Microtubule-associated Rac

微管相关Rac

• 批准号: 8474833
• 负责人: Anna Birukova
• 金额: $ 36.76万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8474833
• 关键词: Actins; Actomyosin; Acute; Acute Lung Injury; Adaptor Signaling Protein; Adenomatous Polyposis Coli; Adherens Junction; Adult Respiratory Distress Syndrome; Agonist; Animal Model; Attenuated; Automobile Driving; Binding; Biochemical; Biological Preservation; Blood Vessels; Cell Adhesion; Cells; Complex; Complication; Cytoskeleton; Data; Development; Disease; Down-Regulation; Drug Design; Elements; Endothelial Cells; Endothelium; Equilibrium; Functional disorder; Future; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hepatocyte Growth Factor; Human; In Vitro; Inflammatory; Injury; Intercellular Junctions; Kinesin; Knockout Mice; Lead; Link; Lung; Lung Inflammation; Lung diseases; Mechanics; Mediating; Mediation; Microtubule Alteration; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular; Molecular Analysis; Molecular Target; Monomeric GTP-Binding Proteins; Motor; Pathogenesis; Peripheral; Permeability; Pilot Projects; Play; Prevention; Prevention therapy; Property; Protective Agents; Proteins; Publishing; Pulmonary Edema; Regulation; Research Proposals; Role; Sepsis; Signal Pathway; Signal Transduction; Small Interfering RNA; Thrombin; Time; Transducers; Translational Research; Vascular Permeabilities; Ventilator-induced lung injury; base; effective therapy; in vivo; in vivo Model; injured; insight; lung injury; lung preservation; lung repair; monolayer; mortality; multidisciplinary; novel; protective effect; protein complex; restoration; rho; rho GTP-Binding Proteins; targeted delivery

DESCRIPTION (provided by applicant): Microtubule-associated Rac regulation and acute lung injury. Compromised lung endothelial cell (EC) permeability leads to pulmonary edema, a serious complication observed in various lung diseases and associated with high mortality. Small GTPases Rac and Rho play opposing roles in EC cytoskeletal remodeling and cell junction dynamics, the critical mechanisms of endothelial barrier regulation. Precise regulation of small GTPase activities in different subcellular compartments may be a key mechanism of Rac-dependent enhancement of peripheral actin, cell junction complexes and EC barrier protective potential. It is known that increased microtubule (MT) disassembly leads to EC barrier dysfunction. Our published studies show that edemagenic and pro-inflammatory agonists thrombin, TGF2 and TNF1 increase EC permeability via Rho-dependent alteration of MT dynamics leading to MT- dependent actomyosin contraction. In contrast, effects of barrier-protective agents on crosstalk between MT, cell adhesions, and actin cytoskeleton are not clear, nor the role of MT changes in Rac regulation is described. Our novel data show that MT-mediated signaling is directly involved in the EC barrier preservation by hepatocyte growth factor (HGF), a circulating molecule with proven protective effects in the models of acute ling injury (ALI). These results have led us to a hypothesis that microtubules may mediate barrier-protective effects of HGF and attenuate acute pulmonary EC dysfunction via MT-associated signaling complexes. We hypothesize that MT-associated adaptor protein APC (adenomatous polyposis coli) can bind and deliver a novel Rac-specific guanine nucleotide exchange factor Asef along MT to the cell periphery, where Asef switches its cytoskeletal localization from MT to actin and targets actin-associated Rac effector IGQAP1. This mechanism may lead to a novel paradigm of microtubule- dependent enhancement of peripheral actin cytoskeleton and cell-cell junctions via targeted delivery of Rac activator Asef, which promote EC barrier properties and lung repair mechanisms in ALI. Specific Aim #1 will examine the role of APC and Asef in the Rac GTPase activation and HGF-induced barrier enhancement in pulmonary endothelial cells. Specific Aim #2 will study the role of MT in APC/Asef activation and intracellular localization. Specific Aim #3 will examine HGF-induced APC/Asef interactions with IQGAP1 and study the role of APC/Asef/IQGAP1 complex in HGF-induced EC barrier enhancement. Specific Aim #4 will use siRNA-based Asef knockdown in vivo, Asef knockout mice, and rescue strategies to evaluate Asef role in the lung protective mechanisms against ALI. These studies will characterize novel barrier protective mechanisms and identify new protein targets for future therapies aimed at prevention of the pulmonary vascular barrier dysfunction associated with acute lung injury.

描述（由申请人提供）：微管相关的 Rac 调节和急性肺损伤。肺内皮细胞（EC）通透性受损会导致肺水肿，这是各种肺部疾病中观察到的严重并发症，并与高死亡率相关。小 GTP 酶 Rac 和 Rho 在 EC 细胞骨架重塑和细胞连接动力学（内皮屏障调节的关键机制）中发挥相反的作用。不同亚细胞区室中小 GTP 酶活性的精确调节可能是 Rac 依赖性增强外周肌动蛋白、细胞连接复合物和 EC 屏障保护潜力的关键机制。众所周知，微管 (MT) 分解增加会导致 EC 屏障功能障碍。我们发表的研究表明，致水肿和促炎激动剂凝血酶、TGF2 和 TNF1 通过 Rho 依赖性 MT 动力学改变导致 MT 依赖性肌动球蛋白收缩，从而增加 EC 通透性。相反，屏障保护剂对 MT、细胞粘附和肌动蛋白细胞骨架之间的串扰的影响尚不清楚，也没有描述 MT 变化在 Rac 调节中的作用。我们的新数据表明，MT 介导的信号传导直接参与肝细胞生长因子 (HGF) 的 EC 屏障保护，HGF 是一种循环分子，在急性损伤 (ALI) 模型中具有已被证明的保护作用。这些结果使我们得出一个假设，即微管可能介导 HGF 的屏障保护作用，并通过 MT 相关信号复合物减轻急性肺 EC 功能障碍。我们假设 MT 相关接头蛋白 APC（腺瘤性息肉病大肠杆菌）可以结合新型 Rac 特异性鸟嘌呤核苷酸交换因子 Asef，并将其沿着 MT 传递到细胞外周，其中 Asef 将其细胞骨架定位从 MT 切换到肌动蛋白，并靶向肌动蛋白相关蛋白。 Rac 效应器 IGQAP1。这种机制可能会导致通过靶向递送 Rac 激活剂 Asef 微管依赖性增强外周肌动蛋白细胞骨架和细胞-细胞连接的新范例，从而促进 ALI 中的 EC 屏障特性和肺修复机制。具体目标#1 将检查 APC 和 Asef 在 Rac GTPase 激活和 HGF 诱导的肺内皮细胞屏障增强中的作用。具体目标#2 将研究 MT 在 APC/Asef 激活和细胞内定位中的作用。具体目标#3 将检查 HGF 诱导的 APC/Asef 与 IQGAP1 的相互作用，并研究 APC/Asef/IQGAP1 复合物在 HGF 诱导的 EC 屏障增强中的作用。具体目标 #4 将使用基于 siRNA 的 Asef 体内敲低、Asef 敲除小鼠和救援策略来评估 Asef 在针对 ALI 的肺保护机制中的作用。这些研究将表征新的屏障保护机制，并确定未来治疗的新蛋白质靶点，旨在预防与急性肺损伤相关的肺血管屏障功能障碍。