Dissertations / Theses: 'Carboxypeptidase A'

1

Commeinhes, Frédéric. "Structure tridimentionnelle de la carboxypeptidase A par diffraction des rayons X." Paris 5, 1994. http://www.theses.fr/1994PA05P019.

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2

Rowsell, Sian. "Crystal structure of carboxypeptidase Gâ†2." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362421.

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3

Küchenthal, Christian-Hubertus [Verfasser]. "Synthese neuartiger krebsspezifischer Carboxypeptidase-Liganden / Christian-Hubertus Küchenthal." Gießen : Universitätsbibliothek, 2012. http://d-nb.info/1063954940/34.

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4

Lehouritis, Panos. "Bacterial directed enzyme prodrug therapy using carboxypeptidase G2." Thesis, Institute of Cancer Research (University Of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429988.

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5

Bleakman, Alexandra. "Processing of the oxytocin precursor by carboxypeptidase E." Thesis, University of Bath, 1990. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279168.

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6

Nakase, Hiroshi. "Action Mode and Subsite Properties of Carboxypeptidase Y." Kyoto University, 2000. http://hdl.handle.net/2433/151622.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第8627号
農博第1154号
新制||農||814(附属図書館)
学位論文||H12||N3472(農学部図書室)
UT51-2000-R33
京都大学大学院農学研究科応用生命科学専攻
(主査)教授林力丸, 教授池田篤治, 教授加藤暢夫
学位規則第4条第1項該当

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7

Arndt, Joseph W. "Characterization and structural determination of metalloenzymes DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-A decarbonylase/synthase /." Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061312369.

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Thesis (Ph. D.)--Ohio State University, 2003.
Title from first page of PDF file. Document formatted into pages; contains xxii, 172 p. : ill., some col. Includes abstract and vita. Advisor: Michael K. Chan, Dept. of Chemistry. Includes bibliographical references (p. 165-172).

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8

Transfiguracion, Julia de la Cruz. "Purification and characterization of carboxypeptidase Y from Kluyveromyces fragilis." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22527.

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Carboxypeptidase Y (E.C. 3.4.12.1) was produced from Kluyveromyces fragilis ATCC 28244. The maximum growth and enzyme production were obtained during 24 hr of growth at the late logarithmic phase with optimized conditions (25$ sp circ $C, 300 rpm, pH 5) using YPD (1% yeast extract, 2% peptone, 2% dextrose, w/v) broth medium. A Fast Protein Liquid Chromatography (FPLC) was used for the enzyme purification. The enzyme was purified to 216 fold over the crude extract with a recovery of 18%. The apparent molecular weight of the purified enzyme was estimated to be 120 kDa on Native-PAGE and 56 kDa on SDS-PAGE suggesting that carboxypeptidase Y from Kluyveromyces fragilis consists of two subunits. The pH and temperature optima of the enzyme were pH 6.0 and 35$ sp circ$C, respectively. The enzyme activity was strongly inhibited by diisopropylphosphofluoridate (DIPF) and phenylmethylsulfonylfluoride (PMSF), and caused an average 50% loss of activity when incubated with various metal cations.
The apparent K$ sb{ rm m}$ and V$ sb{ rm max}$ values obtained for n-benzoyl-L-tyrosine-p-nitroanilide (BTPNA) and carboxybenzoxyphenylalanylalanine (Cbz-Phe-Ala) were 5.1 mM and 13.4 $ mu$mole/min/mg and 2.98 mM and 22.58 $ mu$mole/min/mg, respectively. Carboxypeptidase Y hydrolysis on the tryptic digests of $ alpha sb{ rm s1}$- and $ beta$-casein showed that the enzyme randomly removed five and three hydrophobic peptides, respectively and greatly reduced the size and heights of the other peptides analysed on Reversed Phase-High Performance Liquid Chromatography (RP-HPLC).

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9

Möller, Carsten. "Carboxypeptidase Z und Lix1 in der Embryogenese der Vertebraten." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969730136.

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10

Fonrose, Xavier. "Recherche et caractérisation d'inhibiteurs de la tubuline carboxypeptidase (TCP)." Université Joseph Fourier (Grenoble), 2008. http://www.theses.fr/2008GRE10072.

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Au cours du cycle de détyrosination tyrosination, la tyrosine C-terminale de la sous-unité alpha de la tubuline est clivée par une tubuline carboxypeptidase (TCP) inconnue, puis re-ajoutée par la tubuline tyrosine ligase (TIL). L'accumulation de tubuline détyrosinée, consécutive à la perte de la TTL, est un événement fréquent et de mauvais pronostic dans plusieurs types de cancers. Cette accumulation peut être supprimée par des inhibiteurs de la TCP. Ces inhibiteurs pourraient interférer avec la progression tumorale, être utilisés pour purifier la TCP. Ces inhibiteurs ont été recherchés par criblage automatisé de molécules chimiques, à l'aide d'un test cellulaire de l'activité TCP. Nous avons pu sélectionner plusieurs molécules chimiques, de structures différentes, inhibant spécifiquement l'activité TCP. Parmi ces molécules figure le parthénolide, connu pour ces activités anti-cancéreuses. Son action inhibitrice vis-à-vis de la TCP pourrait participer à ces propriétés anti-cancéreuses
Tubulin can be cyclically modified on its alpha-subunit by enzymatic removal of the COOH-terminal tyrosine residue by tubulin carboxypeptidase (TCP) and its readdition by tubulin tyrosine ligase (TIL). Accumulation of detyrosinated tubulin is frequent in human cancers of poor prognosis. Thus, TCP could be a target for developing novel therapeutic strategies for cancers. Inhibitors of TCP, by reversing abnormal detyrosinated tubulin accumulation in tumor cells, could impair tumor progression. TCP has never been isolated and this has hampered search of specific inhibitors. We develop a cell-based assay of TCP activity and its use to screen two libraries of chemical compounds for their inhibitory potency. This led to the isolation of several compounds, not structurally related. Among them, parthenolide can efficiently inhibit TCP. Parthenolide is known for its anticancer properties. Thus, TCP inhibition could be one of the underlying mechanisms of these anticancer properties

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11

Cooper, Antony. "Characterisation of the Kex1-encoded processing carboxypeptidase of Saccharomyces cerevisiae." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74650.

