Littérature scientifique sur le sujet « Collagenose »

Collagens are the major structural proteins in animal tissues. Their degradation is essential in embryogenesis and development, while unbalanced collagen breakdown is seen in diseases such as arthritis, atherosclerosis and cancer. Fibril-­forming collagens I, II and III consist of three polypeptide chains forming a triple helix, which is resistant to cleavage by most proteases. Collagenases of the matrix metalloproteinase family (MMP-­1, MMP-­8, and MMP-­13) degrade fibrillar collagens by locally unwinding the helix, followed by cleavage into ¼ and ¾ fragments. They comprise two domains, the catalytic (Cat) domain and the hemopexin (Hpx) domain, which are connected via a flexible linker. Both domains are essential for collagenolysis, but the exact sites of collagenase-­collagen interactions and how they unwind collagen remain elusive. This thesis addresses the roles of individual collagenase domains, and the sites in both the enzyme and the substrate that are involved in collagen binding and unwinding, focusing on the fibril-­forming collagens and human MMP-­1 as a prototype. MMP-­1 bound to immobilised collagen I with markedly higher affinity than its Hpx domain alone. The Cat domain alone failed to bind to collagen, but in the full-­length enzyme it participated in collagen binding. Above 25°C the two-­ domain binding involved the catalytic site cleft. Triple-­helical peptide (THP) Toolkits of collagens II and III were screened for MMP-­1 binding, and the collagenase binding motif has been established. It contains two hydrophobic residues within a 9 residue distance. Finally, hydrogen/deuterium exchange mass spectrometry (H/DXMS) experiments indicated two potential collagen binding sites: 285-­316 and 349-365 in the Hpx domain, and suggested a possibility of a dynamic interaction of the collagenase N-­terminus with collagen. These results imply that the two domains of collagenase bind to collagen in a cooperative manner. Based on the THP binding and H/DXMS data a 3D model of collagenase-­collagen interaction has been proposed. It assumes that collagenase utilises hydrophobic interactions to unwind the collagen helix via perturbation of the hydrogen-­bond network which stabilises the helix.

Le collagene represente 25% des proteines chez les mammiferes et seules les collagenases, qu'elles appartiennent a la famille des metalloproteases ou des proteases a serine, sont capables de le couper dans les conditions physiologiques. Parmi les 19 types de collagenes caracterises a ce jour, le collagene de type vi, compose de trois chaines polypeptidiques 1, 2 et 3, est le seul a former des filaments dits perles par un processus d'autoassociation remarquable. Nous avons etudie par cristallographie le fragment c5 du domaine globulaire c terminal de la chaine polypeptidique 3 du collagene de type vi qui presente 40% d'identite l'appi (amyloid- protein precursor inhibitor). La structure cristallographique de ce fragment c5 a ete resolue par remplacement moleculaire a la resolution de 1,6a puis affinee en utilisant des facteurs de temperature anistropes (shelxl-93) a 1,2a de resolution. Le facteur d'accord final est de 13,6% pour 10762 reflexions superieure a 4. La structure tridimensionnelle montre un repliement appartenant a la famille des inhibiteurs de proteases a serine de type kunitz. Des tests biochimiques ont montre que le fragment c5 ne possede pas d'activite inhibitrice envers les proteases a serine. Sur la base de la structure tridimensionnelle, nous avons propose quelques mutations permettant de restaurer l'activite inhibitrice. Des travaux recents de mutagenese dirigee realises sur un triple mutant asp, thr, phe ont confirme la validite de nos hypotheses. La deuxieme partie de cette these a ete consacree a l'etude de la collagenase (pm 25000 da) d'hypoderma lineatum. Elle est extraite du varon parasite du betail responsable de l'hypodermose. Lorsque nous avons aborde ce travail, aucun substrat ni inhibiteur synthetique n'etait connu. Nous avons caracterise deux substrats synthetiques, ce qui nous a permis de mettre en evidence trois inhibiteurs synthetiques. Par ailleurs, nous avons demontre les proprietes auto-inhibitrice de la collagenase due a la presence d'un coude (31-44) qui peut penetrer dans le site actif. Le dodecapeptide synthetique correspondant a cette structure secondaire a ete synthetise et son activite inhibitrice demontree. Par ailleurs, des anticorps polyclonaux produits contre ce peptide reconnaissent la collagenase. Nous avons clone la collagenase d'hypoderma lineatum dans e. Coli permettant ainsi une etude de mutagenese dirigee.

