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: 10052843
• 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/10052843
• 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 therapeutics; 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.

整合素是一种α/β异二聚体细胞表面受体，通过其结合配体的能力，通过 胞外区和在细胞质尾部附近招募一系列效应蛋白，调节不同的 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，受激活和激活之间的严格控制的平衡调节 停用状态。血小板中αIIbβ3整合素的不适当激活会导致血栓形成，使αIIbβ3a 有效的抗血栓靶点。静止的αIIbβ3呈紧凑的弯曲构象。在激活时诱导 通过与胞外或细胞质表面结合的蛋白质，它经历了一种远程构象。 重新排列的特征是头盔的伸展和打开，腿部区域的分离。 这种构象激活是高亲和力配体结合并使其返回静息状态所必需的，即 去激活，对于维持适当的αIIbβ3功能是重要的。尽管结构研究揭示了 整合素的静态构象，代表非活性、中间和活性状态，它仍然难以捉摸 α和β亚基都由多个结构域组成，共同作用于执行可逆的大- 缩放活动状态和非活动状态之间的结构转换。人们对细胞的贡献知之甚少 调节整合素构象变化的膜。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控的新方面，提示 细胞膜和其他元素以前未被描述的作用。我们已经确定了一系列的配体- 竞争性失活抑制剂，可在不诱导的情况下取代αIIbβ3的激活配体 构象变化。此外，我们还调整了iPS来源的人血小板的体外生产。 αIIbβ3信号研究。基于这些发现，这项资助的目标1试图检查分子 整合素构象激活和失活的机制及细胞调节功能的剖析 膜和其他不确定因素。使用失活的抑制剂作为工具化合物，我们将定义 利用新的组合管理整合素构象失活的内在结构特征 结晶学、生化和生物物理方法。获取的结构信息将用于 探索利用小分子靶向构象变化调控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细胞质尾巴的研究扩展到检测 激动剂后细胞内形成的超分子信号复合体的组成和动力学-和 配体诱导的αIIbβ3激活，这将在基因工程的人血小板中进行，使用 邻近标记和蛋白质组学方法。总的来说，我们提议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，这将促进我们的 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。