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The Saccharomyces cerevisiae KEX1 gene product, Kex1p, has been identified and partially characterised to assess its role in processing secreted protein precursors and to define its intracellular location. Kex1p antiserum identified a 113 kDa protein that was absent in kex1-$ Delta$ cells and more abundant in cells overexpressing KEX1. Kex1p was found to be a type I membrane associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kDa.
After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, $ alpha$-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like activity to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. These results provide biochemical evidence, consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.
Immunological and activity studies indicate that most Kex1p is intracellular and suggests that the enzyme is retained within the secretory pathway. COOH-terminal truncations of the protein indicate that the cytoplasmically exposed domain of Kex1p is responsible for correct localisation of the protein, probably in the late Golgi.
When KEX1 was expressed in Schizosaccharomyces pombe, Kex1p was localised in structures consistent with components of the Golgi. Mammalian cells expressing KEX1 produce a membrane associated activity that is not detected in the medium. In immunofluorescence studies on mammalian cells, Kex1p was localised to the ER and Golgi but not to the plasma membrane. Kex1p in such cells was responsible for completing the processing of the neuropeptide, $ gamma$-lipotropin. This in vivo processing of $ gamma$-lipotropin by Kex1p demonstrates a significant functional homology of the basic prohormone processing machinery in yeast and neuroendocrine cells.

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12

Al-Ajlan, Abdulrahman S. M. "A study of chymotrypsins and carboxypeptidase B from camel pancreas." Thesis, University of Essex, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284582.

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13

Ner, S. K. "The synthesis and testing of cyclopropane inhibitors of Carboxypeptidase A." Thesis, University of Strathclyde, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381719.

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14

Melton, R. G. "Physiological properties of carboxypeptidase G2 : Polymer conjugates and their clinical application." Thesis, Birmingham City University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374679.

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15

Oguntona, Taiwo Shola. "The potential role of a carboxypeptidase B2 inhibitor in renal fibrosis." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/19949/.

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Diabetic nephropathy (DN) is a fibrotic disease from renal injury which is characterized by extracellular matrix (ECM) build-up. Thus, DN leads to disruption of renal architecture and loss of renal function. We hypothesized that renal fibrosis can be improved by increasing plasmin activity through inhibition of carboxypeptidase B2 (CPB2) activity using UK-396082- a selective CPB2 inhibitor. An in vivo rat model of DN was used. Unilateral nephrectomy and induction of type 1 diabetes with 80mg/kg streptozotocin injection. Blood glucose was maintained between 20-25 mM for 8 months with linplants. The rats were divided into 3 groups. Group 1 (diseased) fed on normal chow. Group 3 (treatment) fed on normal chow for four months after which it was changed to chow mixed with 0.3g/kg of UK-396082 for the final four months. Group 2 (prophylaxis) fed on chow mixed with a 0.3g/kg of UK-396082 for 8 month. In an in vitro study CPB2, plasmin, tissue–type (tPA) and urokinase-type (uPA) plasminogen activators were assayed in the presence and absence of UK-396082 in rat kidney epithelial-like, fibroblast-like and mesangial cells cultured on plastic and fibrin gel. Our results confirmed DN in this rat model. Prophylactic administration of UK-396082 gave protection against development of kidney fibrosis and impaired kidney function. However, with advanced DN, UK-396082 appeared to have little protective effect. The In vitro results demonstrated that CPB2, tPA, uPA and plasmin activities were present in rat kidney epithelial-like, fibroblast-like and mesangial cell culture. CPB2, tPA, and plasmin activities were increased when epithelial and mesangial rat kidney cells were cultured on fibrin as opposed to plastic. Both epithelial-like and mesangial cells in the presence of fibrin led to more plasmin generation in the presence of UK-396082.

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16

Jager, Michal. "The role of aortic carboxypeptidase-like protein in epithelial-mesenchymal transition." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12428.

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Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Communication from stromal cells to tumors contributes to the progression of several carcinomas. Stromal fibroblasts, also referred to as cancer associated fibroblasts, in part through their production of secreted factors, promote epithelial-mesenchymal transition (EMT). EMT contributes to cancer progression by disseminating cells from the primary tumor and increasing these cells migratory capacity, an initial step in metastasis. Recently, several microarray studies have identified aortic carboxypeptidase-like protein (ACLP) as being significantly up-regulated in cancers, including prostate cancer and breast cancer, leading to the hypothesis that ACLP may regulate tumor progression and metastasis. To begin to test this hypothesis, this study first examined ACLP expression in a mouse mammary ductal carcinoma model and detected abundant ACLP expression in the cells surrounding the tumor. Cultured fibroblasts, derived from these tumors, readily expressed and secreted ACLP. To explore the functional contribution of ACLP to EMT in vitro we treated normal murine mammary gland epithelial cells (NMuMG) with recombinant ACLP (rACLP). In NMuMG cells, rACLP modulated the expression of epithelial-mesenchymal transition markers, Snail, fibronectin, occludin, and a-smooth muscle actin. Furthermore, rACLP treatment resulted in E-cadherin dissolution from the cell surface when compared with controls. These studies indicate that fibroblasts within a breast carcinoma express and may secrete ACLP, and in vitro data demonstrate that rACLP is capable of promoting EMT in normal epithelial cells. Therefore, ACLP may serve as an important mediator in the progression of cancer.

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17

Abaiian, Kayvan Jasper. "Regulation of aortic carboxypeptidase-like protein (ACLP) during 3T3-L1 adipogenesis." Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9355.

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The 175 kD aortic carboxypeptidase-like protein (ACLP), suggested to be involved in smooth vascular muscle cell differentiation, has been shown to be expressed in 3T3-L1 preadipocytes. In this study we demonstrate that ACLP protein expression is transiently but significantly down-regulated by day 2 of an 8-day 3T3-L1 differentiation. ACLP protein down-regulation correlates with increases in cell number, suggesting a potential link between ACLP and clonal expansion. The transient modulation of ACLP is shown to be partly due to transcriptional regulation. Analysis of the individual components of differentiation cocktail indicate that all components are necessary to induce maximal ACLP downregulation and, as such, this event is differentiation-dependent. Although ACLP overexpression had no apparent effect on adipogenesis, its pattern of expression, unique to post-mitotic proliferation, indicates a potential role for ACLP in adipose tissue development and warrants further investigation to elucidate its function during preadipocyte differentiation.

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18

Wichmann, Karin. "Quantenchemische Untersuchungen zur Hydrolyse von Formamid und zum Mechanismus von Carboxypeptidase A." [S.l.] : [s.n.], 2000. http://archiv.ub.uni-marburg.de/diss/z2003/0154.

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19

Wichmann, Karin. "Quantenchemische Untersuchungen zur Hydrolyse von Formamid und zum Mechanismus von Carboxypeptidase A." [S.l. : s.n.], 2003. http://archiv.ub.uni-marburg.de/diss/z2003/0154/.

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20

North, Stan. "The characterization of the yeast SKN7 gene and the identification of a maize carboxypeptidase homologue /." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68231.

