Investigating the substrate specificity of the metalloprotease ADAMTS13

研究金属蛋白酶 ADAMTS13 的底物特异性

• 批准号: RGPIN-2017-04692
• 负责人: Kretz, Colin
• 金额: $ 4.52万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760821
• 关键词: Investigating; substrate; specificity; metalloprotease; ADAMTS13

The coagulation system consists of plasma proteins and circulating platelets that coordinate to form a blood clot at sites of blood vessel damage. Von Willebrand Factor (VWF) is a large multimeric plasma protein that initiates clot formation by capturing circulating platelets to the injured vessel wall. ADAMTS13 is a metalloprotease that cleaves VWF to regulate its platelet binding activity. Too much ADAMTS13 activity leads to smaller VWF multimers with insufficient platelet binding activity, which can cause bleeding. Too little ADAMTS13 activity causes VWF to have excessive platelet binding activity, which can cause life-threatening thrombosis. Therefore, regulation of ADAMTS13 activity is critical to maintaining proper balance in the blood clotting system. ADAMTS13 is constitutively secreted into the circulation as an active protease and is not inhibited by any known natural protease inhibitors. Despite this seemingly unregulated activity, VWF is currently the only known substrate for ADAMTS13. For these reasons, the regulation of ADAMTS13 activity is poorly understood. This proposed research program seeks to understand the regulation of ADAMTS13 protease activity in an effort to better define its role in biology. Currently, my lab has 3 specific research goals that will contribute to our understanding of ADAMTS13 specificity and regulation. Project 1 will utilize substrate phage display to establish the rules that govern ADAMTS13 specificity. By incorporating high throughput DNA sequencing into our phage display methodology, we can simultaneously measure enrichment and depletion for all 64 million peptides in the library in a single reaction. The resulting substrate recognition motif is highly refined, and will aid in the identification of novel ADAMTS13 substrates. Project 2 seeks to understand the molecular mechanism for ADAMTS13 resistance to natural protease inhibitors. Because ADAMTS13 is the only member of the ADAMTS protease family that is resistant to all natural protease inhibitors, we will engineer (a) deletion constructs of ADAMTS13 and (b) chimeras with other ADAMTS proteases, to define the features of ADAMTS13 that promote resistance to protease inhibitors. Project 3 will utilize a new method called Bio-ID to identify novel physiologic substrates or ligands of ADAMTS13. This project will make a substantial impact on the field by uncovering potential new roles for ADAMTS13 in the cardiovascular system. Overall, our research will address important questions regarding ADAMTS13 regulation as well as break exciting new ground on our understanding of the role of ADAMTS13 in biology. Many of the methods that we are developing to investigate ADAMTS13 are widely applicable, and will make a broad impact to protease research in the natural sciences.

凝血系统由血浆蛋白和循环血小板组成，它们协调在血管损伤部位形成血凝块。血管性血友病因子（VWF）是一种大的多聚体血浆蛋白，其通过将循环血小板捕获到损伤的血管壁来启动凝块形成。ADAMTS 13是切割VWF以调节其血小板结合活性的金属蛋白酶。过多的ADAMTS 13活性导致血小板结合活性不足的较小VWF多聚体，这可能导致出血。太少的ADAMTS 13活性导致VWF具有过度的血小板结合活性，这可能导致危及生命的血栓形成。因此，ADAMTS 13活性的调节对于维持凝血系统的适当平衡至关重要。ADAMTS 13作为活性蛋白酶组成型分泌到循环中，并且不被任何已知的天然蛋白酶抑制剂抑制。尽管这种看似不受调节的活性，VWF是目前已知的ADAMTS 13的唯一底物。由于这些原因，对ADAMTS 13活性的调节知之甚少。 这项拟议的研究计划旨在了解ADAMTS 13蛋白酶活性的调节，以更好地定义其在生物学中的作用。目前，我的实验室有3个具体的研究目标，这将有助于我们了解ADAMTS 13的特异性和调控。项目1将利用底物噬菌体展示来建立ADAMTS 13特异性的规则。通过将高通量DNA测序结合到我们的噬菌体展示方法中，我们可以在单个反应中同时测量文库中所有6400万肽的富集和耗尽。所得到的底物识别基序是高度精制的，并将有助于鉴定新的ADAMTS 13底物。项目2旨在了解ADAMTS 13对天然蛋白酶抑制剂耐药性的分子机制。由于ADAMTS 13是ADAMTS蛋白酶家族中唯一对所有天然蛋白酶抑制剂具有抗性的成员，因此我们将工程化（a）ADAMTS 13的缺失构建体和（B）与其他ADAMTS蛋白酶的嵌合体，以定义ADAMTS 13促进对蛋白酶抑制剂的抗性的特征。项目3将利用称为Bio-ID的新方法来鉴定ADAMTS 13的新生理底物或配体。该项目将通过揭示ADAMTS 13在心血管系统中的潜在新作用对该领域产生重大影响。总的来说，我们的研究将解决有关ADAMTS 13调控的重要问题，并在我们对ADAMTS 13在生物学中的作用的理解上开辟令人兴奋的新天地。我们正在开发的许多研究ADAMTS 13的方法具有广泛的适用性，并将对自然科学中的蛋白酶研究产生广泛的影响。