Controlling allbB3 function by altering its energy landscape

通过改变其能量格局来控制 allbB3 功能

• 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
• 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

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：整联蛋白是一个普遍存在的跨膜异二聚体的家族，介导需要细胞 - 基质和细胞细胞相互作用的基本过程，并驻留在静息分子和活性的配体结合分子之间平衡的细胞表面上。像其他蛋白质一样，整合素也是“软”氨基酸聚合物，可连续采样构象状态的集合。当二价阳离子MN [2+]或细胞骨架蛋白塔林（Talin）将构象的分布从一个预先存在的种群转移到另一个人时，将从其静止状态转化为活性状态。静息整联蛋白构象通过涉及其膜细胞质，跨膜和叶膜外细胞外域的非共互相互作用来实现。当整联蛋白转移到其活性构象时，这些相互作用会破坏。该项目的重点是血小板整合素AllBB3，这是一种大分子配体的受体，例如纤维蛋白原和von Willebr和von Willebr和因子（VWF）。这些配体与AllBP3的结合是负责血小板聚集的，并且是形成止血血小板塞和病理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，并利用这种理解来开发新型的变构调节剂来减轻AllBP3功能。在AIM 1中，我们将生成用于AllBP3激活的热力学模型，并测试了以下假设：ALLB和B3之间的地形差异相互作用差异地调节了AllBP3激活。膜透明质细胞质，跨膜和ALLB或B3的叶膜外域对维持AllBB3在其静止构象中维持ALLBB3的相对贡献将使用新型的基于ARAC的细菌细菌转录器系统，以及基于光学镊子的基于光学镊子。数据将用于得出AllBB3激活的热力学模型。然后将筛选化学文库和分子数据库，以筛选与ALLB或B3相关的细胞外热点区域结合的潜在ALLBB3抑制剂。在AIM 2中，我们将使用可溶性抗跨膜结构域肽来修改原位单个AllBB3分子的激活状态。通过破坏AllBB3异二聚体的稳定，我们发现抗甲基跨膜结构域肽会导致B3结合C-SRC的配体结合非依赖性反式反式激活，这意味着单独的AllBB3激活足以引起快速的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的AllBB3拮抗剂。因此，我们建议使用计算方法设计稳定AllBB3 TM结构域异二聚体的肽，以防止AllBB3激活并用作开发新型抗强制性剂的铅化合物。