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The Saccharomyces cerevisiae SKN7 gene has been identified through a search for genes which, at a high copy number, could restore the growth of the $kre9 Delta$ disrupted strain showing a cell wall $ beta$-glucan defect. SKN7 was mapped to the right arm of chromosome VIII, and is predicted to encode a 70 kDa protein, Skn7p, with a region of homology to the DNA binding domain of the yeast heat shock transcription factor, Hsf1p. Skn7p also has a domain which shows similarity to the prokaryotic receiver modules found on an extensive family of two-component response regulators, including the product of the rcsC gene. While restoring the growth rate to near wild type levels, SKN7 does not appear to restore the $ beta$-glucan levels of the $kre9 Delta$ mutant. However, SKN7-suppressed cells show a partially restored cellular morphology, and a restored cell wall resistance to mechanical stress. SKN7 does not suppress other mutations in the ($1 rightarrow6$)-$ beta$-glucan biosynthetic pathway, suggesting that it does not act as a general bypass suppressor of this glucan.

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21

Fougère, Aurélie. "Caractérisation des carboxypeptidases B d'Anopheles gambiae et analyse de leurs rôles sur le développement de Plasmodium falciparum et sur la reproduction des moustiques." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00829524.

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Mes travaux de thèse portent sur deux gènes cpbAg1 et cpbAg2 qui codent des enzymes digestives : les carboxypeptidases digestives du moustique Anopheles gambiae, vecteur majeur africain de Plasmodium falciparum, parasite responsable des formes graves du paludisme. La contribution de CPBAg1 dans le développement de P. falciparum chez An. gambiae a été démontrée ainsi qu'un rôle potentiel dans la production des oeufs du moustique (Lavazec et al., 2005 & 2007). Cependant le rôle potentiel de CPBAg2 dans ces deux phénotypes n'avait pas été abordé. Nos travaux ont permis de montrer des propriétés enzymatiques différentes pour chaque carboxypeptidase, avec CPBAg1 qui libère de l'arginine et de la lysine tandis que CPBAg2 est spécifique de l'arginine. La cinétique d'apparition in vivo des protéines montre une production précoce de CPBAg1 et une production constante de CPBAg2 au cours du processus de digestion du repas sanguin. L'analyse fonctionnelle de CPBAg1 et CPBAg2 utilisant l'inactivation génique par ARN interférence a permis de comprendre leurs rôles respectifs. Ainsi, CPBAg1 est impliquée dans le développement de P.falciparum chez An. gambiae, suggérant une plus forte dépendance du parasite pour les ressources en lysine. A l'inverse, CPBAg2 intervient au cours de la vitellogénèse du moustique et son inactivation induit également un retard du développement ovarien. Ainsi, ce dernier semble être plus dépendant de l'arginine, produit principalement par CPBAg2. CPBAg1 et CPBAg2 pourraient de ce fait être utilisées comme les cibles de nouvelles stratégies pour diminuer la transmission du paludisme, en bloquant le parasite chez le moustique tout en diminuant la production d'oeufs.

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22

Dalle, Ore-Dedeyan Florence. "Purification et caractérisation de la carboxypeptidase basique membranaire de la muqueuse intestinale de porc." Aix-Marseille 3, 1999. http://www.theses.fr/1999AIX30077.

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La muqueuse intestinale de porc contient une activite carboxypeptidasique qui est apparemment due a deux enzymes differentes responsables de la liberation des acides amines basiques a partir de l'extremite c-terminale de petits peptides. En effet, nous avons detecte une carboxypeptidase membranaire de ph optimum neutre, activee par le cocl 2 et inhibee par l'acide guanidinoethylmercaptosuccinique, l'o-phenanthroline, l'acide ethylenediamine tetraacetique et l'acetate de cadmium, ainsi qu'une carboxypeptidase soluble possedant une activite optimale a ph 5 et peu affectee par le cocl 2, l'o-phenanthroline, l'acide ethylenediamine tetraacetique, le nicl 2 et le cdcl 2. Cette derniere activite serait due a la cathepsine b lysosomale qui est effectivement presente dans la fraction soluble de la muqueuse intestinale de porc. Nous n'avons donc pas caracterise cette enzyme qui n'est pas une metallocarboxypeptidase. L'activite de la carboxypeptidase membranaire est distribuee de facon homogene le long de l'intestin grele. Contrairement a la carboxypeptidase m presente dans de nombreux tissus et organes, la carboxypeptidase membranaire de l'intestin de porc n'est pas ancree par un segment phosphatidylinositol car elle est resistante a la phospholipase c phosphatidylinositol-specifique, mais egalement solubilisee en memes proportions par differents detergents. La purification de la forme solubilisee par la trypsine permet d'obtenir une glycoproteine de 200 kda, ce qui la differencie de la carboxypeptidase m purifiee a partir de placenta humain (62 kda), resultat confirme par l'analyse immunologique. De plus, cette enzyme libere preferentiellement la lysine a l'arginine, contrairement a la carboxypeptidase m. La carboxypeptidase membranaire de la muqueuse intestinale de porc est donc differente de la carboxypeptidase m. Les caracteristiques de cette enzyme sont semblables a celles de la carboxypeptidase d, un nouveau membre de la famille des metallocarboxypeptidases decrit recemment.

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23

Taxis, Christof. "Proteinqualitätskontrolle im endoplasmatischen Retikulum : Variationen im Abbaumechanismus von löslicher und membrangebundener missgefalteter Carboxypeptidase (CPY*) /." [S.l. : s.n.], 2002. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10111673.

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24

Ladds, Graham Robert. "Adaptation with the fission yeast Schizosaccharomyces pombe : the characterisation of the secreted serine carboxypeptidase Sxa2." Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322675.

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25

NORMANT, EMMANUEL. "Caracterisations d'isoformes de carboxypeptidase a ainsi que d'un nouvel inhibiteur proteique endogene dans le cerveau." Paris 6, 1995. http://www.theses.fr/1995PA066418.

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Les localisations, dans d'autres tissus que le pancreas, des isoformes cpa1 et cpa2 de la carboxypeptidase a (cpa, ec 3. 4. 17. 1) ont ete realisees. Trois approches differentes, permettant la recherche i) de l'activite enzymatique avec un substrat radiomarque, ii) des proteines, grace aux anticorps polyclonaux, ou iii) des transcrits codant pour ces proteines ont ete envisagees. Ces trois methodes ont apporte des resultats coherents et complementaires permettant, in fine, de definir de nouveaux organes, tels que le tractus digestif, le cur, le cerveau, le poumon et le testicule comme producteur de ces enzymes. Elles ont notamment permis de localiser les cellules nerveuses produisant la cpa1 au niveau des noyaux arque et supraoptique de l'hypothalamus. Elles ont egalement permis de caracteriser un transcrit court, provenant d'epissage alternatif du gene de la cpa2, et codant pour la forme mature de l'enzyme (sans son peptide d'activation), prive de ses 20 residus n-terminaux. L'activite enzymatique correspondante a ete caracterisee grace a l'obtention de la proteine recombinante, et detectee dans le cerveau. C'est une metallocarboxypeptidase, qui possede des constantes d'inhibition, vis-a-vis de certains inhibiteurs, differentes de celles de la cpa2 pancreatique. Enfin ces techniques ont permis de caracteriser et de localiser le premier inhibiteur proteique de mamifere de metallocarboxypeptidases. Cette proteine de 26 kda, est essentiellement localisee dans le cerveau, le poumon et l'estomac. Elle inhibe de facon quasi irreversible les cpa1 et cpa2 (mais pas la cpa2 forme courte) ainsi que la cpa mastocytaire et la cpb. L'adnc de son transcrit a ete clone et la proteine exprimee. Celle-ci ne possede aucune homologie avec les sequences des banques de donnees

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26

Gusinjac, Arjeta. "The role of aortic carboxypeptidase-like protein (ACLP) in 3T3-L1 preadipocyte and adipocyte function." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28384.

