Controlling allbB3 function by altering its energy landscape

通过改变其能量格局来控制 allbB3 功能

基本信息

批准号：
9062482
负责人：
Joel S. Bennett
金额：
$ 74万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9062482
关键词：
Adhesions Adhesives Affinity Amino Acids Atherosclerosis Attenuated Binding Blood Platelets Blood Vessels Cell Communication Cell physiology Cell surface Cells Collagen Complex Computing Methodologies Cytoskeletal Proteins Data Databases Development Divalent Cations Electron Microscope Endothelial Cells Equilibrium Excess Mortality Extracellular Domain Family Fibrin Fibrinogen Fibrinogen Receptors Fibrinolytic Agents Genetic Goals Hemorrhage Hemostatic Agents Hot Spot In Situ Individual Integrins Intravenous Lead Ligand Binding Ligands Measures Mechanics Mediating Membrane Membrane Proteins Modeling Molecular Molecular Conformation Myocardial Infarction Oral Pathogenesis Pathologic Peptides Pharmacology Physiology Platelet aggregation Play Polymers Population Process Proteins Regulation Reporter Rest Role SRC gene Sampling Spectrum Analysis Stroke Structure Surface System Talin Testing Thermodynamics Thrombus Transactivation Transmembrane Domain Trauma base clinical application cohesion design extracellular inhibitor/antagonist laser tweezer microscopic imaging nanomechanics novel prevent receptor research study small molecule libraries

项目摘要

Project 1: Integrins are a family of ubiquitous transmembrane heterodimers that mediate fundamental processes requiring cell-matrix and cell-cell interactions and reside on cell surfaces in an equilibrium between resting inactive molecules and active ligand-binding molecules. Like other proteins, integrins are "soft" amino acid polymers that continuously sample ensembles of conformational states. Conversion from their resting to active states occurs when allosteric modulators such the divalent cation Mn[2+] or the cytoskeletal protein talin shift the distribution of conformations from one pre-existing population to another. The resting integrin conformation is enforced by non-convalent interactions involving their membraneproximal cytoplasmic, transmembrane, and juxtamembrane extracellular domains; these interactions are disrupted when an integrin shifts to its active conformation. This project is focused on the platelet integrin allbB3 , a receptor for macromolecular ligands such as fibrinogen and von Willebr and factor (VWF) following platelet stimulation. Binding of these ligands to allbp3 is responsible for platelet aggregation and is a critical step in the formation of hemostatic platelet plugs and pathologic arterial thrombi. The overall goals of the project are gain a thermodynamic understanding of allbp3 regulation and to use this understanding to develop novel allosteric modulators to attenuate allbp3 function. In Aim 1, we will generate a thermodynamic model for allbp3 activation, testing the hypothesis that topographically-distinct interactions between allb and B3 differentially regulate allbp3 activation. The relative contribution of the membrane-proximal cytoplasmic, transmembrane, and juxtamembrane extracellular domains of allb or B3 to maintaining allbB3 in its resting conformation will be quantitated using a novel AraC-based bacterial transcriptional reporter system, as well as optical tweezers-based force spectroscopy. The data will be used to derive a thermodynamic model of allbB3 activation. Chemical libraries and molecular databases will then be screened for potential allbB3 inhibitors that bind to the relevant extracellular hot spot regions of either allb or B3. In Aim 2, we will use soluble anti-transmembrane domain peptides to modify the activation state of individual allbB3 molecules in situ. By destabilizing the allbB3 heterodimer, we found that an anti-allb transmembrane domain peptide causes ligand binding-independent transactivation of B3-bound c-Src, implying that allbB3 activation alone is sufficient to cause rapid allbB3 oligomerization. Conversely, by stabilizing the allbB3 heterodimer, transmembrane-domain targeted peptides would act as novel allbB3 antagonists. Thus, we propose using computational methods to design peptides that stabilize the allbB3 TM domain heterodimer, preventing allbB3 activation and serving as lead compounds for the development of novel antithrombotic agents.

项目 1：整合素是一类普遍存在的跨膜异二聚体，它介导需要细胞-基质和细胞-细胞相互作用的基本过程，并以静止非活性分子和活性配体结合分子之间的平衡存在于细胞表面。与其他蛋白质一样，整合素是“软”氨基酸聚合物，可以连续采样构象状态的集合。当二价阳离子 Mn[2+] 或细胞骨架蛋白 talin 等变构调节剂将构象分布从一种预先存在的群体转移到另一种群体时，就会发生从静止状态到活性状态的转换。静息整合素构象是通过涉及近膜胞质、跨膜和近膜胞外结构域的非共价相互作用来加强的；当整合素转变为其活性构象时，这些相互作用就会被破坏。该项目的重点是血小板整合素 allbB3，它是血小板刺激后纤维蛋白原和血管性血友病因子 (VWF) 等大分子配体的受体。这些配体与 allbp3 的结合负责血小板聚集，并且是形成止血血小板栓塞和病理性动脉血栓的关键步骤。该项目的总体目标是获得对 allbp3 调节的热力学理解，并利用这种理解开发新型变构调节剂以减弱 allbp3 功能。在目标 1 中，我们将生成 allbp3 激活的热力学模型，测试 allb 和 B3 之间拓扑上不同的相互作用差异调节 allbp3 激活的假设。 allb 或 B3 的近膜细胞质、跨膜和近膜胞外结构域对维持 allbB3 处于静止构象的相对贡献将使用新型基于 AraC 的细菌转录报告系统以及基于光镊的力谱进行定量。该数据将用于推导 allbB3 激活的热力学模型。然后将筛选化学文库和分子数据库，寻找与 allb 或 B3 的相关细胞外热点区域结合的潜在 allbB3 抑制剂。在目标 2 中，我们将使用可溶性抗跨膜结构域肽原位修饰单个 allbB3 分子的激活状态。通过破坏 allbB3 异二聚体的稳定性，我们发现抗 allb 跨膜结构域肽会导致 B3 结合的 c-Src 发生配体结合独立的反式激活，这意味着仅 allbB3 激活就足以引起快速的 allbB3 寡聚化。相反，通过稳定 allbB3 异二聚体，跨膜域靶向肽将充当新型 allbB3 拮抗剂。因此，我们建议使用计算方法来设计能够稳定 allbB3 TM 结构域异二聚体的肽，防止 allbB3 激活并作为开发新型抗血栓药物的先导化合物。