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

项目摘要 该MIRA提案旨在揭示在细胞中金属蛋白酶的调节机制和底物， ADAMTS（一种具有血栓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 参与许多生物过程，已知一些成员对重要的人类活动有贡献。 疾病目前的研究主要集中在ADAMTS家族的两个成员，ADAMTS 13和ADAMTS 7。 项目1将研究ADAMTS 13的变构调节，ADAMTS 13是血液凝固的重要调节剂。我 最近发现ADAMTS 13采用静止的闭合构象并变构， 由其底物血管性血友病因子（VWF）激活。这项建议概述了一种机械的方法来研究 ADAMTS 13如何维持变构调节，并通过与VWF的相互作用破坏变构调节。利用 通过功能、动力学、结构和生物信息学分析，本项目确定了关键的结构要素 ADAMTS 13的变构调节。这项工作的结果将提高我们对遗传性疾病的理解。 以及ADAMTS 13缺陷引起的获得性凝血障碍。我还预计，我们的调查 ADAMTS 13将作为研究其他ADAMTS蛋白酶调控的模型。项目二重点是 已知ADAMTS 7通过促进动脉粥样硬化而导致冠状动脉疾病， 炎症然而，ADAMTS 7的底物靶点及其生物化学性质仍然很差， 明白以前试图解决这些研究问题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质量较差。为了规避这些 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绘 是ADAMTS 7的理想底物。新确定的基质将构成 研究ADAMTS 7活性的新生物化学工具，是未来研究目标筛选的必要条件。 对于ADAMTS 7的小分子抑制剂，可能具有翻译应用。Summer，the study of ADAMTS 13和ADAMTS 7的酶催化和调控为我们了解血栓形成的机制奠定了基础。 以及出血性疾病和心血管及炎症性疾病，这些结果可以应用于 研究其他ADAMTS蛋白酶。更重要的是，MIRA为首席研究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学员。

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.