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Aortic carboxypeptidase-like protein (ACLP) is a 175kDa secreted protein that is downregulated with adipogenesis. I engineered 3T3-L1 preadipocytes that overexpressed ACLP protein and sustained the overexpression with differentiation. I assessed the role of sustained ACLP overexpression on 3T3-L1 adipogenesis, with a focus on possible ACLP interaction with collagen-I during differentiation. ACLP overexpression did not affect 3T3-L1 adipogenesis under standard cell culture conditions ACLP overexpression did not affect subconfluent preadipocyte proliferation, apoptosis in serum-supplemented preadipocytes, or serum-deprived preadipocyte cell death. Sustained ACLP overexpression did not affect collagen I/III protein expression, or the activity of matrix metalloproteinase (MMP)-2 and MMP-9 extracellular degrading enzymes. However, sustained ACLP overexpression on collagen-I coated dishes inhibited the expression of three key differentiation markers, peroxisome proliferator-activated receptor gamma, CCAAT enhancer-binding protein alpha and fatty acid synthase and decreased insulin-stimulated Akt/protein kinase B phosphorylation. This suggests that a collagen-I-rich environment is necessary for ACLP to alter preadipocyte function.

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27

Bonis, Mathilde. "Validation des hydrolases du peptidoglycane comme nouvelles cibles thérapeutiques contre l'infection à Helicobacter pylori." Paris 11, 2010. http://www.theses.fr/2010PA114856.

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L’infection à Helicobacter pylori touche près de 50 % de la population mondiale, et peut être associée à des pathologies sérieuses. De plus, l’émergence de résistances bactériennes ne cesse d’accroître le taux d’échec thérapeutique, justifiant pleinement la recherche de nouvelles stratégies thérapeutiques. Dans ce sens, ce travail de thèse a eu pour objet de rechercher de nouvelles cibles thérapeutiques contre H. Pylori, via la caractérisation d’acteurs du métabolisme du peptidoglycane et de leur rôle dans la virulence bactérienne. Ainsi, nos travaux ont permis d’une part, la caractérisation d’une nouvelle peptidase bifonctionnelle du PG, HdpA, impliquée dans la virulence de H. Pylori, via la régulation de sa forme bâtonnet, et d’autre part, la mise en évidence de l’action inhibitrice de la bulgécine sur la transglycosylase lytique Slt, engendrant une altération de la mobilité de H. Pylori, ainsi qu’une forte réduction de sa croissance par action synergique avec l’amoxicilline
Helicobacter pylori is a major pathogen since it colonizes around 50 % of the world population and it can be associated to serious diseases. Moreover the emergence of bacterial resitances leads to more and more therapeutic failures, requiring the search for new therapeutic strategies. Consequently, the aim of that PhD work was to identify new therapeutic targets against H. Pylori, via the characterization of peptidoglycan metabolism proteins, and their role in the bacterial virulence. Thereby, our work led firstly, to the characterization of a novel PG peptidase, involved in the virulence of H. Pylori via its shape regulation, and secondly, to the study of the bulgecin, an inhibitor of the lytic transglycosylase Slt, provoking a bacterial motility defect and a strong growth decrease in presence of amoxicillin

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28

Prasad, Ankur. "The role of aortic carboxypeptidase-like protein in adipose-derived mesenchymal stem cell adipogenesis and fibrosis." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12193.

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Thesis (M.A.)--Boston University
The prevalence of obesity and obesity related diseases are increasing worldwide. Obesity is characterized by the pathological expansion of white adipose tissue. Previous studies on white adipose tissue of obese individuals have detected inflammation and fibrosis. These conditions may cause dysregulation of the tissue, leading to negative outcomes, including type II diabetes and metabolic syndrome. Aortic carboxypeptidase-like protein (ACLP) is a secreted extracellular matrix protein that is upregulated in fibrotic lung tissue. Importantly ACLP knockout mice are protected from experimentally induced lung fibrosis. ACLP is expressed in adipose tissue and is downregulated as stem cells undergo adipogenesis. Its overexpression increases α smooth muscle actin expression and impairs adipogenesis in preadipocyte lines; however, its role in white adipose tissue fibrosis has not been fully explored. The studies presented in this thesis aimed to investigate the hypothesis that ACLP overexpression in fibrotic white adipose tissue would promote a fibroblast to myofibroblast transition and repress adipogenesis. To determine if ACLP promotes a fibroblast to myofibroblast transition, we tested the capacity of ACLP to induce α smooth muscle actin and collagen I protein expression and increase contractility of primary stromal vascular cells. To assess the effects of ACLP on adipogenesis, we tested the ability of 10T1/2 fibroblasts and stromal vascular cells to undergo adipogenesis in collagen I gels under ACLP treatment. Results presented herein demonstrate ACLP is a potent inhibitor of adipogenesis and induces an upward trend in myofibroblast proteins and RNA expression. Significantly, these studies used murine adipose-derived cells to show the effects of ACLP, suggesting these results might be reflected in adipose tissue. These experiments support a model where ACLP potentiates adipose tissue fibrosis by inhibiting adipogenesis, resulting in fewer developing adipocytes, and stimulating myofibroblast differentiation, resulting in further collagen deposition and tissue compaction. This contribution to adipose tissue dysfunction also gives ACLP a possible role in the development of obesity related diseases, including diabetes and metabolic syndrome, identifying it as a possible target for therapeutics.

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29

Berne, Pierre-François. "Marquage carboxyl-terminal de protéines : deux approches complémentaires utilisant la transpeptidation catalysée par la carboxypeptidase y." Palaiseau, Ecole polytechnique, 1991. http://www.theses.fr/1991EPXX0008.

