Functional Involvement of IntegrinB4/ITGB4 and Kindlin/FERMT2 in Focal Adhesion Dynamic Remodeling in ARDS

IntegrinB4/ITGB4 和 Kindlin/FERMT2 在 ARDS 粘着动态重塑中的功能参与

基本信息

批准号：
10871783
负责人：
ANNE E CRESS
金额：
$ 40.52万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-20 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10871783
关键词：
3-Dimensional Acceleration Actins Acute Respiratory Distress Syndrome Address Adhesions Alternative Splicing Anti-Inflammatory Agents Automobile Driving Binding Blood Vessels COVID-19 pandemic Cell Adhesion Molecules Cell membrane Cells Chimeric Proteins Code Complex Cytoplasmic Tail Cytoskeletal Proteins Cytoskeleton DNA Methylation EMS1 gene Endothelial Cells Endothelium Erinaceidae Family suidae Focal Adhesions Functional disorder Generations Genes Genetic Genetic Polymorphism HIF1A gene Inflammation Inflammatory Integrin Binding Integrin beta4 Integrins Intercellular Junctions Laminin Liposomes Lung Mechanical Stress Mechanical ventilation Mediating Modeling Molecular Multiple Organ Failure PTK2 gene Peripheral Phosphorylation Post-Translational Protein Processing Pre-Clinical Model Proteins Pulmonary Circulation Rattus Reactive Oxygen Species Regulation Role Signal Transduction Signaling Molecule Simvastatin Site Stimulus Structure Therapeutic Variant Vascular Permeabilities Viral epigenetic regulation health disparity inhibitor insight link protein live cell imaging lung injury mechanotransduction mortality mutant nitration novel paxillin pharmacologic plectin porcine model pre-clinical promoter recruit response restoration transcription factor translational approach ubiquitin-protein ligase

项目摘要

ABSTRACT: The elevated ARDS mortality observed in the COVID-19 pandemic has highlighted the contribution of excessive mechanical stress produced by mechanical ventilation in promoting lethal increases in lung vascular permea- bility. Integrin β4 (ITGB4 gene) and kindlin-2 (FERMT2 gene) are essential adhesion molecules in endothelial cell (EC) focal adhesions (FAs), structures critical for mechano-sensing, for bidirectional signaling between the EC cytoskeleton and the cell-matrix interface, and for EC barrier regulation. The mechanistic basis for dy- namic FA coordination during inflammatory EC barrier dysfunction and subsequent barrier restoration is a fun- damental question that remains unresolved. We speculate that coordinate control of FA structures requires the dynamic interactions of integrin β4 (ITGB4) and kindlin-2 with key PPG cytoskeletal effectors (nmMLCK, cortactin, Dock1, lamellipodin, paxillin) to efficiently assemble functional FAs during EC barrier responses (pe- ripheral cytoskeletal remodeling, lamellipodial formation, gap closure). ITGB4 is a unique mechano-sensing, laminin-binding integrin, and kindlin-2 is a multi-domain mechano-sensing adapter FA protein that recruits struc- tural and signaling molecules to FAs in concert with cytoskeletal rearrangement. We speculate that these EC responses are highly influenced by ITGB4 and kindlin-2 post-translational modifications (PTMs) and coding pol- ymorphisms (SNPs). As reactive oxygen species (ROS) is an important stimulus for FA dynamics and loss of EC barrier integrity, with Core B, SA #1 will characterize the role of three ROS-sensing transcription factors (NRF2, HIF1α/HIF2α), ITGB4/FERMT2 SNPs and DNA methylation in genetic/epigenetic regulation of ITGB4/FERMT2 expression and the influence on generation of the unique ITGB4 alternatively-spliced, barrier- regulatory variant, Integrin β4E (ITGB4E), that we identified as involved in mechano-sensing and EC barrier regulation. With Core D, SA #2 will conduct in depth structure/function studies including 3D live cell imaging of mutant ITGB4 and kindlin-2 fusion proteins (SNPs, PTMs) to characterize ITGB4/kindlin-2 function in spatially- specific EC cytoskeletal rearrangements driving EC barrier-disruption and barrier-restoration. SA #3 will examine the functionality of ITGB4/kindlin-2 interactions within lamellipodia with known and novel FA-binding cytoskeletal partners, including cortactin, Dock1, lamellipodin and highly novel interactions with nmMLCK (Project #1). Finally, utilizing elegant rat and porcine models of LPS/VILI (Core C), SA #4 will assess the therapeutic utility of the SMURF inhibitor A01, or SRI-38832 to augment kindlin-2 expression as cargo in TySIPonate-conjugated liposomes (TySIPosomes, Project #4). Project #3 studies will determine the structure/function and molecular basis for the dynamic FA control by ITGB4 and kindlin-2 and yield important insights into functional relevance of this unique FA signaling axis in restoration of EC barrier function. Our highly translational approaches will also provide actionable EC barrier-regulatory strategies that restore the integrity of the injured pulmonary circulation while yielding insights into ITGB4/FERMT2 variant participation in ARDS health disparities.

