No description available.

No description available.

Reproduction de : Thèse doctorat : Chimie physique : Université de Cergy-Pontoise : 2005. / Titre provenant de l'écran titre. Bibliogr. p.200-213.

Les yapsines représentent une famille d'aspartyl peptidases fongiques nouvellement identifiées qui ont la propriété particulière d'être ancrées aux membranes par un groupement glycosylphosphatidylinositol (GPI). Ces enzymes localisées à la surface cellulaire sont importantes pour le maintien de l'intégrité de la paroi cellulaire et contribueraient même à la virulence des levures pathogènes. Nous avons décidé d'utiliser Yps1p, première yapsine découverte chez la levure Saccharomyces cerevisiae, comme modèle d'étude afin de mieux comprendre le rôle et le mécanisme d'action de ces enzymes. Dans ce but, trois objectifs spécifiques ont été posés : 1) mieux comprendre les événements de protéolyse limitée qui régulent l'activité de Yps1p, 2) étudier la fonction de Yps1p en identifiant ses substrats naturels et 3) étudier le transport intracellulaire de Yps1p. En réponse à ces objectifs, nous avons premièrement identifié par immunobuvardage et par séquençage en N-terminal des sites précis où Yps1p subit des clivages protéolytiques pour enlever son propeptide et pour générer une enzyme à deux sous-unités. Nous avons établi que ces événements protéolytiques avaient lieu à la surface cellulaire et qu'il existait une corrélation directe entre le choix des sites de clivage et le pH environnemental. Deuxièmement, en effectuant des tests d'activité in vivo, nous avons mis en évidence un rôle régulateur important pour la boucle de Yps1p. En effet, la mutation de résidus basiques contenus dans cette région augmente la capacité de Yps1p à corriger les phénotypes d'un mutant présentant des défauts de paroi cellulaire. Troisièmement, par séquençage en N-terminal de protéines retrouvées dans le milieu extracellulaire, nous avons observé que Yps1p était responsable de sa propre relâche de la surface cellulaire et nous avons identifié un premier substrat naturel pour l'enzyme : Gas1p, une enzyme impliquée dans le remodelage de la paroi cellulaire. Enfin, en comparant différentes formes de Yps1p dont l'association aux membranes a été modifiée (soluble, transmembranaire et GPI), nous avons pu établir que l'ancre GPI conférait un mode de transport particulier à Yps1p qui permet son acheminement à la membrane plasmique sans passer par le réseau trans-Golgien et les endosomes. Ce contournement est essentiel pour réguler les événements de maturation de Yps1p précédemment décrits et pour assurer son rôle à la surface cellulaire.

