Structural Transition of Cellular Integrins and Applications Thereof

细胞整合素的结构转变及其应用

基本信息

批准号：
10198990
负责人：
Jieqing Zhu
金额：
$ 56.09万
依托单位：
VERSITI WISCONSIN, INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10198990
关键词：
Address Affinity Agonist Antibodies Attenuated Autoimmune Diseases Binding Binding Proteins Biochemical Biological Process Biology Blood Platelets Cell Adhesion Cell Line Cell Surface Receptors Cell membrane Cell model Cell physiology Cell surface Complex Coupled Cytoplasmic Tail Disease Elements Environment Equilibrium Event Exhibits Extracellular Domain Face Failure Family Family member Genetic Engineering Goals Grant Hemorrhage Hemostatic function Human Human Engineering Individual Inflammation Inherited Integrin alpha Chains Integrin beta Chains Integrins Label Lead Leg Ligand Binding Ligands Malignant Neoplasms Mediating Megakaryocytes Methodology Methods Modeling Molecular Molecular Conformation Mutation Myocardial Infarction Neoplasm Metastasis PTK2 gene Pathogenesis Pathway interactions Pharmaceutical Preparations Pharmacology Phosphotransferases Platelet Activation Platelet aggregation Play Process Production Property Proteins Proteomics RGD (sequence)Receptor Signaling Regulation Rest Role Series Signal Transduction Specificity Stroke Structure Talin Technology Therapeutic Thrombocytopenia Thrombosis base biophysical techniques clinical application clinically relevant clinically translatable design extracellular genetic regulatory protein glycosylation human disease induced pluripotent stem cell inhibitor/antagonist innovation insight member mimetics mouse model new therapeutic target novel novel strategies novel therapeutic intervention platelet function prevent prototype recruit small molecule spatiotemporal therapeutic target tool

项目摘要

SUMMARY - Integrins are α/β heterodimeric cell surface receptors, which, via their ability to bind ligands through extracellular domains and to recruit a series of effector proteins in proximity to cytoplasmic tails, regulate diverse biological processes and play critical roles in many human diseases. Normal biological functions of integrins, such as αIIbβ3-mediated hemostasis, are regulated by a tightly-controlled balance between activated and deactivated states. Inappropriate activation of αIIbβ3 integrin in platelets causes thrombosis, making αIIbβ3 a validated anti-thrombotic target. Resting αIIbβ3 assumes a compact bent conformation. Upon activation induced by proteins binding to either the extracellular or cytoplasmic face, it undergoes a long-range conformational rearrangement characterized by an extension and opening of the headpiece and separation of the leg domains. Such conformational activation is required for high-affinity ligand binding and returning it to the resting state, i.e. deactivation, is important for maintaining appropriate αIIbβ3 function. Although structural studies revealed multiple static conformations of integrin, representing the inactive, intermediate, and active states, it remains elusive how the α and β subunits, both of which consist of multiple domains, act in concert to perform the reversible large- scale structural transitions between the active and inactive states. Little is known about the contribution of cell membrane in regulating integrin conformational changes. Our recent structural and functional studies of β3 in the absence of α subunit revealed novel aspects of integrin conformational regulation on the cell surface, suggesting previously uncharacterized roles of the cell membrane and other elements. We have identified a series of ligand- competitive inactivating inhibitors, which can displace the activating ligand from αIIbβ3 without inducing conformational changes. Furthermore, we have adapted the ex vivo production of iPS-derived human platelets to αIIbβ3 signaling studies. Based on these findings, the Aim 1 of this grant seeks to examine the molecular mechanism of integrin conformational activation and deactivation and dissect the regulatory functions of the cell membrane and other undefined factors. Using the inactivating inhibitors as tool compounds, we will define the intrinsic structure features that govern integrin conformational deactivation utilizing a combination of novel crystallographic, biochemical and biophysical approaches. The acquired structural information will be used to explore novel concepts of modulating integrin function by targeting conformational changes using small-molecule and antibody regulators. The second aim will extend our studies on integrin cytoplasmic tails to examining the composition and dynamics of the supramolecular signaling complex formed intracellularly following agonist- and ligand-induced αIIbβ3 activation, which will be performed in genetically engineered human platelets using proximity labeling and proteomics approaches. Collectively, our proposed extracellular and intracellular studies will provide new molecular insights into structural and signaling regulation of integrins, which will advance our understanding of integrin biology and may identify novel therapeutic targets for modulating integrin function.

摘要 - 整合素是 α/β 异二聚体细胞表面受体，通过其结合配体的能力细胞外结构域并在细胞质尾部附近招募一系列效应蛋白，调节不同的整合素的生物过程并在许多人类疾病中发挥关键作用。例如 αIIbβ3 介导的止血作用，是通过激活和激活之间严格控制的平衡来调节的。血小板中 αIIbβ3 整合素的不适当激活会导致血栓形成，使 αIIbβ3 成为一种状态。经验证的抗血栓靶标，静息 αIIbβ3 在激活后呈现紧凑的弯曲构象。通过蛋白质与细胞外或细胞质表面结合，它经历了长程构象重新排列的特点是头件的延伸和打开以及腿部区域的分离。这种构象激活是高亲和力配体结合并将其恢复到静止状态所必需的，即，尽管结构研究揭示了多种原因，但失活对于维持适当的 αIIbβ3 功能非常重要。整合素的静态构象，代表非活性、中间和活性状态，但它如何仍然难以捉摸 α 和 β 亚基均由多个结构域组成，协同作用以执行可逆的大- 关于细胞在活性和非活性状态之间的尺度结构转变的贡献知之甚少。我们最近对 β3 的结构和功能进行了研究。 α亚基的缺失揭示了细胞表面整合素构象调节的新方面，表明我们已经确定了一系列以前未表征的细胞膜和其他元件的作用。竞争性失活抑制剂，可以取代 αIIbβ3 中的激活配体而不诱导此外，我们还调整了 iPS 衍生的人血小板的离体生产。基于这些发现，本次资助的目标 1 旨在检查分子。整合素构象激活和失活机制并剖析细胞的调节功能使用失活抑制剂作为工具化合物，我们将定义利用新颖的组合控制整合素构象失活的内在结构特征所获得的结构信息将用于晶体学、生物化学和生物物理方法。探索通过使用小分子靶向构象变化来调节整合素功能的新概念第二个目标是将我们对整合素细胞质尾部的研究扩展到检查。激动剂和后细胞内形成的超分子信号复合物的组成和动力学配体诱导的 αIIbβ3 激活，将在基因工程人类血小板中使用总的来说，我们提出的细胞外和细胞内研究。将为整合素的结构和信号调节提供新的分子见解，这将推动我们的研究了解整合素生物学并可能确定调节整合素功能的新治疗靶点。