Investigating the substrate specificity of the metalloprotease ADAMTS13

研究金属蛋白酶 ADAMTS13 的底物特异性

基本信息

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：
2020
资助国家：
加拿大
起止时间：
2020-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=709129
关键词：
Investigating substrate specificity metalloprotease ADAMTS13

项目摘要

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，它们协调作用在血管损伤部位形成血凝块。血管性血友病因子 (VWF) 是一种大型多聚体血浆蛋白，它通过将循环血小板捕获到受损的血管壁来启动凝块形成。 ADAMTS13 是一种金属蛋白酶，可裂解 VWF 以调节其血小板结合活性。 ADAMTS13 活性过高会导致 VWF 多聚体变小，血小板结合活性不足，从而导致出血。 ADAMTS13 活性过低会导致 VWF 具有过多的血小板结合活性，从而导致危及生命的血栓形成。因此，ADAMTS13 活性的调节对于维持凝血系统的适当平衡至关重要。 ADAMTS13 作为活性蛋白酶组成型分泌到循环中，并且不受任何已知的天然蛋白酶抑制剂的抑制。尽管这种活性看似不受监管，但 VWF 是目前 ADAMTS13 唯一已知的底物。由于这些原因，人们对 ADAMTS13 活性的调节知之甚少。该拟议研究计划旨在了解 ADAMTS13 蛋白酶活性的调节，以便更好地确定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 ADAMTS13 的特异性和监管。项目 1 将利用底物噬菌体展示来建立控制 ADAMTS13 特异性的规则。通过将高通量 DNA 测序纳入我们的噬菌体展示方法，我们可以在一次反应中同时测量文库中所有 6400 万个肽的富集和消耗。由此产生的底物识别基序经过高度精炼，将有助于识别新型 ADAMTS13 底物。项目 2 旨在了解 ADAMTS13 对天然蛋白酶抑制剂产生耐药性的分子机制。由于 ADAMTS13 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，因此我们将设计 (a) ADAMTS13 的缺失构建体和 (b) 与其他 ADAMTS 蛋白酶的嵌合体，以确定 ADAMTS13 促进对所有天然蛋白酶抑制剂产生抗性的特征。蛋白酶抑制剂。项目 3 将利用一种称为 Bio-ID 的新方法来鉴定 ADAMTS13 的新型生理底物或配体。该项目将通过揭示 ADAMTS13 在心血管系统中的潜在新作用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，并为我们对 ADAMTS13 在生物学中的作用的理解开辟令人兴奋的新天地。我们正在开发的许多用于研究 ADAMTS13 的方法都具有广泛的适用性，并将对自然科学中的蛋白酶研究产生广泛的影响。