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: $ 2.26万
• 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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=656001
• 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中的血小板组成，它们相互协调，在血管损伤的部位形成血块。血管性血友病因子(VWF)是一种大的多聚体血浆蛋白，通过将循环中的血小板捕获到受损的血管壁而启动血栓形成。ADAMTS13是一种金属蛋白酶，能裂解VWF，调节其与血小板的结合活性。过多的ADAMTS13活性会导致较小的VWF多聚体与血小板结合活性不足，这可能会导致出血。ADAMTS13活性过低会导致VWF的血小板结合活性过高，这可能会导致危及生命的血栓形成。因此，调节ADAMTS13的活性对于维持凝血系统的适当平衡至关重要。ADAMTS13作为一种活性蛋白酶被结构性地分泌到循环中，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控的活性，VWF目前是唯一已知的ADAMTS13底物。由于这些原因，人们对ADAMTS13活性的调控知之甚少。这项拟议的研究计划试图了解ADAMTS13蛋白酶活性的调节，以努力更好地确定其在生物学中的作用。目前，我的实验室有三个具体的研究目标，这将有助于我们理解ADAMTS13的特异性和调节。项目1将利用底物噬菌体展示来建立管理ADAMTS13特异性的规则。通过将高通量DNA测序整合到我们的噬菌体展示方法学中，我们可以在一次反应中同时测量文库中所有百万肽的富集度和耗尽率。得到的底物识别基序是高度精炼的，将有助于鉴定新的ADAMTS13底物。项目2试图了解ADAMTS13对天然蛋白水解酶抑制剂产生抗性的分子机制。由于ADAMTS13是ADAMTS蛋白水解酶家族中唯一能抵抗所有天然蛋白水解酶抑制剂的成员，我们将设计(A)ADAMTS13的缺失结构和(B)与其他ADAMTS蛋白酶的嵌合体，以确定ADAMTS13促进对蛋白水解酶抑制剂的抗性的特征。项目3将利用一种名为Bio-ID的新方法来识别ADAMTS13的新生理底物或配体。该项目将通过揭示ADAMTS13在心血管系统中潜在的新角色，对该领域产生重大影响。总体而言，我们的研究将解决有关ADAMTS13调控的重要问题，并在我们对ADAMTS13在生物学中的作用的理解上开辟令人兴奋的新领域。我们正在开发的许多研究ADAMTS13的方法都是广泛适用的，并将对自然科学中的蛋白酶研究产生广泛的影响。