Mestrado em Biologia Molecular e Celular<br>Collagen is an abundant protein in all animals and is the main component of numerous tissues, having also structural and regulatory roles. Collagen can be found in various numerous locations presenting specific functions, such as the establishment of the cellular shape, its strength and structural integrity, and is also involved in tissue maintenance. Moreover, collagen has an important role in the formation of the extracellular matrix of the connective tissue, by contributing to its physical properties. Collagenases are enzymes, more specifically metalloproteinases, with a zincbinding domain. These enzymes are able to digest the triple helical region of native collagen, cleaving its peptide bonds. In recent years, bacterial collagenases have been gaining interest for industry proposes, by offering a variety of applications. In the clinical area, collagenases are used as therapy for diseases associated with excessive deposition of collagen, as for example, in patients suffering from Dupuytren’s disease. The enzymes can also be used to help the debridement of wounds and burns, and also for cancer gene therapy applications. The objective of this work is the comparison and optimization of two methods of recombinant collagenase purification, evaluating which method provides the most cost-effective purification. For that purpose, a simple and high resolution method for purification of histidine-tagged recombinant protein was used. Immobilized metal ion affinity chromatography (IMAC) with a Ni Sepharose 6 Fast Flow, through a column HisTrap FF 1ml or in batch were used. Results obtained through protein quantification by densitometry technique, showed that the purification using a column has higher yield. By zimography was possible determine the gelagenolytic activity of ColAh, and was also possible quantify the collagenolytic activity of ColAh, using a synthetic peptide N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGA). Obtained results also showed that the obtained recombinant protein is stable to storage over time and when submitted to different temperatures (room temperature; 4°C; -20°C).<br>O colagénio é uma proteína abundante em todos os animais, exercendo funções estruturais e reguladoras. Está presente em diversos locais e executa funções específicas, nomeadamente na manutenção dos tecidos e na conservação da sua estrutura celular, da sua força e integridade estrutural. Possui uma elevada importância na formação da matriz extracelular do tecido conjuntivo ao contribuir para as suas propriedades físicas. Colagenases são enzimas, mais especificamente metaloproteinases, com um característico domínio de ligação a zinco característico Atualmente, as colagenases bacterianas possuem diversas aplicações industriais. Por exemplo, na clínica são utilizadas no tratamento de doenças associadas a acumulação excessiva de colagénio, como a Doença de Dupuytren, no desbridamento de queimaduras e feridas e também em terapia génica oncológica. O objetivo deste trabalho é a comparação e a otimização de dois métodos de purificação, para avaliar qual dos processos será mais rentável. Para tal, foi utilizado um método de purificação de proteínas recombinantes, que foram marcadas com histidinas, utilizando um método de cromatografia de afinidade com ião metálico imobilizado (IMAC). Para comparação, a purificação foi estabelecida utilizando um método em coluna, HisTrap FF e um método em batch. Os resultados obtidos através da quantificação de proteína, usando técnica de quantificação por densitometria demonstraram que a purificação quando realizada por coluna, apresenta maior rendimento. Através de ensaios realizados posteriormente foi possível demonstrar a atividade da ColAh por zimografia de gelatina, e quantificar a atividade colagenolitica contra o péptido sintético N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA). Os resultados também demonstram que a proteína recombinante apresentou estabilidade, quando armazenada ao longo do tempo, a diferentes temperaturas (temperatura ambiente; 4°C; -20°C).

A polarimetric method for monitoring the rate of soluble collagen breakdown by collagenase enzyme action has been developed. The method represents an extension of previous physicochemical techniques based on viscometry, but is simpler and easier to carry out, particularly in the case of reaction rate studies. The method was developed arising from reports of collagenase activity measurement on inappropriate substrates such as gelatin, modified collagens and synthetic polypeptides. The optical method depends on measurement of the loss in optical rotation in solutions of soluble calfskin collagen resulting from initial enzymic cleavage of the collagen trip1e-helix, followed by spontaneous unwinding of the resultant unstable helical fragments. Specific assay conditions were chosen to ensure that the loss in optical rotation following enzymic cleavage was rapid and complete. The method is specific since in the absence of collagenase, non-specific proteinases produce only a limited decrease in solution optical activity. The method has also been compared with established physicochemical assay techniques and compares favourably with both viscometric and titrimetric collagenase assays. The availability of a rapid, sensitive and quantitative procedure for measurement of collagenase activity provides a convenient means for detecting the presence of collagenase in solution and examination of hide bacterial cultures for collagenase production. In addition, a study of biocidal compounds of potential interest in hide preservation for possible inhibitory effects on collagenase is conveniently carried out with the method. Fundamental research into synergistic action in enzymic hydrolysis of collagen is now possible, providing valuable insight into the mechanism of raw hide biodeterioration.