Investigating the regulation and substrate processing of ADAMTS13 and ADAMTS7 metalloproteases.

项目摘要 这项Mira提案旨在揭示金属蛋白酶的调节机制和底物。 ADAMTS(一种带有血栓反应蛋白基序的去整合素和金属蛋白酶)家族。这些蛋白酶被认为是 参与了许多生物过程，其中一些成员对人类 疾病。目前的研究主要集中在ADAMTS家族的两个成员，ADAMTS13和ADAMTS7。 项目1将研究ADAMTS13的变构调节，ADAMTS13是一种重要的凝血调节因子。我 最近发现ADAMTS13采用静止的闭合构象，并成为变构 被其底物Von Willebrand因子(VWF)激活。这项建议概述了一种机械式的研究方法。 变构调节如何在ADAMTS13内维持，并被其与VWF的相互作用所扰乱。利用 功能、动力学、结构和生物信息学分析，该项目旨在确定关键的结构元素 在ADAMTS13中的变构调节。这项工作的结果将提高我们对遗传的理解 以及由ADAMTS13缺陷引起的获得性凝血障碍。我还预计我们对此的调查 ADAMTS13将作为研究其他ADAMTS蛋白水解酶调控的模型。项目2的重点是 ADAMTS7，已知通过促进动脉粥样硬化和 发炎。然而，ADAMTS7的底物靶点及其生化性质仍然较差 明白了。先前解决这些研究问题的尝试导致了文献中相互矛盾的报告， 可能是由于可用于研究这种新型蛋白酶的试剂和工具的质量较差。为了绕过这些 挑战，我们将利用蛋白质组学、酶学和蛋白质-蛋白质相互作用的新方法来描述 ADAMTS7的生化特性和理想底物。新确定的底物将成为 研究ADAMTS7活性的新生化工具，是筛选未来研究目标所必需的 用于可能具有翻译应用的ADAMTS7的小分子抑制剂。总而言之，对 ADAMTS13和ADAMTS7酶的催化和调节为我们理解血栓形成奠定了基础 以及出血性疾病、心血管疾病和炎症性疾病，这些结果可以应用于 研究其他ADAMTS蛋白酶。更重要的是，Mira为首席调查员提供了一个令人难以置信的 有机会招募和指导来自不同背景的学生和其他实习生。

Project Summary This MIRA proposal aims to uncover the regulatory mechanisms and substrates for metalloproteases in the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin motifs) family. These proteases are thought to be involved in many biological processes and some members are known to contribute to important human diseases. The current study focuses mainly on two members of the ADAMTS family, ADAMTS13 and ADAMTS7. Project 1 will investigate the allosteric regulation of ADAMTS13, an important regulator of blood clotting. I recently discovered that ADAMTS13 adopts a quiescent closed conformation and becomes allosterically activated by its substrate, Von Willebrand Factor (VWF). This proposal outlines a mechanistic approach to study how allosteric regulation is maintained within ADAMTS13 and disrupted by its interaction with VWF. Leveraging functional, kinetic, structural and bioinformatic analyses, this project sets to identify the key structural elements of allosteric regulation in ADAMTS13. The outcomes from this work will improve our understanding of inherited and acquired blood clotting disorders caused by defects in ADAMTS13. I also anticipate that our investigation of ADAMTS13 will serve as a model to study the regulation of other ADAMTS proteases. Project 2 focuses on ADAMTS7, which is known to contribute to coronary artery disease by promoting atherosclerosis and inflammation. However, the substrate targets of ADAMTS7 and its biochemical properties remain poorly understood. Previous attempts to address these research questions led to conflicting reports in the literature, likely due to the poor quality of reagents and tools available to study this novel protease. To circumvent these challenges, we will utilize novel methods in proteomics, enzymology, and protein-protein interactions to delineate biochemical properties and ideal substrates for ADAMTS7. Newly identified substrates will form the basis of novel biochemical tools to investigate ADAMTS7 activity and are necessary for future research goals to screen for small molecule inhibitors of ADAMTS7 that may have translational applications. Summarily, the study of ADAMTS13 and ADAMTS7 enzyme catalysis and regulation lay a foundation for our understanding of thrombotic and bleeding disorders and cardiovascular and inflammatory diseases, and these outcomes can be applied to study other ADAMTS proteases. More importantly, the MIRA offers the principal investigator an incredible opportunity to recruit and mentor students and other trainees from diverse backgrounds.