Protein Regulation and Cytoskeletal Dynamics of Pulmonary Endothelial Barrier Function

肺内皮屏障功能的蛋白质调节和细胞骨架动力学

• 批准号: 9223148
• 负责人: Patrick Belvitch
• 金额: $ 17.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9223148
• 关键词: Actins; Acute Lung Injury; Adaptor Signaling Protein; Adult Respiratory Distress Syndrome; Air; Alveolar; Area; Binding; Binding Sites; Biochemical; Biological Assay; Blood Vessels; Cell Shape; Cell membrane; Cells; Cessation of life; Complex; Cytoskeletal Proteins; DNA; Development; EMS1 gene; Endothelial Cells; Endothelium; Event; Extravasation; Fiber; Functional disorder; Generations; Goals; Human; Hypoxemia; Imaging Techniques; Individual; Infection; Inflammation; Kinetics; Knowledge; Kymography; Lead; Lipids; Liquid substance; Lung; Measurement; Measures; Mediating; Membrane; Microscopy; Modeling; Molecular; Morbidity - disease rate; Mus; Mutate; Myosin ATPase; Myosin Light Chain Kinase; Oranges; Pathologic; Patients; Peripheral; Permeability; Play; Pre-Clinical Model; Process; Proline; Proline-Rich Domain; Proteins; RNA Interference; Reagent; Recovery; Regulation; Research; Resolution; Respiratory Failure; Role; Scaffolding Protein; Site; Small Interfering RNA; Stimulus; Stress Fibers; Structure; Techniques; Time; Translating; ataxia telangiectasia mutated protein; clinically relevant; electric impedance; genetic regulatory protein; improved; in vivo; inhibitor/antagonist; insight; interstitial; lung injury; mortality; mouse model; new therapeutic target; non-muscle myosin; novel; overexpression; polymerization; protein complex; protein protein interaction; response; sphingosine 1-phosphate

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.

抽象的 肺内皮屏障功能的丧失是导致富含蛋白质的液体从肺中渗漏的原因。 血管间隙进入正常充满空气的肺泡，导致低氧血症、呼吸衰竭和 急性呼吸窘迫综合征（ARDS）患者的发病率和死亡率显着。一个专业 这种屏障功能的决定因素是细胞骨架重排和力的产生，进而改变细胞 形状和膜动力学导致细胞内间隙形成。该提案将机械地 表征 3 种关键细胞骨架蛋白的功能作用，这些蛋白整合起来决定肺 内皮细胞 (EC) 通透性：非肌肉肌球蛋白轻链激酶 (MLCK)，一种效应子 通过其在肌动蛋白-肌球蛋白棘轮中的催化作用实现细胞骨架重排和力产生 债券； cortactin (CTTN)，一种多功能接头蛋白和支架蛋白，在 许多动态细胞骨架过程，并且已知在以下领域与 MLCK 形成稳定的关联： 外周肌动蛋白聚合和重排响应屏障保护刺激；和肌动蛋白 相关蛋白质复合物 Arp 2/3，肌动蛋白聚合和这些外周分支的关键效应器 已知与 cortactin 相互作用的位点。这些蛋白质的具体作用及其作为一部分的相互作用 细胞骨架动力学的复杂性尚未完全表征。此外，后果是 细胞骨架动力学对膜动力学和最终屏障完整性的影响仍然不明确 内皮细胞，特别是肺血管系统中的内皮细胞。我们假设 CTTN、MLCK 和 Arp2/3 形成 调节肺 EC 细胞骨架结构和膜动力学的整合复合物 确定屏障功能。该提案将使用互补且复杂的分子、生物化学、 和成像技术来表征这些蛋白质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 在生成中的综合作用 在屏障增强或条件下调节肺 EC 通透性的关键肌动蛋白结构 扰乱。具体目标 2 将探索这些结构对膜的功能影响 动力学和屏障完整性，采用新型 MLCK 结构，在 cortactin 的假定位点发生突变 结合以及蛋白质抑制剂和沉默 RNA。具体目标 3 将使用这些新型试剂来 通过表征 MLCK 的综合功能效应，将我们的观察结果转化为临床前模型， LPS 诱导的急性肺损伤 (ALI) 小鼠模型中的 CTTN 和 Arp2/3。这些研究将提供新颖的 对 ALI/ARDS 细胞机制的深入了解和更好的理解将有助于 开发新的治疗靶点来扭转或减轻这些破坏性病理的影响 患有这些疾病的患者的状况。