Mechanical Regulation of Selectin-Ligand Binding Kinetics

描述（由申请人提供）：本提案的目的是阐明选择素和糖缀合物配体之间分子相互作用的机械调节，其介导循环白细胞在组织损伤或感染部位附着并迁移穿过血管内皮的多步粘附和信号级联的第一步。这些相互作用是至关重要的，因为它们的功能障碍可能导致许多炎症和血栓性疾病。选择素-配体相互作用在循环的流体动力学环境中发生时受到机械调节。我们的假设是，机械调节选择素配体结合动力学的结果，从特定的原子水平的相互作用，是由这些分子的结构。力通过改变这些相互作用的能量景观和/或在力诱导的配合和/或构象变化期间形成新的相互作用来调节键解离，从而引发滑动和捕获键。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性。由于选择素-配体结合动力学决定了流动下的细胞功能，包括束缚、滚动和聚集，因此改变原子水平相互作用的相对较小的结构差异可能对生理学和病理学产生重大影响。这一广泛的假设将在三个综合的具体目标进行测试：1）定义选择素和配体的结构变化对它们的相互作用的影响，2）通过分子动力学模拟定义选择素-配体在原子水平上的相互作用，和3）定义L-选择素依赖性粘附在体内的配体特异性改变的后果。这三个具体目标结合了联合收割机实验、理论和计算方法，包括计算机模拟、体外和体内研究，并跨越了从原子级机制、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。本系统研究将阐明选择素-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粘附于血管壁。解码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见解。因此，这些数据可能提供新的治疗方法来抑制炎症和血栓形成过程中的病理性细胞粘附。

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.