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Unraveling the mechanism of ADAMTS13 resistance to protease inhibitionSingh, Kanwal January 2022 (has links)
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.
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The Role of TIMP3 in Models of Inflammation and ImmunitySmookler, David 01 September 2010 (has links)
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.
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The Role of TIMP3 in Models of Inflammation and ImmunitySmookler, David 01 September 2010 (has links)
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.
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Physical exercise training but not metformin attenuates albuminuria and shedding of ACE2 in type 2 diabetic db/db miceSomineni, Hari Krishna 05 June 2013 (has links)
No description available.
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Fonction de la glycoprotéine Golgi apparatus protein 1 (GLG1) dans la différenciation des adipocytes et l'effet de la forme de type sauvage et la forme tronquée de GLG1 sur le métabolisme des lipidesKatbe, Alisar 08 1900 (has links)
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.
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