Mechanical Regulation of Binding and Cleavage of VWF by ADAMTS-13

ADAMTS-13 对 VWF 结合和裂解的机械调节

• 批准号: 8274715
• 负责人: Cheng Zhu
• 金额: $ 36.77万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8274715
• 关键词: ADAMTS; Accounting; Adhesions; Atomic Force Microscopy; Behavior; Binding; Biological Process; Blood Circulation; Blood Platelets; Blood Vessels; Blood flow; Chemistry; Cleaved cell; Data; Disease; Disintegrins; Dissociation; Environment; Hemorrhage; Hemostatic function; Individual; Intervention; Kinetics; Measurement; Measures; Mechanics; Metalloproteases; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mutagenesis; Mutation; Pathology; Pathway interactions; Physiology; Poly A; Process; Proteolysis; Regulation; Site; Stretching; Testing; Thrombosis; Thrombotic Thrombocytopenic Purpura; Time; Triad Acrylic Resin; Variant; Von Willebrand Factor A Domain; base; insight; meetings; novel therapeutic intervention; protein structure; research study; single molecule; von Willebrand Disease; von Willebrand Factor

PROJECT SUMMARY We will employ a biophysical approach combined with mutagenesis to study the structural bases and biophysical mechanisms that regulate the molecular interaction between von Willebrand factor (VWF) and VWF-cleaving metalloprotease ADAMTS-13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs). We will focus on the processes of VWF domain unfolding, conformational changes, binding to and proteolytic cleavage by ADAMTS-13 at the level of single molecules or single pairs of molecules. The objective is to elucidate how mechanics regulate these molecular processes in order to understand how their functions are regulated by the blood flow in the circulation. These studies are organized into two specific aims. Aim 1 is to elucidate the regulatory mechanisms and structural bases of ADAMTS-13/VWF binding. Our hypotheses include: The CUB domains and the spacer domain of ADAMTS-13 bind to separate sites on the A domains of VWF. Initial binding involves either the CUB domains or the spacer domain and is regulated by separation distance. Subsequent binding of the second site is induced by the binding of the first site and regulated by applied force. We will determine how distance regulates formation, and how force regulates dissociation, of ADAMTS-13/VWF bonds, measure the effects of structural variations on distance-dependent formation and force-dependent dissociation of ADAMTS-13/VWF bonds, and develop a multi-site and multi-state binding model for the ADAMTS-13/VWF interaction. Aim 2 is to investigate the structural stability of VWF, its structural determinants, its regulation by ADAMTS-13 binding, and its regulation of ADAMTS-13 proteolysis. Our hypotheses include: Force regulates VWF-cleavage by ADAMTS-13 via disrupting noncovalent interactions between and/or within the A domains. This destabilizes the protein structure and induces catastrophic structural changes, which exposes the cryptic cleavage site in the A2 domain, allowing proteolysis by ADAMTS-13. The forced-induced structural changes in the A domains may also be regulated by binding of ADAMTS-13 to the A domains, which may change the A domain conformations. We will determine the kinetics of force-induced structural changes in A domains, its regulation by ADAMTS-13 binding, and its regulation of ADAMTS-13 proteolysis. We will also measure the effects of structural variations on forced-destabilization of A domains and on the force-regulated VWF proteolytic cleavage by ADAMTS-13. This project will clarify how mechanics regulates the chemistry of binding and cleavage of VWF by ADAMTS-13 to meet the stringent requirements for them to carry out their biological functions in the stressful environment of the circulation of rapidly flowing blood. Decoding how molecular structures determine these regulatory mechanisms will provide crucial insights into vascular physiology and pathology. Information thus obtained will also help develop new therapeutic approaches to inhibiting pathological platelet adhesion during thrombosis and/or intervention to thrombotic thrombocytopenic purpura (TTP) and/or the bleeding disorder von Willebrand diseases (VWD).

项目概要 我们将采用生物物理方法与诱变相结合来研究结构基础和 调节血管性血友病因子 (VWF) 和 VWF 裂解金属蛋白酶 ADAMTS-13（一种具有 ThromboSpondin 类型的解整合素和金属蛋白酶 1 主题）。我们将重点关注 VWF 结构域的展开、构象变化、结合和结合的过程。 ADAMTS-13 在单分子或单对分子水平上进行蛋白水解切割。目标 是阐明力学如何调节这些分子过程，以了解它们的功能 受循环中血流的调节。这些研究分为两个具体目标。目标 1 是 阐明 ADAMTS-13/VWF 结合的调节机制和结构基础。我们的假设 包括： ADAMTS-13 的 CUB 结构域和间隔结构域结合到 A 结构域上的单独位点 VWF。初始结合涉及 CUB 结构域或间隔结构域，并受分离调节 距离。第二个位点的后续结合是由第一个位点的结合诱导的，并受以下因素调节： 施加的力。我们将确定距离如何调节形成，以及力如何调节解离， ADAMTS-13/VWF 键，测量结构变化对距离依赖性形成的影响和 ADAMTS-13/VWF 键的力依赖性解离，并形成多位点和多状态结合 ADAMTS-13/VWF 交互的模型。目标 2 是研究 VWF 的结构稳定性，其结构 决定簇、ADAMTS-13 结合的调节以及 ADAMTS-13 蛋白水解的调节。我们的 假设包括： 力通过破坏非共价相互作用来调节 ADAMTS-13 对 VWF 的裂解 A域之间和/或内部。这会破坏蛋白质结构的稳定性并引发灾难性的后果 结构变化，暴露 A2 结构域中的神秘切割位点，允许通过以下方式进行蛋白水解： ADAMTS-13。 A结构域中强制诱导的结构变化也可以通过结合 ADAMTS-13 到 A 结构域，这可能会改变 A 结构域的构象。我们将确定动力学 力诱导的 A 结构域结构变化、ADAMTS-13 结合的调节及其对 ADAMTS-13 蛋白水解。我们还将测量结构变化对 A 的强制失稳的影响 结构域以及 ADAMTS-13 强制调节的 VWF 蛋白水解裂解。该项目将阐明如何 力学通过 ADAMTS-13 调节 VWF 的结合和裂解化学，以满足严格的要求 它们在循环压力环境下发挥其生物学功能的要求 快速流动的血液。解码分子结构如何决定这些调节机制将提供 对血管生理学和病理学的重要见解。由此获得的信息也将有助于开发新的 抑制血栓形成过程中病理性血小板粘附的治疗方法和/或干预 血栓性血小板减少性紫癜 (TTP) 和/或血管性血友病出血性疾病 (VWD)。