Os venenos de serpentes são complexas misturas de proteínas e peptídeos que apresentam uma variedade de atividades biológicas. Estudos apontam para uma rica diversidade de moléculas bioativas de baixa massa molecular nos venenos, como a crotamina, miotoxina A, peptídeos potenciadores de bradicinina (BPPs) inibidores do tipo Kunitz de serinopeptidases e tripeptídeos inibidores de metalopeptidases. O interesse nestas moléculas está relacionado ao potencial uso como agentes terapêuticos contra diversas patologias, como distúrbios da coagulação e modulação da atividade de metalopeptidases, moléculas estas envolvidas com tumorigenêse e outros processos patológicos como inflamação crônica e distúrbios neurológicos. O veneno da serpente Bitis gabonica rhinoceros provoca alterações fisiopatológicas como severa desordem na coagulação sanguínea e danos teciduais seguidos de necrose. No presente estudo foram isoladas e caracterizadas metalopeptidases e serinopeptidases, além de componentes de baixa massa molecular como inibidor do tipo Kunitz e BPPs. Estes peptídeos foram testados quanto a sua capacidade inibitória frente as peptidases endógenas e sequenciados por espectrometria de massa. Os nossos dados mostram que as peptidases isoladas degradam caseína e não tem atividade sobre colágeno. A serinopeptidase tem atividade β-fibrinogenolítica e o inibidor tipo Kunitz isolado apresenta maior capacidade de inibir a quimotripsina, com valor de Ki= 0,07 μM, mostrando-se um promissor substituto ao fármaco aprotinina. Este peptídeo apresentou também atividade citotóxica em células B16F10 e tênue atividade antimicrobiana. Dentre os BPPs identificados, o peptídeo que possui sequência não canônica apresentou a capacidade de potencializar a ação da bradicinina tanto em ensaio edematogênico quanto de inibição da atividade enzimática da enzima conversora de angiotensina. Esses resultados indicam o potencial de peptídeos de venenos animais para o desenvolvimento de novos agentes terapêuticos para o tratamento de enfermidades como hipertensão e distúrbios de coagulação. / Snake venoms are complex mixtures of proteins and peptides with a wide array of activities. Some studies point towards a vast diversity of low molecular mass bioactive molecules in venoms such as crotamine, myotoxin A, bradykinin potentiating peptides (BPPs), Kunitz type serine peptidase inhibitors and tripeptides inhibiting metallopeptidases. The interest on these molecules is related to their potential use as therapeutic drugs against several pathologies such as coagulation disturbs and modulation of the activity of metallopeptidases, involved in tumorigenesis and other disease like chronical inflammation and neurological disorders. The venom of Bitis gabonica rhinoceros promotes severe blood clotting disorders and tissular damages followed by necrosis. In the present study we isolated and characterized metallo and serine-peptidases, as well as as low molecular mass components such as Kunitz inhibitors and BPPs. Those peptides were assayed for their ability to inhibit the venom ednogenous peptidases and were sequenced by mass spectrometry. Our data indicate that the isolated peptidases hydrolyze casein, but not gelatin, indicating that they have no activity on collagen. The isolated serine protase has β-fibrinogenolytic activity and is not inhibited by the endogenous Kunitz peptide isolated from the venom. The Kunitzlike peptide inhibits preferentially chymotrypsin with a Ki of 0.07 μM and appears as a promising substitute for the commercial drug aprotinin. Among the three bradykinin potentiating peptides, two displayed non-canonical sequences, a fact that might represent an interesting field for new studies for the development of new anti-hypertensives. Although displaying mutations in highly conserved regions, the non-canonical BPP potentialized bradykinin in both edematogenic and angiotensin converting enzyme inhibition assays. These results indicate the potential of animal venom peptides for the development of new drugs against Diseases such as hypertension and coagulopathies.

