The Molecular Basis for Integrin-Mediated Bidirectional Signaling

白细胞是保护身体免受外来侵略者侵袭的基本免疫成分。 黏附、迁移、外渗和细胞间的通讯是通过 β2整合素的双向信号转导，整合素是存在于 白细胞表面。由于其复杂和多功能的作用，β-2整合素的调节失调 与自身免疫、心脏和肺部病理以及传染病和 几种癌症。尽管整合素是主要的治疗靶点，但药物开发一直是 由于我们在理解上的巨大差距而产生的意外副作用而受到阻碍 驱动特异性和整合素激活的机制。我将破译其分子基础 β2整合素激活、配体识别和双向信号转导 接近。我将利用我在低温EM方法开发方面的专业知识来捕获高分辨率 分离的β-2整合素和配体结合复合体的构象快照以揭示 与信号转导相关的动态结构重排和识别关键残基 调节配基专一性。使用细胞表面表达的整合素，我将评估功能 突变这些残基对配体结合和构象特异性的影响 抗体以及黏附、吞噬和细胞运动。对整合素有广泛的了解 在近乎自然的背景下，我将开发一个膜模拟系统，使用NEXT- 一代苯乙烯-马来酸共聚物。膜脂影响整合素的活化和 配体结合，是形成稳定络合物的关键。这些聚合物将提供一股- 预包埋整合素配体纳米盘的衬里提取纯化方法 在它们的自然环境中的复合体。我将利用这个系统来研究整合素在复杂的 Talin，中央整合素激活蛋白，结合细胞质上的保守基序 整合素上的区域。这将在分子细节上揭示整合素是如何被激活以传递信号的 质膜上的变构并定义了双向的分子基础 并提供了设计生化、生物物理和机械的框架。 研究更大的复合体并全面了解整合素的敏感实验 功能。最终，这项工作将为合理的设计提供结构蓝图 治疗自身免疫性疾病，这是该实验室的长期目标。

Leukocytes are essential immune components protecting the body against foreign invaders. Adhesion, migration, extravasation, and cell-cell communication are mediated though the bidirectional signaling of β2 integrins, which are integral membrane proteins found on the leukocyte surface. Due to their complex and multifunctional roles, dysregulation of β2 integrins is linked to autoimmune, cardiac and pulmonary pathologies as well as infectious diseases and several cancers. Although integrins are prime therapeutic targets, drug development has been hindered due to unanticipated side effects that arise from large gaps in our understanding of the mechanisms that drive specificity and integrin activation. I will decipher the molecular basis for β2 integrin activation, ligand recognition, and bidirectional signaling using an integrative approach. I'll build on my expertise in cryoEM method development to capture high- resolution conformational snapshots of isolated β2 integrin and ligand-bound complexes to reveal the dynamic structural rearrangements associated with signal transduction and identify key residues mediating ligand specificity. Using cell-surface expressed integrins, I'll assess the functional consequences of mutating these residues on binding of ligands and conformation- specific antibodies and on adhesion, phagocytosis, and cell motility. To gain broad insight into integrin allostery in a near-native context, I'll develop a membrane mimetic system using next- generation styrene maleic acid copolymers. Membrane lipids influence integrin activation and ligand binding and are key to forming stable complexes. These polymers will afford a stream- lined method to extract and purify nanodiscs embedded with pre- formed integrin-ligand complexes in their native environment. I will use this system to study integrin in complex with talin, the central integrin- activator protein that binds a conserved motif on the cytoplasmic region on integrin. This will reveal in molecular detail how integrins are activated to relay signals allosterically across the plasma membrane and define a molecular basis for bidirectional signaling as well as provide a framework for designing biochemical, biophysical, and mechano- sensitive experiments to study larger complexes and gain comprehensive insight into integrin function. Ultimately, this work will provide a structural blueprint for the rational design of therapeutics for autoimmune diseases, which is a long-term goal of the lab.