Mechanical Regulation of Selectin-Ligand Binding Kinetics

描述(申请人提供)：这项建议的目的是阐明选择素和糖共轭配体之间分子相互作用的机械调节，这是循环白细胞在组织损伤或感染部位附着和跨越血管内皮细胞的多步黏附和信号级联反应的第一步。这些相互作用是至关重要的，因为它们的故障可能会导致一些炎症和血栓性疾病。选择素-配体的相互作用是在循环的流体动力环境中发生的，是机械调节的。我们的假设是，选择素-配体结合动力学的机械调节是由这些分子的结构决定的特定的原子水平相互作用的结果。力通过改变这些相互作用的能量格局和/或在力诱导的配位和/或构象变化中形成新的相互作用来调节键的解离，从而引发滑移键和捕获键。运输通过影响相互作用分子之间的碰撞频率和相遇时间来调节键的形成，调节缔合动力学对内在对接的依赖。由于选择素-配体结合动力学决定了细胞在流动下的功能，包括系留、滚动和聚集，相对较小的结构差异改变了原子水平的相互作用，可能会对生理和病理产生重大影响。这一广泛的假设将在三个综合的具体目标中得到验证：1)确定选择素和配体结构变化对它们相互作用的影响，2)通过分子动力学模拟在原子水平上定义选择素-配体相互作用，以及3)确定体内L-选择素依赖的黏附中配体特异性改变的后果。这三个具体目标结合了实验、理论和计算方法，包括硅学、体外和体内研究，并跨越了从原子水平的机制、单细胞和单分子动力学/力学实验到整个动物生理学的多个尺度。这项系统的研究将阐明选择素-配体结合动力学的机械调节如何使白细胞在循环的流体动力学环境中附着在血管壁上。破译分子结构如何决定这种调节将为血管生理学和病理学提供关键的见解。因此，这些数据可能为抑制炎症和血栓形成期间的病理性细胞黏附提供新的治疗方法。

DESCRIPTION (provided by applicant): The objective of this proposal is to elucidate the mechanical regulation of molecular interactions between selectins and glycoconjugate ligands, which mediate the first step of a multistep adhesion and signaling cascade for circulating leukocytes to attach to and migrate across vascular endothelium at sites of tissue injury or infection. These interactions are crucial, because their malfunction can result in a number of inflammatory and thrombotic disorders. Selectin-ligand interactions are regulated mechanically as they take place in the hydrodynamic environment of the circulation. Our hypothesis is that mechanical regulation of selectin-ligand binding kinetics results from specific atomic-level interactions that are dictated by the structures of these molecules. Force regulates bond dissociation by changing the energy landscape of these interactions and/or forming new interactions during force-induced fit and/or conformational changes, thereby eliciting slip and catch bonds. Transport regulates bond formation by influencing collision frequency and encounter time between interacting molecules, modulating the dependence of association kinetics on intrinsic docking. Since selectin-ligand binding kinetics determines cellular function under flow, including tethering, rolling, and aggregation, relatively minor structural differences that alter atomic-level interactions may have major consequences for physiology and pathology. This broad hypothesis will be tested in three integrated specific aims: 1) Define impact of structural variations in selectins and ligands on their interactions, 2) Define selectin-ligand interactions at the atomic level by molecular dynamics simulations, and 3) Define the consequences of ligand-specific alterations in L-selectin-dependent adhesion in vivo. The three specific aims combine experimental, theoretical and computational approaches, include in silico, in vitro, and in vivo studies, and span multiple scales from atomic-level mechanisms, single-cell and single-molecule kinetic/mechanics experiments, to whole animal physiology. This systematic study will clarify how the mechanical regulation of selectin-ligand binding kinetics enables leukocytes to adhere to blood vessel wall in the hydrodynamic environment of the circulation. Decoding how molecular structure determines this regulation will provide key insights into vascular physiology and pathology. As a result, the data may offer new therapeutic approaches to inhibiting pathological cell adhesion during inflammation and thrombosis.