vector's innate immune system to viral infection. © 2016 Elsevier Ltd

Background: Fish is one of the most allergenic foods. While clinical cross-reactivity among different fishes is a widely accepted feature of fish allergy, associations with other food allergies are not well understood. This study aims at analyzing the relevance of clinical cross-reactivity between fish and chicken meat in patients with allergy to chicken meat without sensitization to hen's eggs. Methods: Patients with food allergy to fish and chicken meat (n = 29) or chicken meat only (n = 7) were recruited. IgE-reactive chicken proteins were identified (Edman, MS analysis) and quantified (ELISA). Allergens were used in IgE ELISA and skin testing. Results: Chicken parvalbumin and two new allergens, aldolase and enolase, were identified at 12, 40, and 50 kDa, respectively. They were recognized by sIgE of 61%, 75%, and 83% of all patient sera which were in the majority of the cases positive for the fish homologues as well. Fish and chicken meat allergens were highly cross-reactive while high inhibition rates with fish or chicken allergens correlated with the patients' primary sensitization to fish or chicken. In cooked or roasted foods, enolase and aldolase were detectable in chicken breast while parvalbumin was detectable in chicken legs and wings. Conclusions: Fish and chicken meat are cross-reactive foods; both fish-allergic and chicken meat-allergic patients might be at risk of developing a food allergy to chicken meat or to fish, respectively. This clinical phenomenon is proposed to be termed ‘fish–chicken syndrome’ with cross-reactive allergens involved being parvalbumins, enolases, and aldolases., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Invasive candidiasis (IC) adds significantly to the morbidity and mortality of non-neutropenic patients if not diagnosed and treated early. To uncover serologic biomarkers that alone or in combination could reliably detect IC in this population, IgG antibody-reactivity profiles to the Candida albicans intracellular proteome were examined by serological proteome analysis (SERPA) and data mining procedures in a training set of 24 non-neutropenic patients. Despite the high interindividual molecular heterogeneity, unsupervised clustering analyses revealed that serum 22-IgG antibody-reactivity patterns differentiated IC from non-IC patients. Univariate analyses further highlighted that 15 out of the 22 SERPA-identified IgG antibodies could be useful candidate IC biomarkers. The diagnostic performance of one of these candidates (anti-Hsp90 IgG antibodies) was validated using an ELISA prototype in a test set of 59 non-neutropenic patients. We then formulated an IC discriminator based on the combined immunoproteomic fingerprints of this and another SERPA-detected and previously validated IC biomarker (anti-Eno1 IgG antibodies) in the training set. Its consistency was substantiated using their ELISA prototypes in the test set. Receiver-operating-characteristic curve analyses showed that this two-biomarker signature accurately identified IC in non-neutropenic patients and provided better IC diagnostic accuracy than the individual biomarkers alone. We conclude that this serum IgG antibody signature directed against C. albicans Hsp90 and Eno1, if confirmed prospectively, may be useful for IC diagnosis in non-neutropenic patients., Unión Europea, Comunidad de Madrid, Ministerio de Economía, Comercio y Empresa (España), Spanish Network for Research in Infectious Diseases, Marie Curie Initial Training Networks, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

As opposed to other oilseeds, developing sunflower seeds do not accumulate starch initially. They rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Glycolysis is the principal source of carbon skeletons and reducing power for lipid biosynthesis. In this work, glycolytic initial metabolites and enzyme activities from developing seed of two different sunflower lines, of high and low oil content, were compared during storage lipid synthesis. These two lines showed different kinetic lipid accumulation in the developing embryos. Fatty acids levels during the initial and final stage of lipid synthesis were higher in CAS-6 than in ZEN-8. The analysis of the photosynthate and sugars content suggests that, although the hexoses levels were quite similar in both lines, the amount of sucrose produced by the mother plant and available for lipid synthesis was higher in CAS-6. Although, a smaller amount of sucrose is available in the ZEN-8 line, its seeds maintain the levels of intermediate sugars in the initial steps of glycolysis due to an increase in the levels of the invertase, hexokinase and phosphoglucose isomerase activities in ZEN-8, with respect to CAS-6. Also, a readjustment in the final part of this metabolic route took place, with the activities of phosphoglycerate kinase and enolase in CAS-6 being higher, allowing increased synthesis of phosphoenolpiruvate, the intermediate carbon donor for fatty acid synthesis. In addition, recently, it has been shown that Arabidopsis mutants with a lower fat content in their seeds have a higher amount of sucrose. These data together point to these last two enzymatic activities, phosphoglycerate kinase and enolase, as being responsible for the lower fat content in the ZEN-8 line.