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En dépit de nombreuses tentatives, aucune méthode de caractérisation non ambigue de l'extrémité c-terminale des protéines ne s'est encore imposée. Une solution pourrait être fournie par le marquage radioactif spécifique des protéines a cette extrémité, les applications possibles d'un tel outil sont de deux sortes: détermination de la structure primaire des polypeptides et obtention d'informations conformationnelles. Nous avons développé deux approches distinctes du marquage c-terminal répondant séparément a ces deux problèmes et reposant, toutes deux, sur la transpeptidation catalysée par la carboxypeptidase y. Cette réaction consiste en la substitution de la liaison normalement hydrolysée par la carboxypeptidase y par une nouvelle liaison, mettant en jeu un nucléophile exogène. Dans le cadre d'une première approche, nous avons étudié la transpeptidation de substrats peptidiques modèles a ph neutre en présence d'une variété de nucléophiles. Cette étude a révelé que l'obtention de rendements de transpeptidation satisfaisants était subordonnée a la présence d'un résidu proline en position penultieme ou antepenultieme du peptide substrat. Pour de tels peptides, il a été montré que la transpeptidation se produit spécifiquement du cote c-terminal de la proline, ce qui suggère un rôle déterminant de ce résidu pour la transpeptidation. Nous avons ensuite transpose ces résultats a une protéine. Pour cela nous avons utilisé un variant de la methionyl-arnt synthetase d'escherichia coli obtenu par mutagenese dirigée, avec la séquence c-terminale glu-pro-met. Nous avons pu marquer spécifiquement le variant a son extrémité c-terminale avec de la methionine tritiee alors que la forme non modifiée de la methionyl-arnt synthetase n'est pas un substrat convenable. La seconde approche repose sur l'estérification par voie chimique du substrat, suivie d'une transpeptidation catalysée par la cpy a ph b

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30

Jeyaharan, Dhadchayini. "Biophysical study into the structure and substrate binding properties of peptido-mimetic ligands to carboxypeptidase G2." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/78855/.

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The antibody directed enzyme pro-drug therapy (ADEPT) is a new anticancer treatment, where pre-clinical experiments concluded that an intermediate step involving the inhibition of carboxypeptidase G2 (CPG2) from the circulatory system prior to pro-drug administration is crucial to prevent systemic toxicity (Bagshawe et al. 1991). The research described in this thesis sought to better understand the mode of binding of these inhibitors to CPG2 using solution state nuclear magnetic resonance (NMR) spectroscopy. A high-yield expression of active and soluble mature CPG2 (in the absence of the leader peptide) in E. coli suitable for NMR studies and co-crystallisation screening is reported. We have used this method to routinely produce milligrams quantities of 1H/13C/15N isotopically-labelled protein suitable for NMR studies. The second aim of this thesis is interactions; interactions between CPG2 and selected inhibitors provided by our industrial partner Mologic Ltd. (Bedford, UK). Different structural parts of the inhibitors were identified by NMR to directly interact with CPG2: the naphthalene and the glutamate groups. Chemical shift perturbations studies show different patterns for CP06 and CP67 inhibitors suggesting that they have different binding mechanisms. Site-directed mutagenesis of residues in P1 pocket of CPG2 reveal no activity against methotrexate (MTX), suggesting that they are key players in substrate recognition, while H285A and E200A mutant proteins display similar activity to wild type CPG2 protein. Although the NMR data described here for CPG2 were incomplete and thus did not yield resonance assignment, we show attempts at a "divide-and-conquer" approach. The CPG2CAT construct shows great promise for downstream NMR studies as it has favourable solution properties and retains key properties of the parent protein, namely enzymatic activity and the ability to self associate.

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31

Lanthier, Christopher Michael. "The inhibition of carboxypeptidase A and angiotensin-converting enzyme by target enzyme-activated inhibitors and N-acylhydrazones." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq21362.pdf.

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32

Jung, Giman. "The Charge-Relay System in Enzyme Catalysis : Construction and Function of Active Site Residues in Carboxypeptidase Y." Kyoto University, 1999. http://hdl.handle.net/2433/181885.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7876号
農博第1034号
新制||農||776(附属図書館)
学位論文||H11||N3239(農学部図書室)
UT51-99-G470
京都大学大学院農学研究科農芸化学専攻
(主査)教授林力丸, 教授佐藤文彦, 教授江崎信芳
学位規則第4条第1項該当

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33

Schochter, Fabienne [Verfasser]. "Die Rolle der Mastzelle im murinen Sepsismodell Cecal Ligation and Puncture (CLP) anhand der Carboxypeptidase A / Fabienne Schochter." Ulm : Universität Ulm. Medizinische Fakultät, 2014. http://d-nb.info/1046890115/34.

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34

Dumoulin, Mireille. "High Pressure-Induced Cold Denaturation of Proteins : Structural and Functional Study of Wild Type and Unglycosylated Carboxypeptidase Y." Kyoto University, 1999. http://hdl.handle.net/2433/181908.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7900号
農博第1058号
新制||農||780(附属図書館)
学位論文||H11||N3263(農学部図書室)
UT51-99-G494
京都大学大学院農学研究科農芸化学専攻
(主査)教授林力丸, 教授天知輝夫, 教授加藤暢夫
学位規則第4条第1項該当

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35

Stehle, Felix [Verfasser], Milton T. [Akademischer Betreuer] Stubbs, Dieter [Akademischer Betreuer] Strack, and Dietrich [Akademischer Betreuer] Ober. "Struktur-Funktionsbeziehungen der Serin-Carboxypeptidase-ähnlichen Sinapoylglucose:Malat-Sinapoyltransferase / Felix-Oliver Stehle. Betreuer: Milton T. Stubbs ; Dieter Strack ; Dietrich Ober." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2009. http://d-nb.info/1024896161/34.

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36

Henningsson, Frida. "Mast cell carboxypeptidase A, a secretory granule component : insights to its processing, intracellular sorting and interaction with serglycin proteoglycans /." Uppsala : Dept. of Molecular Biosciences, Swedish University of Agricultural Sciences, 2005. http://epsilon.slu.se/200586.pdf.

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37

Föh, Bandik [Verfasser]. "Carboxypeptidase E moduliert die immunologische Homöostase des Darms und hat protektive Effekte im Rahmen einer experimentellen Colitis / Bandik Föh." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2018. http://d-nb.info/1169355137/34.

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38

Fritz, Loïc. "Etude de l'activité peptidyl synthétase de la carboxypeptidase Y : influence de la structure primaire du fragment C-terminal du substrat et application au radiomarquage 3H de 3 hormones neurohypophysaires." Paris 5, 1989. http://www.theses.fr/1989PA05P617.

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Serval, Valérie. "Synthèse et propriétés pharmacologiques d'inhibiteurs du métabolisme de l'acide N-Acetyl-Aspartyl-Glutamique (NAAG)." Paris 6, 1991. http://www.theses.fr/1991PA066677.

