Kindlin-3 signaling in platelets

血小板中的 Kindlin-3 信号传导

• 批准号: 10366509
• 负责人: Yan-Qing Ma
• 金额: $ 57.09万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-10 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366509
• 关键词: Affinity; Alanine; Avidity; Binding; Binding Proteins; Biochemical; Biological Assay; Biology; Blood Platelets; Bone Marrow Cells; CRISPR/Cas technology; Cardiovascular Diseases; Cell Adhesion; Cells; Complement; Crystallization; Cytoplasmic Tail; Data; Dimerization; Electron Microscopy; Family; Fibrinogen; Functional disorder; Genes; Goals; Gold Colloid; Hematopoietic; Hemorrhage; Hemostatic function; Hereditary Disease; Human; ITGB3 gene; Integrin beta Chains; Integrins; Interferometry; Lead; Lentivirus Vector; Leukocyte Adhesion Deficiency; Leukocytes; Life; Ligands; Mediating; Megakaryocytes; Molecular; Morbidity - disease rate; Mus; Mutagenesis; Mutate; Mutation; Myosin ATPase; Myosin Alkali Light Chains; Myosin Light Chains; N-terminal; Neck; PH Domain; Patients; Platelet aggregation; Process; Proteins; Receptor Signaling; Regulation; Research Project Grants; Risk; Role; Scanning; Signal Transduction; Site; Specificity; Spectrometry; Technology; Therapeutic; Thrombosis; Time; Transplantation; antibody conjugate; base; dimer; induced pluripotent stem cell; insight; loss of function mutation; member; mortality; novel; polypeptide; prevent; protein complex; structural biology; success; thrombotic

Integrin αIIbβ3 activation in platelets is an important step on the road to fibrinogen binding, platelet aggregation, and primary hemostasis. Overreaction of platelet accumulation, however, can lead to life-threatening arterial thrombosis, a hallmark of end-stage cardiovascular disease. Carefully controlled regulation of the activation state of αIIbβ3, therefore, is crucial for the ability of platelets to fulfill their hemostatic function while at the same time limiting thrombotic risk. The activation of αIIbβ3, like other members of the integrin family of cell adhesion and signaling receptors, is carefully regulated by the association of cytosolic proteins that bind in a cellular activation-dependent manner to the integrin β3 subunit cytoplasmic domain. Kindlin-3 is a FERM domain- containing protein that is primarily expressed in cells of hematopoietic origin. Previous studies have shown that members of the kindlin family promote integrin activation primarily by binding to the integrin β cytoplasmic tail and mediating integrin clustering via a process known as avidity modulation. Kindlin-3 has been shown to be essential for integrin activation, as exemplified by the finding that patients with leukocyte adhesion deficiency III, a congenital inherited disorder characterized by leukocyte and platelet dysfunction and severe bleeding, have causative loss-of-function mutations in the kindlin-3 gene. Precisely how kindlin-3 acts mechanistically to support integrin αIIbβ3 activation, however, remains largely unknown. Consistent with the integrin clustering hypothesis, a related kindlin, kindlin-2, has been shown to form a FERM domain-swapped dimer, and we have obtained preliminary data showing that dimerization is also required for kindlin-3 to promote avidity modulation of integrin αIIbβ3. Interestingly, however, dimeric forms of highly purified kindlin-3 have not been observed in either crystals or in solution, suggesting that extra components may be required to facilitate kindlin-3 dimerization and integrin clustering. In this regard, we have recently found that kindlin-3 directly interacts with the myosin essential light chain (ELC), a key component of myosin that exists as a dimer by attaching to the proximal neck regions of myosin. Importantly, preliminary data obtained in our lab has shown that the myosin ELC also contributes to avidity modulation of integrin αIIbβ3 in platelets. Based on these findings, the purpose of this application is to examine the hypothesis that the ability of kindlin-3 to dimerize and modulate αIIbβ3 activation involves a heretofore undescribed interaction with the myosin ECL. In Specific Aim 1, we will determine the molecular basis of kindlin-3 in interacting with the myosin ECL by employing multiple biochemical and structural approaches. In Specific Aim 2, we will determine the role of kindlin-3/ECL interactions in regulating the affinity and avidity modulations of integrin αIIbβ3 in both mouse and human platelets. Together, we believe that the finding from this study will establish the detailed molecular mechanism by which kindlin-3 fine-tunes integrin αIIbβ3 activation in platelets, which may lead to novel opportunities for developing safer and more specific anti-thrombotic strategies.

血小板中整合素αIIBβ3活化是纤维蛋白原结合，血小板聚集的重要步骤， 和原发性止血。但是，血小板积累过度反应会导致威胁生命的动脉 血栓形成，终末期心血管疾病的标志。精心控制激活的调节 因此 时间限制血栓形成风险。与整联蛋白家族的其他成员一样，细胞粘附的其他成员的激活 和信号接收器，通过在细胞中结合的胞质蛋白的关联仔细调节 积分依赖于整联蛋白β3亚基细胞质结构域的方式。 Kindlin-3是一个FERM域 - 含有主要在造血起源细胞中表达的蛋白质。先前的研究表明 Kindlin家族的成员通过与整联蛋白β细胞质尾巴结合来促进整联蛋白的激活。 并通过称为亲身调制的过程中介导整联蛋白聚类。 Kindlin-3已显示为 对于整联蛋白激活至关重要的，这是由白细胞粘附缺乏症患者III的发现所举例的 以白细胞和血小板功能障碍和严重出血为特征的先天性遗传疾病，具有 Kindlin-3基因中的功能丧失突变。恰好是Kindlin-3的机械作用 但是，支持整联蛋白αIIBβ3激活仍然在很大程度上未知。与整联蛋白簇一致 假设是一种相关的Kindlin，Kindlin-2，已显示出形成FERM域折叠的二聚体，我们有 获得的初步数据表明，Kindlin-3也需要二聚化来促进自动调制 整联蛋白αIIBβ3。然而，有趣的是，在 要么晶体或溶液中，这表明可能需要额外的组件来促进Kindlin-3 二聚化和整联蛋白聚类。在这方面，我们最近发现Kindlin-3直接与 肌球蛋白必需轻链（ELC），这是肌球蛋白的关键组成部分，它是通过附加到二聚体的 肌球蛋白的近端颈部区域。重要的是，我们实验室获得的初步数据表明肌球蛋白 ELC还有助于血小板中整联蛋白αIIBβ3的同意调节。基于这些发现，目的 该应用的是检查Kindlin-3二聚体和调节αIIBβ3的能力的假设 激活涉及迄今与肌球蛋白ECL的相互作用。在特定目标1中，我们将 通过使用多种 生化和结构方法。在特定目标2中，我们将确定Kindlin-3/ECL的作用 在确定整合素αIIBβ3的亲和力和亲和力调制方面的相互作用 血小板。我们认为，这项研究的发现将建立详细的分子机制 KINDLIN-3微调整联蛋白αIIBβ3在血小板中激活，这可能导致新的机会 制定更安全，更具体的反栓性策略。