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of S. pneumoniae -enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252+255A, SENK304A, SENK334A, SENK344E, SENK435L and SEN434-435) was used to examine their roles in maintaining structural integrity, enzymatic function and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as determined by circular dichroism spectroscopy and MS. However, ion-mobility MS revealed distinct differences in the stability of several mutant octamers in comparison to wild-type. Enzymatic analysis indicated that SENK344E had lost -enolase activity, which was also reduced in SENK334A and SENΔ434-435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SENK252+255A, SENK435L and SENΔ434-435. The lysine residues at positions 252, 255, 434 and 435, therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

7 p., Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

The incidence of human infections by the fungal pathogen Candida species has been increasing in recent years. Enolase is an essential protein in fungal metabolism. Sequence data is available for human and a number of medically important fungal species. An understanding of the structural and functional features of fungal enolases may provide the structural basis for their use as a target for the development of new anti-fungal drugs. We have obtained the sequence of the enolase of Candida krusei (C. krusei), as it is a significant medically important fungal pathogen. We have then used multiple sequence alignments with various enolase isoforms in order to identify C. krusei specific amino acid residues. The phylogenetic tree of enolases shows that the C. krusei enolase assembles on the tree with the fungal genes. Importantly, C. krusei lacks four amino acids in the active site compared to human enolase, as revealed by multiple sequence alignments. These differences in the substrate binding site may be exploited for the design of new anti-fungal drugs to selectively block this enzyme. The lack of the important amino acids in the active site also indicates that C. krusei enolase might have evolved as a member of a mechanistically diverse enolase superfamily catalying somewhat different reactions.

7 p., Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

7 p., Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

The gene of Leishmania mexicana enolase was cloned and overexpressed in Escherichia coli as an active enzyme; the protein was biochemically analyzed. This enolase shares with enolases from other trypanosomatids the presence of three atypical residues, each with a reactive side group, near the active site, already described for the enzyme from Trypanosoma brucei. The natural enzyme was purified, using a three-step procedure, from a cytosolic fraction of L. mexicana promastigotes. The kinetic properties of the purified recombinant enzyme were similar to those of the natural enzyme. Both the recombinant and natural enzyme were inhibited by inorganic pyrophosphate. Subcellular localization analysis after differential centrifugation showed that the enzyme activity is only associated with the cytosolic fraction. However, an apparently inactive form of enolase was detected by Western blots in the microsomal fraction. Digitonin treatment of parasites and immunofluorescence studies with permeabilized and non-permeabilized parasites showed that enolase is also associated with membranes and it was found at the external face of the plasma membrane.

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

To investigate the effect of different population densities on gene transcription in the sea bream brain (Sparus aurata), the messenger RNA (mRNA) differential display (DD) technique was used to analyse gene expression. Sea bream were held at different densities, 6 or 26 kg m−3, over a period of 14 days. We identified seven differentially expressed sequences of which one sequence was functionally identified. The S. aurata enolase gene homologue (S-enolase), pertaining to the alpha non-neuronal enolase group of the enzyme superfamily, was up-regulated in the brain of fish in the high-density population group. S-enolase mRNA expression was also found in other tissues including heart, liver and head kidney suggesting a ubiquitous nature. Furthermore, brain S-enolase mRNA is highly up-regulated 48 h after intra-peritoneal bacterial lipopolysaccharide (LPS) administration. Therefore, S-enolase gene expression is linked to the incidence of different stressors, density and infective agents, in the sea bream and may be a potential molecular biomarker for stress diagnosis in this fish

In this article, we report the results of an analysis of the glycolytic enzyme enolase (2-phospho-d-glycerate hydrolase) of Trypanosoma brucei. Enolase activity was detected in both bloodstream-form and procyclic insect-stage trypanosomes, although a 4.5-fold lower specific activity was found in the cultured procyclic homogenate. Subcellular localization analysis showed that the enzyme is only present in the cytosol. The T. brucei enolase was expressed in Escherichia coli and purified to homogeneity. The kinetic properties of the bacterially expressed enzyme showed strong similarity to those values found for the natural T. brucei enolase present in a cytosolic cell fraction, indicating a proper folding of the enzyme in E. coli. The kinetic properties of T. brucei enolase were also studied in comparison with enolase from rabbit muscle and Saccharomyces cerevisiae. Functionally, similarities were found to exist between the three enzymes: the Michaelis constant (Km) and KA values for the substrates and Mg2+ are very similar. Differences in pH optima for activity, inhibition by excess Mg2+ and susceptibilities to monovalent ions showed that the T. brucei enolase behaves more like the yeast enzyme. Alignment of the amino acid sequences of T. brucei enolase and other eukaryotic and prokaryotic enolases showed that most residues involved in the binding of its ligands are well conserved. Structure modelling of the T. brucei enzyme using the available S. cerevisiae structures as templates indicated that there are some atypical residues (one Lys and two Cys) close to the T. brucei active site. As these residues are absent from the human host enolase and are therefore potentially interesting for drug design, we initiated attempts to determine the three-dimensional structure. T. brucei enolase crystals diffracting at 2.3 A resolution were obtained and will permit us to pursue the determination of structure.