Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 143 p.; also includes graphics. Includes bibliographical references (p. 106-111). Available online via OhioLINK's ETD Center

Orientador: Gustavo Orlando Bonilla Rodriguez / Banca: Luiz Juliano / Banca: Vitor Marcelo Silveira Bueno Brandão de Oliveira / Banca: Marcos Roberto de Mattos Fontes / Banca: Paula Rahal Liberatore / Resumo: As peptidases ou proteases hidrolisam ligações peptídicas. Apesar de todas terem essa característica funcional comum, elas diferem acentuadamente no seu grau de especificidade. O conhecimento da especificidade das cisteíno-peptidase, nos fornece valiosas informações que podem levar a uma melhor compreensão da relação estrutura-função, do papel fisiológico destas enzimas, ou para o desenho de inibidores seletivos. Pela caracterização realizada, a Fastuosaina, uma cisteíno peptidase extraída de frutos verdes de gravatá (Bromelia fastuosa) possui um pH ótimo próximo do neutro, semelhante à Bromelina do talo e do fruto, por enquanto para a Papaína, que possui um pH ótimo de 6,3. Em relação à estabilidade térmica, a Fastuosaina mostrou ser mais resistente à desnaturação, seguida pela Papaína, a Bromelina do fruto e por último a Bromelina do talo. / Abstract: Peptidases, also known as proteases, hydrolyse peptide bonds with different specificities. Knowing their preferences for cleavage sites, gives valuable informations that can lead to a better understanding of the structure-function relationships, their physiological role, or for design of selective inhibitors. We performed a characterization of Fastuosain, a cystein-peptidase isolated from unripe fruits of "gravatá" (Bromelia fastuosa), which showed an optimum pH near 7.0, as found also for stem and fruit bromelains. Papain showed a lower value, at pH 6.3. Concerning its thermal stability, Fastuosain showed higher resistance to denaturation, followed by Papain, Fruit and Stem Bromelain. / Doutor

No description available.

L’ilot génomique pks de Escherichia coli et d’autres Enterobacteriaceae code des synthases depolycétides et de peptides non ribosomaux qui permettent l’assemblage d’un composé hybride polycétidepeptidenon ribosomal putative. Ce composé nommé colibactine induit des cassures double-brin de l’ADN descellules eucaryotes.La machinerie enzymatique codée par l’ilot pks comporte une protéine essentielle ClbP, atypique dansce type d’ilot. Nous avons montré que ClbP possède une partie N-terminale catalytique et périplasmique, et unepartie C-terminale associée à la membrane cytoplasmique. La structure cristalline de ClbP et des expériences demutagenèse ont révélé un site actif à sérine et des caractéristiques structurales originales, qui sont compatiblesavec une activité peptidase, confirmée par des analyses biochimiques. Dix homologues de ClbP ont été identifiésin silico dans des ilots génomiques de synthases de peptides non ribosomaux d’espèces bactériennes proches etéloignées. Tous les homologues testés ont présenté une promiscuité fonctionnelle avec ClbP. ClbP est donc leprototype d’une nouvelle sous-famille de peptidases, qui sont probablement impliquées dans la maturation decomposés peptidiques non ribosomaux.Par ailleurs, nous avons réalisé deux études épidémiologiques sur la prévalence de l’ilot pks dansl’espèce E. coli dans deux contextes physiopathologiques, l’urosepsis et les cancers coliques et rectaux. L’ilotpks était significativement associé aux souches issues d’urosepsis comparé à des souches commensales, et auxsouches issues de biopsies de tumeurs coliques comparé à des souches commensales ou issues de biopsies detumeurs rectales, de diverticuloses et de lésions iléales de maladie de Crohn. / The pks genomic island of Escherichia coli and other Enterobacteriaceae encodes polyketide andnonribosomal peptide synthases that build a putative hybrid PK-NRP compound. This compound designatedColibactin induces DNA double-strand breaks in eukaryotic cells.The pks-encoded enzymatic machinery comprises an essential protein ClbP, atypical for this type ofgenomic islands. We report that ClbP harbors a catalytic and periplasmic N-terminal part, and a C-terminal partassociated to the cytoplasmic membrane. ClbP crystal structure and mutagenesis experiments revealed a serineactivesite and original structural features, which are compatible with peptidase activity confirmed bybiochemical assays. Ten ClbP homologs were identified in silico in NRPS-encoding genomic islands of closeand distant-related bacterial species. All tested ClbP homologs showed functional promiscuity with ClbP. ClbPis therefore a prototype of a new subfamily of peptidases, which are probably involved for the maturation ofNRP compounds.Furthermore, we undertook two epidemiological studies on the prevalence of pks island in E. coli in twopathophysiology contexts; urosepsis and colorectal cancers. The pks island was significantly associated withurosepsis strains compared to commensal strains, and strains isolated from biopsies of colon tumors comparedwith commensal strains or strains isolated from biopsies of rectal tumors, diverticulosis and ileal lesions ofCrohn disease.

