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61	Isolierung, Identifizierung und funktionelle Charakterisierung der Metallo-Aminopeptidase CaApe2 — Ein experimenteller Beitrag zur Beurteilung des kariogenen Potentials von Candida albicans Pönisch, Roman 09 December 2008 (has links) Die Hefe Candida albicans ist ein fakultativ humanpathogener Mikroorganismus, der insbesondere bei immungeschwächten Patienten schwere Erkrankungen der Haut und Schleimhäute sowie der inneren Organe hervorrufen kann. Seit langer Zeit wird eine Beteiligung des Hefepilzes an der Ätiopathogenese der Zahnkaries diskutiert, vor allem aufgrund der Säure-bildung, die zur Demineralisation der Zahnhartsubstanz beitragen kann. Hydrolytische Enzyme ermöglichen vermutlich die Gewebeinvasion von Candida albicans. In der vorliegenden Arbeit wurde ein sezerniertes peptidolytisches Enzym aus der Zellwand des Mikroorganismus isoliert, identifiziert und funktionell charakterisiert. Die mittels massenspektrometrischer Analyse der tryptischen Peptide und Datenbankrecherche ermittelte Primärstruktur und die Ergebnisse der funktionellen Charakterisierung ließen eine Identifi-zierung des peptidolytischen Enzyms als neutrale Arginin/Alanin/Leucin-spaltende Metallo-Aminopeptidase (CaApe2) zu, die durch den ORF CaO19.5197 (GenBank RefSeq XM 705313) kodiert wird. Mithilfe der Proteinanalytik wurde Serin-88 als N-terminale Aminosäure ermittelt. Die Aminosäuren 88 bis 954 des hypothetischen Genprodukts ergeben eine nominale Molekularmasse von 97,607 kDa. CaApe2 weist gleich hohe Ähnlichkeit mit den paralogen Genprodukten ScAap1 und ScApe2 auf, was eine Duplikation und Subfunktionalisierung des phylogenetischen Vorläufergens in Saccharomyces cerevisiae nahe legt. Die fehlende kollagenolytische Wirksamkeit von CaApe2 spricht gegen eine direkte Rolle des Enzyms in der Pathogenese der Dentinkaries von Candida albicans, schließt aber eine unterstützende Funktion nicht aus. Die Kollagendegradation durch aufgeschlossene Zellen und Kulturüberstand einer Flüssigkultur von Candida albicans wurde im sauren und neutralen Milieu mithilfe der Hydroxyprolin-Bestimmung untersucht. Dabei war keine Kollagenolyse mit Aktivitätsmaximum im neutralen Bereich nachweisbar. Im sauren pH-Bereich konnte eine deutliche Hydrolyse von säureunlöslichem Typ-I-Kollagen und auch von demineralisierter Dentinmatrix durch Kulturmedium gezeigt werden. Diese Kollagenolyse kann auf die bereits umfangreich charakterisierten sezernierten Aspartylproteinasen zurückgeführt werden. Die in der Literatur beschriebene Korrelation zwischen dem Ausmaß des Kariesbefalls und der Quantität der Besiedelung mit Candida albicans legt eine Beteiligung des Hefepilzes an der Kariogenese nahe. Auch die in der vorliegenden Arbeit gezeigte Fähigkeit von Candida albicans zur Dentinkollagendegradation unterstützt die Hypothese einer Kariogenität der Hefe. / The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2 which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. The isolated CaApe2 protein shares equally high similarity with the gene products ScAap1 and ScApe2 suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans. info:eu-repo/classification/ddc/610 ddc:610
62	Host cell responses to Helicobacter pylori secreted factors Garcia Lobato Tavares, Raquel January 2017 (has links) The infection of the human gastric mucosa by the bacterium Helicobacter pylori can lead to the development of gastritis, gastroduodenal ulcers, and cancer. The factors that determine disease development in a small percentage of infected individuals are still not fully understood. In this thesis, we aimed to identify and functionally characterize novel virulence factors of H. pylori and to understand their effect on host cell responses. In Paper I, we found that JHP0290, an uncharacterized secreted protein of H. pylori, induced macrophage apoptosis concomitant to the release of pro-inflammatory cytokine TNF via the regulation of the Src family of kinases and ERK MAPK pathways. In paper II, we demonstrated that JHP0290 exhibits both proliferative and anti-apoptotic activity, together with a faster progression of the cell cycle in gastric epithelial cells. During these responses, ERK MAPK and NF-κB pathways were activated. Paper III revealed a pro-apoptotic effect of another H. pylori-secreted protein HP1286 in macrophages via the TNF-independent and ERK-dependent pathways. No apoptosis was observed in HP1286-treated T cells or HL60 neutrophil-like cells, suggesting cell-type specific effect of HP1286. In Paper IV, we observed the pro-inflammatory activity of H. pylori secreted protein HP1173 in macrophages. The protein was found to induce TNF, IL-1β, and IL-8 in macrophages through MAPKs, NF-κB, and AP-1 signaling pathways. Furthermore, differential expression and release of JHP0290, HP1286, and HP1173 homologues was observed among H. pylori strains (papers II, III, IV). Due to their ability to regulate multiple host cell responses, proteins JHP0290, HP1286, and HP1173 could play an important role in bacterial pathogenesis. Helicobacter pylori Secreted proteins Host cell responses Macrophages Apoptosis Pro-inflammatory cytokines MAPKs
63	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760
64	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760

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