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：
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
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/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

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 - 整联蛋白是α/β异二聚体细胞表面受体，它们通过结合配体的能力细胞外结构域并募集一系列在与细胞质尾部接近的效应蛋白，调节多样生物过程并在许多人类疾病中起关键作用。整联蛋白的正常生物学功能，例如αIIBβ3介导的止血，受激活和激活之间的紧密控制平衡调节停用状态。血小板中αIIBβ3整合素的不当激活会导致血栓形成，从而使αIIBβ3A 经过验证的抗栓性靶标。静止的αIIBβ3假定弯曲的考虑。激活后通过与细胞外或细胞质面结合的蛋白质，它经历了远距离构象重排的特征是延伸和打开头饰和腿部域的分离。高亲和力的配体结合需要这种构象激活，并将其归还静止状态，即停用，对于维持适当的αIIBβ3功能很重要。尽管结构研究揭示了多个整联蛋白的静态构象，代表非活性，中间和活性状态，仍然难以捉摸 α和β亚基都由多个域组成，共同执行可逆的大型主动状态和非活性状态之间的结构过渡。关于细胞的贡献知之甚少控制整联蛋白构象变化方面的膜。我们最近对β3的结构和功能研究缺乏α亚基揭示了整联蛋白构象调节在细胞表面的新方面，这表明以前未表征的细胞膜和其他元素的作用。我们已经确定了一系列配体 - 竞争性灭活抑制剂，可以使αIIBβ3的活化配体移位而不诱导构象变化。此外，我们已经改编了IPS衍生的人血小板的体内生产进行αIIBβ3信号研究。基于这些发现，本赠款的目标1试图检查分子整联蛋白会议激活和失活的机制，并剖析细胞的调节功能膜和其他未定义因素。使用灭活抑制剂作为工具化合物，我们将定义固有结构特征，利用新颖的组合来控制整联蛋白构象失活晶体学，生化和生物物理方法。获得的结构信息将用于通过使用小分子靶向概念变化来探索调节整联蛋白功能的新颖概念和抗体调节剂。第二个目标将使我们对整合素细胞质尾巴的研究扩展到检查超分子信号传导复合物的组成和动力学在激动剂和激动剂后形成细胞内形成配体诱导的αIIBβ3激活，该激活将在基因工程的人血小板中进行接近标记和蛋白质组学方法。总体而言，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，这将推动我们的了解整联蛋白生物学，并可能识别用于调节整联蛋白功能的新型治疗靶标。