Investigation of the Cellular and Molecular Mechanisms of Thrombocyte Integrin Signaling

血小板整合素信号传导的细胞和分子机制研究

基本信息

批准号：
10616738
负责人：
Zhao Wang
金额：
$ 39.66万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-05 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10616738
关键词：
3-Dimensional Adaptor Signaling Protein Affinity Agonist Architecture Binding Biochemical Biological Assay Biology Blood Coagulation Disorders Blood Platelets Cell Surface Receptors Cell surface Cells Cessation of life Cholesterol Classification Clinical Complex Cryo-electron tomography Cryoelectron Microscopy Cytoskeleton Defect Detergents Development Disease Dose Drug Design Drug Targeting Drug usage EGF gene Equilibrium Event Family Feasibility Studies Fibrinogen Future Goals Hematological Disease Hemorrhage Hour Human Hybrids Imaging Techniques Immunotherapy In Situ In Vitro Inflammation Integral Membrane Protein Integrins Investigation Ions Kinetics Laboratories Length Licensing Ligands Link Macrophage-1 Antigen Malignant Neoplasms Maps Membrane Modeling Molecular Molecular Conformation Molecular Probes Monoclonal Antibodies Myocardial Infarction Pathology Pharmaceutical Preparations Physiological Platelet Activation Platelet aggregation Process Property Protein Structure Initiative Proteins Regulation Reporting Research Resolution Roentgen Rays Role Shapes Signal Transduction Specimen Stroke Structure Surface Techniques Therapeutic Thermodynamics Thick Thigh structure Thrombasthenia Thrombocytopenia Thrombosis Time Vesicle Visualization antagonist biophysical techniques clinical effect clinically relevant effective therapy experimental study improved in situ imaging inhibitor insight microvesicles nanodisk nanometer resolution novel particle platelet function pre-clinical receptor reconstitution skills small molecule structural biology success targeted treatment therapeutic target thrombotic tomography von Willebrand Factor

项目摘要

Abstract Platelet integrin αIIbβ3 is central to platelet activation, which occurs through highly complex molecular interactions and structural alteration. Ultimately, αIIbβ3 matures its affinity for fibrinogen and von Willebrand factor. Dysregulation in αIIbβ3 affinity maturation events causes Glanzmann's thrombasthenia or thrombocytopenia. Therefore, fine-tuning these interactions through the αIIbβ3 receptor is a proven and effective strategy for both therapeutic thrombotic pathology targeting and immunotherapy. Currently, there are no αIIbβ3 inhibitors for long-term clinical use, while current inhibitors for short-term use may cause serious thrombocytopenia. The overall goal of our proposed research is to understand the molecular basis of the αIIbβ3 shapeshifting and the mechanochemistry of αIIbβ3 activation and inhibition by small molecules, including clinical- drugs. Our guiding hypothesis is that multiple forms of αIIbβ3 exist in a dynamic conformational equilibrium that is temporally and spatially regulated by cell signaling, association with the cytoskeleton, and interactions with exocellular ligands. We will determine, for the first time, the single-particle cryoEM structure of intact αIIbβ3, characterize structural transitions at the atomic level as they relate to physiological ligands (e.g., fibrinogen), and determine the effects of clinically relevant antagonists on αIIbβ3 conformational equilibrium both in situ and in vitro. All specific aims will elaborate integrin αIIbβ3 events at the molecular level pertaining to these objectives. Aim 1 experiments will characterize shapeshifting structural changes of αIIbβ3 induced by ligands or clinical- relevant drugs. We will investigate the kinetics of the conformational dynamics of the purified full-length αIIbβ3 using a single-particle cryoEM approach and wide-ranging biochemical techniques for the protein in solution. Aim 2 experiments will study architectural changes in intact αIIbβ3 by probing its conformational states on the cell surfaces using conformation-reporting mAbs and by directly solving in situ structures using cutting-edge advanced cryoET imaging techniques. Collectively, our proposed studies will provide unique molecular insights into structural and bidirectional signaling regulation of αIIbβ3, which will advance our understanding of integrin biology and may identify new antagonists for modulating αIIbβ3 function.

抽象的血小板整合素αIIBβ3是血小板激活的核心，该血小板通过高度复杂的分子发生相互作用和结构改变。最终，αIIBβ3将其对纤维蛋白原和von Willebrand的亲和力成熟因素。 αIIBβ3亲和力成熟事件的失调会导致Glanzmann的血栓杆菌或血小板减少症。因此，通过αIIBβ3受体进行微调这些相互作用是一种经过证明且有效的治疗性血栓性病理靶向和免疫疗法的策略。目前，没有αIIBβ3 长期临床使用的抑制剂，而当前用于短期使用的抑制剂可能会导致严重血小板减少症。我们提出的研究的总体目标是了解αIIBβ3的分子基础变形和αIIBβ3激活的机理和小分子抑制，包括临床毒品。我们的指导假设是，在动态构象平衡中存在多种形式的αIIBβ3，受细胞信号，与细胞骨架的关联以及与细胞外配体。我们将首次确定完整αIIBβ3的单粒子冷冻结构特征在原子水平上与物理配体（例如纤维蛋白原）和确定临床相关拮抗剂对原位和IN的αIIBβ3构象平衡的影响体外。所有具体目的都将在与这些目标有关的分子水平上详细说明整联蛋白αIIBβ3事件。 AIM 1实验将表征由配体或临床 - 诱导的αIIBβ3的结构变化相关药物。我们将研究纯化的全长αIIBβ3的构象动力学动力学使用单粒子冷冻方法和溶液中蛋白质的大范围生化技术。 AIM 2实验将通过探测其构象在上面的构象状态来研究完整的架构变化。使用会议报告的mAB并直接使用尖端求解原位结构的细胞表面高级冷冻成像技术。总的来说，我们提出的研究将提供独特的分子见解进入αIIBβ3的结构和双向信号调节，这将提高我们对整合素的理解生物学，并可能识别用于调节αIIBβ3功能的新拮抗剂。