Functional Characterization of ALI-Associated MYLK SNPs

ALI 相关 MYLK SNP 的功能表征

基本信息

批准号：
8968757
负责人：
Alicia N Rizzo
金额：
$ 4.81万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-02 至 2019-09-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8968757
关键词：
Actins Actomyosin Acute Acute Lung Injury Affect African Agonist Asses Asthma Cells Code Complex Confocal Microscopy Disease Endothelial Cells Endothelium Extravasation Fluorescence Resonance Energy Transfer Focal Adhesions Functional disorder Genes Genetic Polymorphism Haplotypes Human Incidence Individual Inflammation Inflammatory Injury Investigation Kymography Liquid substance Lung Maintenance Measurement Mechanical Stress Membrane Modeling Myosin Light Chain Kinase Patients Phosphorylation Pre-Clinical Model Predisposition Proteins Recovery Regulation Research Resistance Risk Role Sampling Severities Stress Stress Fibers Structure Techniques Therapeutic Vascular Endothelial Growth Factors Ventilator Ventilator-induced lung injury Work clinically relevant design genetic regulatory protein health disparity in vivo mortality novel pre-clinical prolyl-serine public health relevance response restoration trafficking

项目摘要

DESCRIPTION (provided by applicant): Acute lung injury (ALI) is a disorder affecting over 200,000 U.S. patients per year and has an unacceptably high mortality of 30-35%. The pathophysiology of ALI centers on inflammation-induced disruption of the endothelial cell (EC) barrier lining the pulmonary vasculature, causing leakage of fluid, protein, and cells into the airspaces. Pulmonary endothelial barrier maintenance and recovery from injury is dependent on EC cytoskeletal dynamics. Myosin light chain kinase (MLCK) is a multi-functional cytoskeletal effector protein that is a key regulator of barrier function and has been a focus of the Garcia lab for over 20 years. This work has clearly established that barrier-disrupting edemagenic agonists produce spatially localized MLC phosphorylation within cytoplasmic contractile stress fibers, resulting in actomyosin contraction, tension, and formation of paracellular gaps. In contrast, barrier-protective agonists induce nmMLCK translocation to cortical actin networks and into lamellipodial membrane protrusions designed to close paracellular gaps and restore barrier integrity. Additionally, nmMLCK regulates lung trafficking of inflammatory cells and is robustly activated by biophysical forces such as excessive mechanical stress, which underlies its implications in ventilator induced lung injury (VILI). Furthermore, levels of nmMLCK expression and enzymatic activity contribute to risk and severity of ALI and VILI in preclinical models and human samples. Previously, the Garcia lab has identified a haplotype of three coding SNPs (Pro21His, Ser147Pro, Val261Ala, "3XSNP") in the nmMLCK gene (MYLK) that is highly associated with increased ALI susceptibility and severe asthma. This three SNP haplotype is significantly more common in individuals of African descent (AD) and likely contributes to the significant health disparities in ALI incidence and mortality. We hypothesize that these ALI-associated MYLK SNPs alter the structure of nmMLCK and that these structural changes impair lamellipodial activity and paracellular gap closure in response to inflammatory injury. We plan to perform functional studies of known MYLK SNPs using sophisticated biophysical and ultrastructural techniques including trans-endothelial resistance (TER) measurement, confocal microscopy and co-localization, kymography and fluorescence resonance energy transfer (FRET). Subsequently, we plan to validate the clinical relevance of these SNPs in established preclinical models of ALI and VILI and clarify nmMLCK antagonism as a novel ALI therapeutic strategy.

描述（由申请人提供）：急性肺损伤 (ALI) 是一种每年影响超过 200,000 名美国患者的疾病，其死亡率高达 30-35%，令人难以接受。 ALI 的病理生理学集中于炎症引起的肺血管内皮细胞 (EC) 屏障破坏，导致液体、蛋白质和细胞渗漏到气腔中。肺内皮屏障的维持和损伤恢复取决于 EC 细胞骨架动力学。肌球蛋白轻链激酶 (MLCK) 是一种多功能细胞骨架效应蛋白，是屏障功能的关键调节因子，一直是 Garcia 实验室的研究重点 20多年来。这项工作明确证实，破坏屏障的水肿激动剂会在细胞质收缩应力纤维内产生空间局部的 MLC 磷酸化，导致肌动球蛋白收缩、张力和细胞旁间隙的形成。相比之下，屏障保护激动剂诱导 nmMLCK 易位至皮质肌动蛋白网络和片状足膜突起，旨在关闭细胞旁间隙并恢复屏障完整性。此外，nmMLCK 调节肺部炎症细胞的运输，并被过度机械应力等生物物理力强力激活，这也是其在呼吸机引起的肺损伤 (VILI) 中的影响的基础。此外，nmMLCK 表达和酶活性水平会影响临床前模型和人类样本中 ALI 和 VILI 的风险和严重程度。此前，Garcia 实验室已在 nmMLCK 基因 (MYLK) 中鉴定出三个编码 SNP（Pro21His、Ser147Pro、Val261Ala、“3XSNP”）的单倍型，该单倍型与 ALI 易感性增加和严重哮喘高度相关。这三个 SNP 单倍型在非洲人后裔 (AD) 个体中更为常见，并且可能导致 ALI 发病率和死亡率的显着健康差异。我们假设这些 ALI 相关的 MYLK SNP 改变了 nmMLCK 的结构，并且这些结构变化损害了响应炎症损伤的板状足活性和细胞旁间隙闭合。我们计划使用复杂的生物物理和超微结构技术对已知的 MYLK SNP 进行功能研究，包括跨内皮电阻 (TER) 测量、共焦显微镜和共定位、kymography 和荧光共振能量转移 (FRET)。随后，我们计划在已建立的 ALI 和 VILI 临床前模型中验证这些 SNP 的临床相关性，并阐明 nmMLCK 拮抗作用作为一种新型 ALI 治疗策略。