Functional Characterization of ALI-Associated MYLK SNPs

ALI 相关 MYLK SNP 的功能表征

• 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
• 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

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%，令人无法接受。ALI的病理生理学集中在炎症诱导的肺血管内皮细胞（EC）屏障破坏，导致液体，蛋白质和细胞泄漏到空气空间。肺内皮屏障的维持和损伤后的恢复依赖于EC细胞骨架动力学。肌球蛋白轻链激酶（MLCK）是一种多功能的细胞骨架效应蛋白，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多年了这项工作已经清楚地确定，屏障破坏性水肿激动剂在细胞质收缩应力纤维内产生空间定位的MLC磷酸化，导致肌动球蛋白收缩、张力和细胞旁间隙的形成。与此相反，屏障保护激动剂诱导nmMLCK易位到皮质肌动蛋白网络，并进入旨在关闭细胞旁间隙和恢复屏障完整性的片状脂质膜突起。此外，nmMLCK调节炎性细胞的肺运输，并被生物物理力如过度机械应力强烈激活，这是其在呼吸机诱导的肺损伤（VILI）中的意义的基础。此外，nmMLCK表达水平和酶活性有助于临床前模型和人体样本中ALI和VILI的风险和严重程度。此前，Garcia实验室已经确定了nmMLCK基因（MYLK）中三个编码SNP（Pro21 His，Ser 147 Pro，Val 261 Ala，“3XSNP”）的单倍型，该基因与ALI易感性增加和严重哮喘高度相关。 这三个SNP单倍型在非洲裔（AD）个体中明显更常见，并可能导致ALI发病率和死亡率的显著健康差异。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炎症损伤时的板脂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，包括跨内皮电阻（TER）测量，共聚焦显微镜和共定位，记波法和荧光共振能量转移（FRET）进行已知MYLK SNPs的功能研究。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的ALI治疗策略。