Colorado potato beetle (Leptinotarsa decemlineata) is an economically important herbivorous pest. Cathepsin D-like aspartic peptidase (LdCD) plays an important role during protein degradation in the midgut of Colorado potato beetle. This work describes the preparation of two expression systems, namely in Escherichia coli and Pichia pastoris, for the production of recombinant LdCD. The protocol for refolding of denatured LdCD was designed and optimized. Activation of the inactive LdCD zymogen and cleavage of the propetide (activation peptide) were investigated. This process proceeds autocatalytically at acidic pH or with the assistance of the cysteine peptidase legumain. The proteolytic activity of LdCD was characterized using fluorogenic peptidic substrate and protein substrates, and kinetic parameters and pH optimum were determined. The inhibition specificity of LdCD was analyzed using a panel of peptidase inhibitors. LdCD was significantly inhibited by PDI (potato cathepsin D inhibitor), a protein inhibitor produced in potato leaves. This suggests that PDI is a natural defense protein, which is directed against LdCD in the midgut of Colorado potato beetle in order to block the digestion. The potential application of PDI in the construction of transgenic crops resistant against insects is discussed.

Os fungos filamentosos são utilizados em larga escala na produção de produtos biotecnológicos na indústria devido a sua versatilidade e um dos exemplos são as peptidases que representam uma das principais classes de enzimas hidrolíticas. As peptidases são hidrolases que catalisam a quebra das ligações peptídicas das proteínas e que nos microrganismos são responsáveis por funções fisiológicas e patológicas, além de ter muitas aplicações em diversos campos industriais. Neste estudo foram analisados diferentes bioprocessos fermentativos para produção de peptidases pelo fungo Aspergillus terreus. Este microrganismo foi capaz de produzir peptidases em ambos os bioprocessos, sólido ou submerso, obtendo melhor performance e o pico de produção no bioprocesso fermentativo sólido de valor 677U/mL, nas condições 5g de farelo de trigo, 72 horas, 30°C e 75% de umidade. Utilizando o bioprocesso fermentativo submerso também obtivemos resultados satisfatórios com pico de atividade de 360U/mL, nas condições de meio padrão suplementado com Caseína 0,5%, 72 horas e 35°C. A caracterização bioquímica parcial dos extratos dos dois bioprocessos mostrou semelhanças entre algumas características das enzimas produzidas como a faixa extensa de pH ótimo abrangendo regiões ácidas, neutra e alcalinas, temperatura ótima pontual de 55°C e perfil de inibição pelo PMSF e EDTA. Contudo, os perfis de estabilidade (pH e temperatura) e comportamento frente a adição de íons apresentaram respostas diferentes entre si, o que sugere a produção de enzimas diferentes produzidas pelo mesmo fungo em meios distintos. A microencapsulação por Spray Drying como processo de estabilização foi satisfatória obtendo rendimentos de 37,5-58,2% e com níveis acima de 50% de atividade enzimática. Em contrapartida, a imobilização enzimática demonstrou ser eficaz na etapa de ligação ao suporte, mas não foi capaz de estabilizar a enzima presente no extrato, o que ficou caracterizado pela perda de atividade proteolítica. / Filamentous fungi are extensively used in the production of biotechnological products in industry because of their versatility and one of the examples are peptidases which constitute a major class of hydrolytic enzymes. The peptidases are hydrolases which catalyze the cleavage of peptide bonds of proteins and microorganisms that are responsible for the physiological and pathological roles, in addition to having many applications in various industrial fields. This study evaluated various bioprocesses for fermentative production of peptidases by the fungus Aspergillus terreus. This microorganism was able to produce peptidase in both bioprocess, solid or submerged, achieving better performance in the solid bioprocess with peak of production