ADAMTS13 resistance to protease inhibition / Background: ADAMTS13 is a metalloprotease that regulates the delicate balance between VWF multimeric length and its platelet capturing capacity. Unlike other ADAMTS and coagulation proteases, ADAMTS13 exhibits a prolonged half-life of several days as an active protease, suggesting that it is protected from inhibitors of metalloproteases in blood. Here, we investigate the mechanism by which ADAMTS13 is resistant to protease inhibition. Methods: C-terminal domain truncations of ADAMTS13 (MDTCS and MD) and chimeras with ADAMTS5 (MD13/TCS5, M13/DTCS5, MD5/TCS13, and MD5(TCS-CUB13)) were generated. Metalloprotease domain segments from ADAMTS5 were swapped into MDTCS13 corresponding to the gatekeeper triad (R193, D217, and D252) (MDTCS-G), the variable loop (G236-S263) (MDTCS-V5), and the calcium-binding loop (R180-R193) (MDTCS-C5). MDTCS-GVC5 was generated to study these features simultaneously. Alpha 2-macrogloublin (A2M), tissue inhibitors of metalloproteinases (TIMPs), and small molecule inhibitor (Marimastat) were used as inhibitors, and tested using FRETS-VWF73 and Western blot. Results: MDTCS, MD, MD13/TCS5, M13/DTCS5, MDTCS-G, MDTCS-V5, and MDTCS-C5 constructs were resistant to all inhibitors, whereas MD5/TCS13 was inhibited. The presence of the closed conformation attenuated MD5(TCS-CUB13) proteolysis by 50-fold, while displaying a slower rate of inhibition compared to MD5/TCS13. We report the kinetic parameters of the unique features of the metalloprotease domain (the gatekeeper triad, the variable loop, and the calcium-binding loop). Moreover, simultaneously swapping these features sensitized MDTCS-GVC5 to Marimastat. Conclusion: Our findings reveal that the closed conformation confers global latency, while the metalloprotease domain confers local latency of ADAMTS13. The local latency is maintained by the flexibility of the variable loop and the calcium-binding loop, which fold across the active site cleft to restrict inhibitor and substrate access. Extensive engagement of exosites by VWF can readily displace these loops, thereby activating ADAMTS13 from its latent form. Altogether, we present novel insight into the mechanism by which ADAMTS13 is resistant to protease inhibition. / Thesis / Doctor of Philosophy (PhD) / Hemostasis is the body’s natural process to prevent bleeding and maintain blood flow. The ability of a blood protein, called VWF, to stop bleeding upon injury is regulated by the protein ADAMTS13. ADAMTS13 circulates in the blood for days, but its function cannot be stopped by inhibitors. Here, we investigate the mechanism by which ADAMTS13 is resistant to inhibition. We found that several structures of ADAMTS13, called domains and loops, protect it from inhibitors. Folding of the distal domains to the centre of ADAMTS13 partially protected ADAMTS13 from inhibitors. Further investigation revealed that two flexible loops close to the active site of ADAMTS13 were primarily responsible for protecting ADAMTS13 from inhibitors. We suggest that the flexibility of these loops guard against inhibition by folding across the active site. These results are important because advances have been made to use ADAMTS13 therapeutically in many clotting illnesses, such as strokes.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

No description available.