Os venenos de serpentes são complexas misturas de proteínas e peptídeos que apresentam uma variedade de atividades biológicas. Estudos apontam para uma rica diversidade de moléculas bioativas de baixa massa molecular nos venenos, como a crotamina, miotoxina A, peptídeos potenciadores de bradicinina (BPPs) inibidores do tipo Kunitz de serinopeptidases e tripeptídeos inibidores de metalopeptidases. O interesse nestas moléculas está relacionado ao potencial uso como agentes terapêuticos contra diversas patologias, como distúrbios da coagulação e modulação da atividade de metalopeptidases, moléculas estas envolvidas com tumorigenêse e outros processos patológicos como inflamação crônica e distúrbios neurológicos. O veneno da serpente Bitis gabonica rhinoceros provoca alterações fisiopatológicas como severa desordem na coagulação sanguínea e danos teciduais seguidos de necrose. No presente estudo foram isoladas e caracterizadas metalopeptidases e serinopeptidases, além de componentes de baixa massa molecular como inibidor do tipo Kunitz e BPPs. Estes peptídeos foram testados quanto a sua capacidade inibitória frente as peptidases endógenas e sequenciados por espectrometria de massa. Os nossos dados mostram que as peptidases isoladas degradam caseína e não tem atividade sobre colágeno. A serinopeptidase tem atividade β-fibrinogenolítica e o inibidor tipo Kunitz isolado apresenta maior capacidade de inibir a quimotripsina, com valor de Ki= 0,07 μM, mostrando-se um promissor substituto ao fármaco aprotinina. Este peptídeo apresentou também atividade citotóxica em células B16F10 e tênue atividade antimicrobiana. Dentre os BPPs identificados, o peptídeo que possui sequência não canônica apresentou a capacidade de potencializar a ação da bradicinina tanto em ensaio edematogênico quanto de inibição da atividade enzimática da enzima conversora de angiotensina. Esses resultados indicam o potencial de peptídeos de venenos animais para o desenvolvimento de novos agentes terapêuticos para o tratamento de enfermidades como hipertensão e distúrbios de coagulação. / Snake venoms are complex mixtures of proteins and peptides with a wide array of activities. Some studies point towards a vast diversity of low molecular mass bioactive molecules in venoms such as crotamine, myotoxin A, bradykinin potentiating peptides (BPPs), Kunitz type serine peptidase inhibitors and tripeptides inhibiting metallopeptidases. The interest on these molecules is related to their potential use as therapeutic drugs against several pathologies such as coagulation disturbs and modulation of the activity of metallopeptidases, involved in tumorigenesis and other disease like chronical inflammation and neurological disorders. The venom of Bitis gabonica rhinoceros promotes severe blood clotting disorders and tissular damages followed by necrosis. In the present study we isolated and characterized metallo and serine-peptidases, as well as as low molecular mass components such as Kunitz inhibitors and BPPs. Those peptides were assayed for their ability to inhibit the venom ednogenous peptidases and were sequenced by mass spectrometry. Our data indicate that the isolated peptidases hydrolyze casein, but not gelatin, indicating that they have no activity on collagen. The isolated serine protase has β-fibrinogenolytic activity and is not inhibited by the endogenous Kunitz peptide isolated from the venom. The Kunitzlike peptide inhibits preferentially chymotrypsin with a Ki of 0.07 μM and appears as a promising substitute for the commercial drug aprotinin. Among the three bradykinin potentiating peptides, two displayed non-canonical sequences, a fact that might represent an interesting field for new studies for the development of new anti-hypertensives. Although displaying mutations in highly conserved regions, the non-canonical BPP potentialized bradykinin in both edematogenic and angiotensin converting enzyme inhibition assays. These results indicate the potential of animal venom peptides for the development of new drugs against Diseases such as hypertension and coagulopathies.

The N-terminally pyroglutamyl-modified β-amyloid (Aβ) peptides are found in abundance in the pathological Alzheimer disease (AD) brain deposits. Such modification not only increases the hydrophobic properties of a given molecule, but also plays a protecting role against proteolytic degradation. This project involved the study of the human type I and type II pyroglutamyl peptidases and their involvement in Aβ processing in AD. Human PcpI has been successfully overexpressed in Escherichia coli strain and purified to homogeneity. The protein displayed significant instability in vitro. To overcome this problem a number of methods were employed such as screening for an optimal protein expression system and buffer composition, site-directed mutagenesis and chemical modification of selected surface residues. This resulted in the selection of the HEPPS buffer system as providing the most stabilising conditions for human PcpI. Improvement in the protein stability enabled initial crystallisation experiments and the identification of favourable conditions for crystal production. Further optimization of this process is needed in order to obtain good quality crystals which are required for structural study. The study on human PcpII involved an extensive screening for optimal expression conditions in bacterial, baculovirus/insect and mammalian systems. The truncated PcpII isoform PcpII/S62-H1024, which lacks the N-terminal transmembrane domain, was successfully expressed and secreted from the HEK 293T cell line using three different pOPIN-based constructs. Moreover, homology modelling of human PcpII catalytic domain was performed, which helped to gain an insight into the three-dimensional structure of the protein and its mode of substrate binding. Lastly, immunohistochemical staining of the human AD brain tissue sections was performed to compare the level and distribution of PcpI and PcpII enzymes between diseased and control cases. The results confirmed that the neurodegenerative conditions lead to the increased synthesis of both enzymes in the cortical AD tissues. Additionally PcpI was shown to be able to participate in the degradation of pGlu-modified Aβ peptides.

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. 51-61. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thèses (Ph.D.)--Université de Sherbrooke (Canada), 2002. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Th. doct.-ing.--Nancy, I.N.P.L., 1981.