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En 1991, trois récepteurs du neurotransmetteur excitateur qu’est l’acide glutamique ont été caractérisés grâce à la synthèse d’agonistes et d’antagonistes sélectifs : les deux récepteurs ionotropiques (ou ionotropes) NMDA et AMPA et le récepteur métabotropique (ou métabotrope). Le dipeptide NAAG (N alpha Acetyl-L-aspartyl-L-glutamique) endogène du système nerveux central de vertébrés est aussi un candidat potentiel au rôle de neurotransmetteur ou bien au rôle de précurseur du neurotransmetteur glutamatergique. Le NAAG est hydrolysé en acide glutamique et en acide N-acétyl-aspartique par une peptidase: la NAALADase. Le but de ce travail a été de synthétiser des inhibiteurs spécifiques de la NAALADase dépourvus d’autres propriétés pharmacologiques en particulier sans effets sur les récepteurs des neurotransmetteurs excitateurs. En effet, l’acide quisqualique, seul inhibiteur connu de la NAALADse à ce jour, est aussi un agoniste des récepteurs glutamatergiques AMPA et métabotropique. Nous avons étudié l’activité enzymatique de la NAALADase in vitro, à partir de synaptosomes de cerveau de rat avec le substrat NAAG radioactif et les analogues non radioactifs en immobilisant les membranes sur un support et in vivo, au niveau du striatum de rat. Nous avons montré que le dipeptide L‐Aspartyl‐L‐glutamate est hydrolysé plus rapidement que le NAAG, ce qui suggère que le groupement acétyl n’est pas essentiel pour la spécificité de l’activité NAALADase. En partant de l’hypothèse que l’enzyme était une métallopeptidase, trois analogues ayant des propriétés inhibitrices ont été synthétisés avec succès, désignés par les nombres 33 (dipeptide beta-NAAG), 73 et 74. Nous avons mis en évidence une protection presque totale par les trois inhibiteurs 33, 73 et 74 de la dégradation in vivo du NAAG. L’affinité des analogues, comme celle du substrat NAAG pour l’enzyme, est de l’ordre du micromolaire. Les analogues contenant un acide glutamique et de nombreuses fonctions acides carboxyliques, les effets de ces molécules sur les trois types de récepteurs NMDA, AMPA et métabotropiques ont été comparés à ceux de l’acide quisqualique. La recherche de meilleurs inhibiteurs de la NAALADase (affinité nanomolaire) nécessite de comprendre comment le dipeptide alpha-NAAG est un substrat tandis que les analogues 33 (beta-NAAG),73 et 74 sont des inhibiteurs. Une préparation d’enzyme purifiée et la connaissance de la séquence de la NAALADase permettront d’y parvenir. Le rôle physiologique de la NAALADase sera clarifié lorsque les réponses biologiques induites par le dipeptide NAAG seront caractérisées puis potentialisées par exemple, en présence des inhibiteurs de l’enzyme
In 1991, NMDA, AMPA and metabotropic receptors of glutamate / glutamic acid, a main excitatory amino acid neurotransmitter of the brain, have been identified following the synthesis of selective agonist and antagonists. The endogenous dipeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) present in vertebrate CNS fulfills several criteria corresponding to a neurotransmitter or glutamate precursor. NAAG inactivation may proceed through enzymatic hydrolysis into N-acetyl-L-aspartate and glutamate by an N-acetylated-alpha-linked acidic dipeptidase (NAALADase). The purpose of our work was to synthesize specific inhibitors of NAALADase with no pharmacological properties on glutamate receptors. Quisqualic acid/ quisqualate, the only known inhibitor of NAALADase to date, is indeed an AMPA and metabotropic glutamate receptor agonist. NAALADase activity has been studied in vitro using immobilized membranes from rat brain synaptosomes and with radioactive NAAG and non radioactive synthetized analogues of NAAG, and in vivo, in the rat striatum. L-Aspartyl-L-glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety was not essential for NAALADase specificity. On the assumption that the peptidase should be a metallopeptidase, three analogues that are inhibitors have been successfully synthesized which have been named 22 (dipeptide beta-NAAG), 73 et 74. Almost full enzymatic in vivo degradation protection of NAAG was achieved by all three inhibitors 33, 73 and 74. Analogues affinities, like NAAG substrate itself, were in the micromolar range. Since analogues contain a glutamic acid and other carboxylic functions, the effect of these on the three types of receptors ie. NMDA, AMPA and metabotropic have been compared to those of quisqualic acid. The search for NAALADase inhibitors with affinity in the nanomolar range requires to understand how alpha-NAAG dipeptide acts as a substrate whereas analogues 33 (beta-NAAG), 73 and 74 act as inhibitors. In the future, this should be achieved thanks to the availability of a purified enzyme preparation and the knowledge of the NAALADase sequence. NAALADase physiological role will be clarified when biological responses induced by NAAG dipeptide will be characterized and then potentiated with enzyme inhibitors

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40

Armento, Angela [Verfasser], and Ulrike [Akademischer Betreuer] Naumann. "Carboxypeptidase E reduces Glioblastoma migration through modulation of motility-associated gene expression and signaling cascades / Angela Armento ; Betreuer: Ulrike Naumann." Tübingen : Universitätsbibliothek Tübingen, 2017. http://d-nb.info/1199545724/34.

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41

Podmore, Adrian Herbert Bailey. "Purification and characterisation of VanXY{SUB}c, a D, D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.622005.

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42

Gotoh, Shimpei. "Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells." Kyoto University, 2015. http://hdl.handle.net/2433/195967.

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Final publication is available at http://dx.doi.org/10.1016/j.stemcr.2014.07.005. Shimpei Gotoh, Isao Ito, Tadao Nagasaki, Yuki Yamamoto, Satoshi Konishi, Yohei Korogi, Hisako Matsumoto, Shigeo Muro, Toyohiro Hirai, Michinori Funato, Shin-Ichi Mae, Taro Toyoda, Aiko Sato-Otsubo, Seishi Ogawa, Kenji Osafune, Michiaki Mishima, Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells, Stem Cell Reports, Volume 3, Issue 3, 9 September 2014, Pages 394-403, ISSN 2213-6711.
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第18681号
医博第3953号
新制||医||1007(附属図書館)
31614
京都大学大学院医学研究科医学専攻
(主査)教授妻木範行, 教授江藤浩之, 教授瀬原淳子
学位規則第4条第1項該当

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43

Hellio, Florence. "Synthèse peptidique par catalyse enzymatique : application nouvelle à la radiosynthèse totale d'une hormone, la leucine-enképhaline tritiée, à l'aide de la carboxypeptidase Y." Compiègne, 1986. http://www.theses.fr/1986COMPD040.

