Structural Transition of Cellular Integrins and Applications Thereof

细胞整合素的结构转变及其应用

• 批准号: 10441416
• 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
• 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/10441416
• 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; antagonist; base; biophysical techniques; clinical application; clinically relevant; clinically translatable; design; extracellular; genetic regulatory protein; glycosylation; human disease; induced pluripotent stem cell; inhibitor; 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

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.

整合素是α/β异二聚体细胞表面受体，其通过其结合配体的能力， 细胞外结构域，并在细胞质尾附近招募一系列效应蛋白，调节多种 生物过程，并在许多人类疾病中发挥关键作用。整合素的正常生物学功能， 如αIIbβ3介导的止血作用，是由活化的和 停用状态。血小板中αIIbβ3整合素的不适当活化引起血栓形成，使αIIbβ3 a 经验证的抗血栓靶点。静息态αIIbβ3呈紧凑的弯曲构象。激活诱导后 通过与细胞外或细胞质表面结合的蛋白质，它经历了一个长距离的构象 重排的特征在于头部的延伸和打开以及腿部结构域的分离。 这种构象活化是高亲和力配体结合并使其返回到静息状态所必需的，即， 失活，对于维持适当的αIIbβ3功能很重要。尽管结构研究显示 整合素的静态构象，代表非活性，中间和活性状态，它仍然是难以捉摸的 α和β亚基都由多个结构域组成，它们协同作用以执行可逆的大- 在活动状态和非活动状态之间缩放结构转换。关于细胞的贡献知之甚少 膜在调节整合素构象变化中的作用。我们最近对β3在细胞中的结构和功能的研究 α亚基的缺失揭示了细胞表面整合素构象调控的新方面，表明 细胞膜和其他元素的先前未表征的作用。我们已经鉴定了一系列的配体- 竞争性失活抑制剂，可以取代αIIbβ3的活化配体，而不诱导 构象变化此外，我们已经调整了iPS衍生的人血小板的离体生产， 到αIIbβ3信号传导研究。基于这些发现，该补助金的目标1旨在检查分子 整合素构象激活和失活的机制，并剖析细胞的调节功能 膜和其他不确定因素。使用失活抑制剂作为工具化合物，我们将定义 本发明涉及一种利用新的组合控制整联蛋白构象失活的内在结构特征， 晶体学、生物化学和生物物理学方法。所获得的结构信息将用于 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 和抗体调节剂。第二个目标是将我们对整合素胞质尾区的研究扩展到检测整合素的细胞质尾区。 激动剂-和 配体诱导的αIIbβ3激活，这将在基因工程人血小板中进行， 邻近标记和蛋白质组学方法。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。 整合素生物学的理解，并可能确定新的治疗目标，调节整合素功能。