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液体从 正常空气中充满空气的血管空间，导致缺氧，呼吸衰竭和 急性呼吸窘迫综合征（ARDS）的患者的发病率和死亡率显着。专业 该障碍功能的决定因素是细胞骨架重排和力产生，从而改变细胞 形状和膜动力学导致细胞内间隙形成。该建议将机械化 表征3种关键细胞骨架蛋白的功能作用，这些蛋白会整合以确定肺 内皮细胞（EC）渗透性：非肌肉肌球蛋白轻链激酶（MLCK），效应子 细胞骨架重排和力通过其在肌动蛋白肌球蛋白棘轮中的催化作用产生 债券； Cortactin（CTTN），一种多功能衔接子和脚手架蛋白，在调节作用中起着调节作用 许多动态细胞骨架过程，已知在 响应屏障保护刺激的外围肌动蛋白聚合和重排；和肌动蛋白 相关的蛋白质复合物ARP 2/3，这是肌动蛋白聚合和分支在这些周围分支的关键效应子 已知与Cortactin相互作用的位点。这些蛋白质的具体作用及其相互作用作为一部分 细胞骨架动力学中的复合物尚未完全表征。此外 膜动力学和最终屏障完整性的细胞骨架动力学在 内皮尤其是在肺脉管系统中。我们假设CTTN，MLCK和ARP2/3形式 调节肺EC细胞骨架结构和膜动力学的综合复合物 确定屏障功能。该建议将使用互补和复杂的分子，生化， 和成像技术以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。特定的目标1将表征MLCK，CTTN和ARP 2/3在生成中的综合作用 在屏障增强条件下，调节肺EC中渗透性的关键肌动蛋白结构或 破坏。特定的目标2将探索这些结构对膜的功能后果 使用新型MLCK构建体的动力和屏障完整性在Cortactin的推定位点突变 结合以及蛋白质抑制剂和沉默的RNA。特定目标3将使用这些新颖的试剂 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 在LPS诱导的急性肺损伤（ALI）的鼠模型中，CTTN和ARP2/3。这些研究将提供新颖 机械洞察力以及对ALI/ARD的细胞机制的了解，这将有助于 开发新的治疗靶标，以逆转或减少这些毁灭性病理的影响 患者患者的条件。