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Dans ce mémoire nous présentons une nouvelle méthode de marquage au tritium d'hormones peptidiques. Cette méthode fait appel à une protéase, la carboxypeptidase Y qui catalyse la formation de liaisons peptidiques, pourvu que les conditions thermodynamiques soient favorables. L'action réversible de la CPD-Y a été appliquée à la synthèse totale d'un pentapeptide, la Leucine-enképhaline, tritié sur chaque acide aminé, et dont la radioactivité spécifique est de 139 Ci/mmole. Le peptide obtenu présente, en outre, des propriétés biologiques (liaison aux récepteurs et immunoréactivité) identiques à celles de Leucine-enképhaline native
In this thesis we present a new tritium-labelling method of peptidic hormones. This method uses a proteolytic enzyme, carboxypeptidase Y, to catalyse peptide bonds. It has been applicated to stepwise synthesis of tritiated Leucine-enkephalin. The pentapeptide, labelled on each amino acid, has a specific radioactivity of 139 Ci/mmole. Moreover its biological properties (immunoreactivity and binding to specific receptors) are identical to native Leucine-enkephalin

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44

Covaleda, Cortés Giovanny. "Kinetic and proteomic identification of protease inhibitors in marine invertebrates. Characterization of a carboxypeptidase inhibitor isolated from the mollusc nerita versicolor." Doctoral thesis, Universitat Autònoma de Barcelona, 2012. http://hdl.handle.net/10803/117662.

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El presente trabajo describe la identificación cinética de actividad inhibidora de carboxipeptidasa A (CPA), carboxipeptidasa B (CPB), pepsina, papaína, tripsina y subtilisina en 30 extractos de invertebrados marinos del Caribe. Los resultados cualitativos mostraron catorce extractos con actividad inhibidora de tripsina, nueve extractos con actividad inhibidora de CPA, CPB, papaína y subtilisina, y sólo cuatro extractos con actividad inhibidora de pepsina. Los extractos más prometedores fueron seleccionados en términos de actividad inhibidora específica, relación dosis-respuesta, IC50 y biodisponibilidad de la especie. Por otra parte, Intensity Fading MALDI-TOF MS, un método proteómico utilizado en este trabajo para completar la estrategia de identificación de inhibidores de proteasas en extractos marinos, resultó ser muy útil para detectar moléculas, capaces de interactuar selectivamente con enzimas diana inmovilizadas, como fue el caso de N. versicolor frente a CPA y CPB, H. carunculata con pepsina, S. helianthus con papaína, N. peloronta y S. helianthus con tripsina y L. isodyctialis con subtilisina. Este trabajo también describe la aplicación de la fragmentación mediante MALDI-TOF MS en una estrategia de caracterización utilizando moléculas resueltas obtenidas en la fracción de elución de IF MALDI-TOF MS. Dos ejemplos son descritos: el primero es con el pico mayoritario obtenido de la interacción entre H. carunculata y pepsina-NHS Sepharose™, fragmentado mediante CID MALDI-TOF/TOF y analizado mediante secuenciación de novo. Otro ejemplo emplea la fragmentación ISD MALDI-TOF MS con la fracción de elución de S. helianthus y tripsina-glioxal Sepharose®. Otro objetivo de este trabajo fue la purificación y caracterización estructural – funcional de NVCI, un inhibidor proteico de carboxipeptidasa aislado del caracol marino N. versicolor. La combinación de técnicas tales como la degradación automatizada de Edman y secuenciación de novo por MALDI-TOF MS permitió el establecimiento de su estructura primaria. La síntesis y clonación del ADNc de NVCI permitió confirmar su secuencia de aminoácido. NVCI es una proteína con 53 residuos de aminoácidos, una masa molecular de 5944 Da y tres puentes disulfuro (código UniProt: P86912). NVCI recombinante fue producido en Pichia pastoris con un alto rendimiento en términos de proteína y actividad. NvCI natural y recombinante mostraron valores de Ki en el intervalo picomolar frente a carboxipeptidasas, tales como bCPA1, hCPA1 y hCPA4. Otros valores de Ki estuvieron en el orden de 1x10-10 M (hCPB1, pCPB1, hTAFI y bTAFI), con excepción de hCPA2 (1x10-9 M). NVCI es un inhibidor competitivo reversible de unión fuerte que representa el inhibidor de carboxipeptidasa más potente de naturaleza proteica descrito actualmente. La estructura de rayos X del complejo NvCI-hCPA4 (código PDB: 4A94) mostró que el inhibidor está formado básicamente por dos hojas β centrales antiparalelas conectadas por tres bucles principales. Las hojas β son estabilizadas mediante tres puentes disulfuro, un pequeño núcleo hidrofóbico situado junto al extremo C-terminal de NvCI y algunos residuos hidrófobos voluminosos expuestos al disolvente. NVCI tiene un área total de contacto de 1875,1 Å2 con la hCPA4. El mecanismo de inhibición de NVCI se debe a una interacción competitiva con el sitio activo de la carboxipeptidasa, por oclusión de los subsitios S1', S1, S2 y S3, tal como se ha observado para la mayoría de los inhibidores proteicos de carboxipeptidasa reportados anteriormente. Estos sitios están ocupados por el extremo C-terminal de NVCI y constituyen la región de contacto primaria del inhibidor. La zona de contacto secundaria está más extendida y cubre casi una cara del inhibidor. Los valores de Ki inferiores observados para NVCI en comparación con otros inhibidores se pueden atribuir tanto a las regiones de interacción "primaria" y "secundaria", que crean una interfaz extendida con la carboxipeptidasa que minimiza la liberación de producto de la reacción catalítica.
The present work described the kinetic identification of carboxypeptidase A (CPA), carboxypeptidase B (CPB), pepsin, papain, trypsin and subtilisin inhibitory activities in 30 Caribbean marine invertebrate extracts. The qualitative results showed fourteen extracts with trypsin inhibitory activity, nine extracts with CPA, CPB, papain and subtilisin inhibitory activities, whilst only four extracts with pepsin inhibitory activity. The most promising extracts were selected in terms of specific inhibitory activity, dose-response relationships, IC50 values and bioavailability of the species On the other hand, Intensity Fading MALDI-TOF MS, a proteomic method used in this work to complete the strategy of identification of inhibitors in marine extracts, was very useful to detect molecules, able to selectively interact with immobilized target enzymes, such as in N. versicolor against CPA and CPB, H. carunculata with pepsin, S. helianthus with papain, N. peloronta and S. helianthus with trypsin and L. isodyctialis with subtilisin. This work also describes the application of MALDI-TOF MS fragmentation in a characterization approach using the resolved molecular species obtained in the elution fraction of IF MALDI-TOF MS. Two examples are described: The first one is the major peak resulting of the interaction between H. carunculata and pepsin-NHS Sepharose™, which was fragmented by CID MALDI-TOF/TOF and analyzed by de novo sequencing. Another example uses the ISD MALDI-TOF MS fragmentation with the elution fraction of S. helianthus and trypsin-glyoxal Sepharose®. Another goal of this work was the purification and structural – functional characterization of NvCI, a proteinaceous carboxypeptidase inhibitor isolated from the marine snail N. versicolor. The combination of techniques such as automated Edman degradation and de novo sequencing by MALDI-TOF MS allowed the establishment of its primary structure. The synthesis and cloning of cDNA encoding NVCI allowed to confirm its amino acid sequence. NvCI is a protein with 53 amino acid residues, a molecular mass of 5944 Da and three intrachain disulfide bridges (UniProt code: P86912). Recombinant NvCI was produced in Pichia pastoris system with a high yield in terms of protein and activity. The kinetic characterization of natural and recombinant NvCI was performed according to the strategy described for tight-binding inhibitors. Natural and recombinant NvCI displayed Ki values in the picomolar range against carboxypeptidases such as bCPA1, hCPA1 and hCPA4. Other Ki values are in the order of 1x10-10 M (hCPB1, pCPB1, hTAFI and bTAFI), except for hCPA2 (1x10-9 M). NvCI is a competitive reversible tight binding inhibitor which represents the most potent carboxypeptidase inhibitor from proteinaceous nature so far described. The X-ray structure of NvCI-hCPA4 complex (PDB code: 4A94) showed that the inhibitor is basically formed by a central anti-parallel β-two-stranded sheet connected by three major loops. The β-strands are stabilized by three disulfide bridges, a small hydrophobic core located next to the C-terminus of NvCI and a few bulky exposed hydrophobic residues to the solvent. NvCI displays a total contact area of 1875.1 Å2 with hCPA4. The inhibition mechanism of NvCI is due to a competitive interaction with the active site of the carboxypeptidase, by occlusion of the subsites S1’, S1, S2 and S3, as it has been observed for most of the reported proteinaceous carboxypeptidase inhibitors. These sites are occupied by the C-terminal tail of NvCI and constitute the primary contact region of the inhibitor. The secondary contact region is more extended and covers almost one face of the inhibitor. The lower Ki values observed for NvCI in comparison to the other inhibitors can be attributed to both, the “primary” and “secondary” interaction regions, which create an extended interface with the carboxypeptidase that minimizes the product release from the catalytic reaction.

