Regulation of Asthmatic Inflammation by Non-Muscle MLCK

描述（由申请人提供）：我们的研究表明，编码多功能细胞骨架蛋白肌球蛋白轻链激酶（MLCK）的基因含有编码多态性，与严重哮喘易感性高度相关。非肌肉同种异构体nmMLCK是一种关键的细胞骨架效应物，它调节EC肌动蛋白细胞骨架参与血管屏障破坏、屏障恢复、肺炎症细胞运输和血管对机械拉伸的反应。继致肿剂之后，MLCK磷酸化MLCs的Ser19和Thr18，产生破坏屏障的细胞质应激纤维、空间定位的肌动球蛋白收缩和细胞旁间隙。相比之下，EC屏障保护激动剂诱导MLCK快速易位到板足膜突起（关闭细胞旁间隙并恢复屏障完整性）和皮质肌动蛋白网络（加强与连接复合物的联系并增加屏障特性）。全长nmMLCK1（及其选择性剪接的变体nmMLCK2）靶向特定细胞位点的机制是完全未知的。此外，哮喘相关的nmMLCK编码snp （Pro21His, Pro147Ser, Val261Ala）对MLCK结构/功能的影响同样未知。我们假设位点特异性的nmMLCK调控涉及翻译后修饰（PTMs），并导致变异和snp特异性的MLCK活性。Specific Aim (SA) #1将对居住在纽约哈莱姆的非裔美国哮喘患者进行病例对照研究，以验证我们早期在芝加哥和巴巴多斯哮喘队列中的观察结果。具体的Aim #2研究将利用激酶和肌动蛋白聚合分析、GFP/YFP-MLCK融合蛋白和细胞骨架结合分析来表征nmMLCK （nmMLCK1、nmMLCK2、mlck编码snp）。SA #3将研究激酶介导的PTMs （Src、Abl和PKA）对位点特异性MLCK反应（nmMLCK1、nmMLCK2、nmMLCK-SNPs）的影响，利用质谱、磷酸肽定位、GFP-MLCK融合蛋白和结合伙伴分析。SA #4将利用现有的新型基因工程小鼠进一步确定nmMLCK （SNPs）在肺炎症损伤中的体内特异性参与。我们相信这些综合的转化研究将导致哮喘病理生物学的机制见解和新型减水肿疗法的发展。

DESCRIPTION (provided by applicant): Our studies have demonstrated that the gene encoding the multi-functional cytoskeletal protein, myosin light chain kinase (MLCK), contains coding polymorphisms which are highly associated with susceptibility to severe asthma. The non-muscle isoform, nmMLCK, is a critical cytoskeletal effector which regulates the participation of the EC actin cytoskeleton in vascular barrier disruption, in barrier restoration, in lung inflammatory cell trafficking and in vascular responses to mechanical stretch. Following edemagenic agents, MLCK phosphorylates MLCs on Ser19 and Thr18, producing barrier-disrupting cytoplasmic stress fibers, spatially-localized actomyosin contraction and paracelular gaps. In contrast, EC barrier-protective agonists induce the rapid translocation of MLCK to lamellipodial membrane protrusions (to close paracellular gaps and restore barrier integrity) and to cortical actin networks (to enhance linkage to junctional complexes and increase barrier properties). The mechanism by which the full length nmMLCK1 (and its alternatively spliced variant nmMLCK2) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of the asthma-associated nmMLCK coding SNPs (Pro21His, Pro147Ser, Val261Ala) on MLCK structure/function are similarly unknown. We hypothesize that site-specific nmMLCK regulation involves post-translational modifications (PTMs) and results in variant- and SNP-specific MLCK activities. Specific Aim (SA) #1 will conduct case control studies in African Americans with asthma who reside in Harlem, NY to validate our earlier observations in asthma cohorts from Chicago and Barbados. Specific Aim #2 studies will characterize nmMLCK (nmMLCK1, nmMLCK2, MLCK-coding SNPs) utilizing kinase and actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #3 will examine the influence of kinase-mediated PTMs (Src, Abl, and PKA) on site-specific MLCK responses (nmMLCK1, nmMLCK2, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptide mapping, GFP-MLCK fusion proteins, and binding partner assays. SA #4 will utilize available and novel genetically-engineered mice to further define the site specific in vivo involvement of nmMLCK ( SNPs) in lung inflammatory injury. We believe these integrated translational studies will lead to mechanistic insights into asthma pathobiology and the development of novel edema-reducing therapies. PUBLIC HEALTH RELEVANCE: Asthma is a disorder affecting over 20 million Americans with unacceptable morbidity and mortality, particularly in under-represented minority populations such as African Americans. Our proposal will offer clues for the involvement and importance of the cytoskeleton in the development and severity of asthma.