of 677U/mL under the conditions 5g wheat bran, 72 hours, 30°C and 75% humidity . Using submerged fermentation bioprocess we also obtained satisfactory results with peak of activity of 360U/mL with conditions of standard medium supplemented with 0.5% casein, 72 hours and 35°C. Biochemical characterization of the two partial purified extracts showed similarities between some characteristics of the enzymes produced, as large optimum pH range spanning regions acidic, neutral and alkaline point temperature optimum of 55 ° C and the inhibition profile of PMSF and EDTA. However, the stability profiles (pH and temperature) and behavior in addition ions showed different responses to each extract, which suggests the production of different enzymes in different ways. Microencapsulation by Spray Drying and stabilization process was obtaining satisfactory yields of 37.5 to 58.2%, with levels above 50% of enzyme activity. In contrast, the enzyme immobilization step was effective in bonding the support, but was not able to stabilize the enzyme present in the extract, which was characterized by the loss of proteolytic activity

La colibactine est une toxine largement distribuée chez Escherichia coli. Sa synthèse est assurée par des enzymes codés par un îlot génomique appelé pks. Elle provoque des cassures double brin de l'ADN, des mutations, d'important remaniements chromosomiques et favorise l'émergence de tumeurs intestinales en modèle murin. Par ailleurs, les E. coli producteurs colonisent fréquemment les tumeurs de patients atteints de cancer colorectal. Nos travaux montrent que les bactéries productrices de colibactine induisent la sénescence cellulaire et stimulent de façon indirect la prolifération cellulaire in vitro et la croissance tumorale in vivo. L'action pro-proliférative des cellules rendues sénescentes par les E. coli producteurs de colibactine est liée à la production du facteur de croissance HGF. L'étude de la signalisation cellulaire responsable montre l'implication du facteur de transcription c-Myc, l'activation de la transcription d'un microARN qui en ciblent la peptidase SENP1, et une modification de la SUMOylation des protéines de l'hôte, notamment p53, un effecteur connu de la sénescence cellulaire. Cette voie de signalisation et les transcripts codant HGF ont été analysés dans des tumeurs de patients atteints de cancer colorectal colonisés ou non par des E. coli producteurs de colibactine. Les résultats obtenus soutiennent les résultats obtenus in vitro et dans le modèle murin. L'ensemble suggère que les bactéries productrices de colibactine favorisent l'émergence d'un micro-environnement tumoral sénescent susceptible de favoriser la croissance tumorale via la sécrétion de HGF. En parallèle, nous avons caractérisé sur le plan structural et fonctionnel la protéine ClbP de l'îlot pks. Les résultats obtenus montrent que ClbP est une peptidase à serine active dont le site actif est extracytoplasmique et indispensable à l'activité biologique de l'îlot pks. Des inhibiteurs « drug-like » de ClbP ont été identifiés à l'aide d'approches structurales, biochimiques, cellulaires et microbiologiques. Ces molécules se lient au site actif de ClbP avec une affinité nanomolaire et bloquent les activités génotoxiques et pro-tumorales des E. coli producteurs de colibactine. ClbP constitue donc une cible thérapeutique potentielle permettant de bloquer les effets délétères des E. coli producteurs de colibactine. / The colibactin toxin is widely distributed in Escherichia coli. Its synthesis is performed by enzymes encoded by the genomic island pks. It causes DNA double-strand breaks, mutations, chromosomal rearrangements in host cells and contributes to tumorigenesis in a mouse model. In addition, colibactin-producing E. coli are frequently isolated from tumors of patients with colorectal cancer. Our work shows that colibactin-producing bacteria induce cellular senescence and, consequently, can indirectly stimulate cell proliferation in vitro and tumor growth in vivo. The pro-proliferative effect