抽象的：在 COVID-19 大流行中观察到的 ARDS 死亡率升高凸显了过度的贡献机械通气产生的机械应力可促进肺血管通透性的致命增加整合素 β4（ITGB4 基因）和 kindlin-2（FERMT2 基因）是内皮细胞中必需的粘附分子。细胞（EC）粘着斑（FA），对于机械传感、之间的双向信号传递至关重要的结构 EC 细胞骨架和细胞基质界面，以及 EC 屏障调节。炎症性 EC 屏障功能障碍和随后的屏障恢复期间的纳米 FA 协调是一个有趣的过程我们推测 FA 结构需要协调控制。整合素 β4 (ITGB4) 和 kindlin-2 与关键 PPG 细胞骨架效应器 (nmMLCK、 cortactin、Dock1、lamellipodin、paxillin）可在 EC 屏障反应期间有效组装功能性 FA（pe- ITGB4 是一种独特的机械传感、 kindlin-2 是一种多结构域机械传感适配器 FA 蛋白，可招募结构域我们推测这些 EC 与 FA 的结合和信号分子与细胞骨架重排相一致。反应很大程度上受到 ITGB4 和 kindlin-2 翻译后修饰 (PTM) 和编码 pol 的影响。活性氧 (ROS) 是 FA 动力学和丧失的重要刺激因素。 EC 屏障完整性，核心 B，SA #1 将表征三个 ROS 感应转录因子的作用 (NRF2、HIF1α/HIF2α)、ITGB4/FERMT2 SNP 和 DNA 甲基化在遗传/表观遗传调控中的作用 ITGB4/FERMT2 表达及其对独特 ITGB4 选择性剪接、屏障-生成的影响调节变体整合素 β4E (ITGB4E)，我们确定其参与机械传感和 EC 屏障借助 Core D，SA #2 将进行深度结构/功能研究，包括 3D 活细胞成像。突变 ITGB4 和 kindlin-2 融合蛋白（SNP、PTM），用于表征 ITGB4/kindlin-2 在空间上的功能 SA #3 将检查驱动 EC 屏障破坏和屏障恢复的特定 EC 细胞骨架重排。板状伪足内 ITGB4/kindlin-2 与已知和新型 FA 结合细胞骨架相互作用的功能合作伙伴，包括 cortactin、Dock1、lamellipodin 以及与 nmMLCK 的高度新颖的相互作用（项目 #1）。最后，利用 LPS/VILI（核心 C）的优雅大鼠和猪模型，SA #4 将评估以下药物的治疗效用： SMURF 抑制剂 A01 或 SRI-38832 增强 Kindlin-2 表达，作为 TySIPonate 缀合的货物脂质体（TySIPosomes，项目#4）。项目#3 研究将确定其结构/功能和分子。 ITGB4 和 kindlin-2 动态 FA 控制的基础，并对功能相关性产生重要见解这种独特的 FA 信号轴在恢复 EC 屏障功能方面也将发挥作用。提供可行的 EC 屏障调节策略，恢复受损肺循环的完整性同时深入了解 ITGB4/FERMT2 变异参与 ARDS 健康差异的情况。