Golgi apparatus protein 1 (GLG1) est une protéine transmembranaire de 160 kDa qui interagit avec l’apolipoprotéine B100 (apoB100), le récepteur des lipoprotéines de basse densité (LDLR) et la proprotein convertase subtilisin/kexin type 9 (PCSK9). Cependant, son mécanisme d’action et sa régulation post-traductionnelle sont inconnus. Des études ont montré que GLG1 subit deux clivages résultant en fragments solubles secrétés de 150 kDa et 55 kDa. Dans cette étude, notre premier objectif est d’identifier les enzymes responsables de la protéolyse de GLG1 ainsi que l’effet du clivage sur sa fonction dans le métabolisme des lipides. De plus, les résultats de nos collaborateurs montrent que les souris adultes déficientes en GLG1 ont un plus grand nombre d’adipocytes mais de taille plus petite que les souris de type sauvage. Notre deuxième objectif est de mesurer la variation de l’expression ainsi qu’identifier l’effet de GLG1 lors de la différentiation des fibroblastes en adipocytes. Pour le premier objectif, les cellules HEK293T surexprimant GLG1 ont été soit transfectées avec des convertases de proprotéines (PCSK) soit incubées avec différents inhibiteurs d’enzymes. Les milieux et les lysats cellulaires ont été analysés par immunobuvardage à la Western. Il n’y a pas eu de nouveaux fragments générés en présence des PCSK. Cependant, en présence d’inhibiteurs des sérines protéases apparentées à la trypsine soit AEBSF et Gabexate mesylate, il y a eu une réduction de la formation du fragment de 55 kDa. Pour identifier la métalloprotéase responsable du clivage de l’ectodomaine générant le fragment de 150 kDa, GLG1 a été transfectée avec les Tissue Inhibitor of Metalloproteinase (TIMP 1-4). Nos résultats ont montré que TIMP3 empêche la relâche de l’ectodomaine de GLG1 dans le milieu de culture. Finalement, nos analyses de plasma de souris par immunobuvardage à la Western ont montré la présence des fragments de 150 kDa et 55 kDa de GLG1 in vivo. Pour le deuxième objectif de l’étude, les fibroblastes préadipocytaires de souris 3T3-L1 ont été différenciés en adipocytes. Des lysats cellulaires et l’isolation d’ARN ont été effectués aux jours 0, 2, 4, 6, 8 et 10 de la différenciation. Des immunobuvardages à la Western ainsi que des RT-qPCR ont été réalisés pour analyser l’expression de GLG1 au cours de la différenciation. Nos résultats ont montré que l’expression de GLG1 augmente durant la différenciation. Bref, nos résultats démontrent que des enzymes trypsin-like clivent GLG1 et génèrent le fragment de 55 kDa. L’inhibition du clivage de l’ectodomaine de GLG1 par TIMP3 suggère que les ADAMs sont impliquées dans la relâche du fragment de 150 kDa. De plus, nous avons montré que l’expression de GLG1 augmente au cours de la différenciation adipocytaire. / Golgi apparatus protein 1 (GLG1) is a 160 kDa transmembrane protein interacting with apolipoprotein B100 (apoB100), low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). However, the protein’s posttranslational regulation and mechanism of action are poorly understood. Previous studies showed that GLG1 is cleaved resulting in two fragments of 150 kDa and 55 kDa secreted at the cell surface and in the extracellular matrix. The first objective of this study is to identify enzymes responsible for GLG1 proteolysis and the effect of cleavage on its function in lipid metabolism. Furthermore, our collaborators showed that mice with GLG1 knockout have a higher number of adipocytes, but those cells are smaller in size compared to those in wild type mice. Therefore, the second objective of the study is to measure the variation of GLG1 expression during adipocytes differentiation and to identify the effects of GLG1 knockout on adipocytes differentiation. For the first objective, HEK293T cells overexpressing GLG1 were either transfected with basic amino acid-specific proprotein convertases (PCSK) or treated with enzyme inhibitors. Media and cell lysates were analyzed by Western blot. No new fragments were detected in media of PCSK-transfected cells. Cell treatment with trypsin-like serine proteases inhibitors, AEBSF and Gabexate mesylate, reduced the secretion of the 55 kDa fragment. To identify the metalloproteinase responsible for GLG1 shedding, GLG1 was co-transfected with Tissue Inhibitors of Metalloproteinase (TIMP1-4). Our results showed that TIMP3 inhibits shedding of the 150 kDa fragment. Finally, wild-type mouse plasma was analyzed by Western blot and showed the presence of both fragments in vivo. For the second objective of the study, fibroblasts 3T3-L1 cells were differentiated into adipocytes and GLG1 mRNA and protein expression were measured at day 0, 2, 4, 6, 8 and 10 by qPCR and Western Blot. Our results showed that GLG1 expression increased during differentiation and a peak was observed at day 4. To conclude, in the first objective of our study, our results showed that trypsin-like enzymes cleave GLG1 and produce a 55 kDa fragment. Shedding of GLG1 is inhibited by TIMP3, which suggests that ADAM10 or ADAM17 are involved in the release of the 150 kDa fragment. In addition, both 55 kDa and 150 kDa fragments were found in normal mouse plasma supporting the relevance of our findings in vivo. In the second objective of our study, GLG1 expression increased during adipocyte differentiation suggesting a role in adipose tissue development and/or morphology. In conclusion, our study will help elucidate how proteolysis of GLG1 impacts its role in the regulation of apoB and PCSK9 secretion and lipid metabolism and how can GLG1 expression affect adipocytes differentiation.