mediated by these senescent cells is due to the secretion of growth factors, in particular HGF. The cell signaling responsible for cellular senescence shows the involvement of the transcription factor c-Myc, the transcription of a microRNA targeting the peptidase SENP1, and a modification of protein SUMOylation, including p53, a well-known effector of cellular senescence. This signaling pathway and HGF-encoding transcripts were analyzed in tumors of patients with colorectal cancer colonized or not by colibactin-producing E. coli. The results support the findings obtained in vitro and in the mouse model. Taken together, the results suggest that, in tumors, colibactin-producing bacteria promote the emergence of a senescent microenvironment, which can stimulate tumor growth via the secretion of HGF. In parallel, we determined the structure and function of the pks-encoded protein ClbP. The results show that ClbP is an active serine peptidase, whose active site is extracytoplasmic and essential to the biological activity of pks island. "Drug-like" inhibitors of ClbP were identified using structural, biochemical, cellular and microbiological approaches. These molecules bind to the active site of ClbP with nanomolar affinity and block the genotoxic and pro-tumoral activities of colibactin-producing E. coli. ClbP is therefore a potential therapeutic target to block the deleterious effects of bacteria-producing colibactin.

The goal of our work was to characterize more precisely the role of ADAMTS2 in physiological and pathological processes, in order to develop potential therapeutic applications. We confirmed that the function of ADAMTS2 is essential during embryogenesis and development, including by its major role in the processing of fibrillar collagens type I and III in skin and lung tissues. Its specific activity was also matched up with that of two other aminoprocollagen peptidases, ADAMTS3 and ADAMTS14. The impact of ADAMTS2 inhibition during pathological formation of scar fibrous tissue was determined in two murine models of hepatic fibrosis and granulomatous reaction. In this context, ADAMTS2 appears as a therapeutic target of interest for the treatment of all process characterized by the deposition of an excessive scar matrix, including liver fibrosis. Finally, the potential anti-angiogenic properties of ADAMTS2 were demonstrated both in vitro and in vivo. Its potent activity during angiogenesis results from its action onto several distinct steps participating to the formation of new blood vessels. Molecular mechanisms by which ADAMTS2 modulates the behaviour of endothelial cells and inhibits tumor growth remain to be confirmed. L'objectif de nos études était de caractériser de manière plus précise le rôle de l'ADAMTS2 dans des processus physiologiques et pathologiques, et d'en déduire d'éventuelles applications en thérapeutique clinique. Nous avons confirmé que l'ADAMTS2 détenait une fonction essentielle au cours de l'embryogenèse et du développement, notamment par son rôle majeur dans la maturation des procollagènes fibrillaires de type I et de type III dans la peau et le poumon. Son activité spécifique a également été mise en perspective avec celle des deux autres aminoprocollagène peptidases, les ADAMTS3 et 14. L'impact de l'inhibition de l'ADAMTS2 au cours de la formation pathologique de tissu fibreux cicatriciel a été démontré dans des modèles murins de fibrose hépatique et de réaction granulomateuse à corps étranger. A ce titre, l'ADAMTS2 apparaît comme une cible thérapeutique d'intérêt pour le traitement de toute affection caractérisée par le dépôt d'une trame cicatricielle excessive, dont la fibrose hépatique. Enfin, le potentiel anti-angiogène de l'ADAMTS2 a été démontré à la fois in vitro et in vivo. Son efficacité remarquable résulte de son action au cours de plusieurs étapes distinctes de la formation des néo-vaisseaux. Les mécanismes moléculaires précis par lesquels l'ADAMTS2 agit sur les cellules endothéliales et l'inhibition de la croissance tumorale restent à préciser.