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45

Lang, Rupert. "Klonierung, Expression, Reinigung und Kristallisation von Dipeptidyl Carboxypeptidase Röntgenstrukturanalyse der katalytischen Domänen von MMP-12, MT3-MMP und MMP-13/TIMP-2 /." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963846914.

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46

Henderson-Haefner, D. L. "Phage display antibodies to native and recombinant Anopheles gambiae midgut carboxypeptidase A : a potential anti-mosquito vaccine strategy for malaria endemic regions." Thesis, University of Aberdeen, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446600.

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Malaria is currently ranked as one of the most deadly human infectious diseases, causing an estimated three million deaths each year with half of these being children under the age of 5 years. Approximately 500 million people are currently infected with one or more species of Plasmodium, with 90% of the infections occurring in sub-Saharan Africa. In regions of the highest endemicity, a significant level of malaria transmission is vectored by the anthropophilic mosquito An. gambiae sensu stricto Giles. Using molecular techniques, native An. gambiae s.s. carboxypeptidase A, a secondary digestive enzyme that breaks down bloodmeal proteins required for egg production, was isolated and two recombinant forms were produced. These proteins were used as antigens in phage display panning to isolate humanised antibodies with high affinity to the enzyme. Two of these phage antibodies, A7 and B10, were used in in vitro assays and in vivo feeding studies. Neither antibody produced enzyme activity in vitro, but A7, which bind specifically to the active site region of the mosquito carboxypeptidase A, affected longevity, egg development, and F1 generation viability. B10, binding outside of the active site region, generally responded in the same patterns as the PBS control. A potential detrimental factor in the in vivo studies was the probable toxicity of imidazole, an eluate solution used in phage display, which may have contributed to the mortality rate of mosquitoes in the A7 and B10 treatment groups. These results have contributed to the redesign of the phage display protocol for use in n vivo mosquito systems.

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47

Antovic, Jovan P. "Thrombin activatable fibrinolysis inhibitor (TAFI) in different hemorrhagic and thrombotic conditions /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-540-9/.

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48

Hellio, Florence. "Synthèse peptidique par catalyse enzymatique application nouvelle à la radiosynthèse totale d'une hormone, la leucine-enkephaline tritiée, à l'aide de la carboxypeptidase Y." Grenoble 2 : ANRT, 1986. http://catalogue.bnf.fr/ark:/12148/cb375982654.

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49

Mombelli, Enrico. "Stabilité conformationnelle et mécanisme de dénaturation de protéines thermostables : étude de la carboxypeptidase et de la ribonucléase Sso7D de l'archéobactérie thermoacidophile "Sulfolobus solfataricus"." Montpellier 2, 1999. http://www.theses.fr/1999MON20165.

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Abstract:

L'etude des determinants au niveau moleculaire de la stabilite des proteines est d'une importance fondamentale en biochimie. La comprehension du chemin de repliement des proteines est aussi une autre question biochimique centrale. Afin d'etudier ces questions, nous avons choisi deux proteines modeles de l'archaebacterie sulfolobus solfataricus : la ribonuclease sso7d et une carboxypeptidase. Ces proteines d'archaebacteries sont connues pour leur extreme stabilite et leurs proprietes catalytiques qui font d'elles des candidates ideales pour ce genre d'etudes. Les experiences menees dans cette these constituent une investigation thermodynamique et cinetique par le biais de la pression afin d'etudier la relation structure-fonction des proteines. La comprehension notamment de l'effet de la pression sur les proteines est parmi les aspects les moins explores en biophysique. Notre travail sur la carboxypeptidase suggere que la pression peut etre employee dans le domaine biotechnologique en tant que facteur thermo-stabilisant des proteines. L'etude effectuee sur la ribonuclease sso7d a demontre l'importance de la juxtaposition des acides amines aromatiques a l'interieur du centre hydrophobe et des liaisons hydrogene afin de stabiliser la structure native des proteines. L'etude des cinetiques de repliement (induites par des sauts de pression) a permis de caracteriser l'etat de transition de la reaction du repliement de sso7d par son volume d'activation. De plus, l'analyse fine des spectres d'absorbance uv par la methode des derivees quatriemes a revele des informations nouvelles concernant la mobilite des residus a l'interieur des proteines.

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50

Serfözö, Peter Daniel [Verfasser]. "Angiotensin-Converting Enzyme 2- and Prolyl Carboxypeptidase-Independent Conversion of Angiotensin II to Angiotensin-(1-7) in Circulation and Peripheral Tissues / Peter Daniel Serfözö." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2020. http://d-nb.info/1218076844/34.

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Dissertations / Theses on the topic 'Carboxypeptidase A'

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