Protein Regulation and Cytoskeletal Dynamics of Pulmonary Endothelial Barrier Function

联系PD/PI：Belvitch，Patrick 摘要 肺内皮细胞屏障功能丧失是富含蛋白质液体渗漏的原因 进入正常充满空气的肺泡的血管空间，导致低氧血症，呼吸衰竭和 急性呼吸窘迫综合征(ARDS)患者的显著发病率和死亡率。一位少校 这种屏障功能的决定因素是细胞骨架重排和力的产生，这反过来又改变了细胞 形状和膜动力学导致细胞内缝隙的形成。这项提议将机械地 鉴定3种关键的细胞骨架蛋白的功能作用，它们整合在一起决定肺 内皮细胞(EC)通透性：非肌肉肌球蛋白轻链激酶(MLCK)，一种 肌动蛋白-肌球蛋白棘轮过程中细胞骨架重排和作用力的催化作用 Cortactin(CTTN)，一种多功能的适配器和支架蛋白，在 许多动态的细胞骨架过程，已知与MLCK在以下区域形成稳定的联系 外周肌动蛋白聚合和重排对屏障保护性刺激的响应；以及肌动蛋白 相关蛋白复合体Arp 2/3，在这些外周肌动蛋白聚合和分支的关键效应因子 已知与Cortactin相互作用的部位。这些蛋白质的特定作用及其相互作用是 细胞骨架动力学中的一个复合体还没有完全被描述出来。更重要的是， 细胞骨架动力学关于膜动力学和最终屏障完整性的定义仍然不明确。 内皮细胞，尤其是在肺血管系统。我们假设CTTN、MLCK和Arp2/3形成 调节肺内皮细胞骨架结构和膜动力学的集成复合体 确定屏障功能。这项提议将使用互补的和复杂的分子，生化， 和成像技术来表征这些蛋白的作用及其在调节肌动蛋白中的相互作用 结构。具体目标1将描述MLCK、CTTN和Arp 2/3在生成过程中的综合作用 屏障增强条件下调节肺内皮细胞通透性的关键肌动蛋白结构 颠覆。具体目标2将探索这些结构在膜上的功能后果 动力学和屏障完整性，采用新的MLCK结构，在皮质蛋白的假定位置突变 结合以及蛋白质抑制剂和沉默RNA。特定目标3将使用这些新型试剂来 通过描述MLCK的综合功能效应，将我们的观察转化为临床前模型， CTTN和Arp2/3在脂多糖诱导的小鼠急性肺损伤(ALI)模型中的表达。这些研究将为我们提供新的 对ALI/ARDS细胞机制的机械性见解和更好的理解将有助于 开发新的治疗靶点以逆转或减轻这些毁灭性的病理影响 对患者的病情进行调查。 项目摘要/摘要第6页

Contact PD/PI: Belvitch, Patrick ABSTRACT Loss of pulmonary endothelial barrier function is responsible for the leakage of protein rich fluid from the vascular space into the normally air filled alveoli which leads to hypoxemia, respiratory failure and significant morbidity and mortality in patients with acute respiratory distress syndrome (ARDS). A major determinant of this barrier function is cytoskeletal rearrangement and force generation which in turn alters cell shape and membrane dynamics leading to intracellular gap formation. This proposal will mechanistically characterize the functional roles of 3 critical cytoskeletal proteins that integrate to determine lung endothelial cell (EC) permeability: non-muscle myosin light chain kinase (MLCK), an effector of cytoskeletal rearrangement and force generation through its catalytic action in the ratcheting of actin-myosin bonds; cortactin (CTTN), a multifunctional adapter and scaffolding protein which serves a regulatory role in many dynamic cytoskeletal processes and is known to form a stable association with MLCK in areas of peripheral actin polymerization and rearrangement in response to barrier protective stimuli; and the actin related protein complex Arp 2/3, a key effector of actin polymerization and branching at these peripheral sites that is known to interact with cortactin. The specific roles of these proteins and their interactions as part of a complex in cytoskeletal dynamics are not yet fully characterized. Furthermore the consequence of cytoskeletal dynamics on membrane kinetics and ultimately barrier integrity remains ill-defined in the endothelium particularly in the lung vasculature. We hypothesize that CTTN, MLCK and Arp2/3 form integrated complexes which regulate pulmonary EC cytoskeletal structure and membrane dynamics to determine barrier function. This proposal will use complementary and sophisticated molecular, biochemical, and imaging techniques to characterize the roles of these proteins and their interactions in regulating actin structure. Specific Aim 1 will characterize the integrated roles of MLCK, CTTN and Arp 2/3 in generating critical actin structures that regulate permeability in lung EC under conditions of barrier enhancement or disruption. Specific Aim 2 will explore the functional consequences of these structures on membrane dynamics and barrier integrity, employing novel MLCK constructs, mutated at the putative site of cortactin binding as well as protein inhibitors and silencing RNA. Specific Aim 3 will use these novel reagents to translate our observations to a pre-clinical model by characterizing the integrated functional effects of MLCK, CTTN, and Arp2/3 in a murine model of LPS-induced acute lung injury (ALI). These studies will provide novel mechanistic insights and an improved understanding of the cellular mechanisms of ALI/ARDS which will aid in the development of new therapeutic targets to reverse or lessen the impact of these devastating pathologic conditions on patients who suffer from them. Project Summary/Abstract Page 6