Os fungos filamentosos são utilizados em larga escala na produção de produtos biotecnológicos na indústria devido a sua versatilidade e um dos exemplos são as peptidases que representam uma das principais classes de enzimas hidrolíticas. As peptidases são hidrolases que catalisam a quebra das ligações peptídicas das proteínas e que nos microrganismos são responsáveis por funções fisiológicas e patológicas, além de ter muitas aplicações em diversos campos industriais. Neste estudo foram analisados diferentes bioprocessos fermentativos para produção de peptidases pelo fungo Aspergillus terreus. Este microrganismo foi capaz de produzir peptidases em ambos os bioprocessos, sólido ou submerso, obtendo melhor performance e o pico de produção no bioprocesso fermentativo sólido de valor 677U/mL, nas condições 5g de farelo de trigo, 72 horas, 30°C e 75% de umidade. Utilizando o bioprocesso fermentativo submerso também obtivemos resultados satisfatórios com pico de atividade de 360U/mL, nas condições de meio padrão suplementado com Caseína 0,5%, 72 horas e 35°C. A caracterização bioquímica parcial dos extratos dos dois bioprocessos mostrou semelhanças entre algumas características das enzimas produzidas como a faixa extensa de pH ótimo abrangendo regiões ácidas, neutra e alcalinas, temperatura ótima pontual de 55°C e perfil de inibição pelo PMSF e EDTA. Contudo, os perfis de estabilidade (pH e temperatura) e comportamento frente a adição de íons apresentaram respostas diferentes entre si, o que sugere a produção de enzimas diferentes produzidas pelo mesmo fungo em meios distintos. A microencapsulação por Spray Drying como processo de estabilização foi satisfatória obtendo rendimentos de 37,5-58,2% e com níveis acima de 50% de atividade enzimática. Em contrapartida, a imobilização enzimática demonstrou ser eficaz na etapa de ligação ao suporte, mas não foi capaz de estabilizar a enzima presente no extrato, o que ficou caracterizado pela perda de atividade proteolítica. / Filamentous fungi are extensively used in the production of biotechnological products in industry because of their versatility and one of the examples are peptidases which constitute a major class of hydrolytic enzymes. The peptidases are hydrolases which catalyze the cleavage of peptide bonds of proteins and microorganisms that are responsible for the physiological and pathological roles, in addition to having many applications in various industrial fields. This study evaluated various bioprocesses for fermentative production of peptidases by the fungus Aspergillus terreus. This microorganism was able to produce peptidase in both bioprocess, solid or submerged, achieving better performance in the solid bioprocess with peak of production of 677U/mL under the conditions 5g wheat bran, 72 hours, 30°C and 75% humidity . Using submerged fermentation bioprocess we also obtained satisfactory results with peak of activity of 360U/mL with conditions of standard medium supplemented with 0.5% casein, 72 hours and 35°C. Biochemical characterization of the two partial purified extracts showed similarities between some characteristics of the enzymes produced, as large optimum pH range spanning regions acidic, neutral and alkaline point temperature optimum of 55 ° C and the inhibition profile of PMSF and EDTA. However, the stability profiles (pH and temperature) and behavior in addition ions showed different responses to each extract, which suggests the production of different enzymes in different ways. Microencapsulation by Spray Drying and stabilization process was obtaining satisfactory yields of 37.5 to 58.2%, with levels above 50% of enzyme activity. In contrast, the enzyme immobilization step was effective in bonding the support, but was not able to stabilize the enzyme present in the extract, which was characterized by the loss of proteolytic activity