Regulation of Asthmatic Inflammation by Non-Muscle MLCK

描述（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，这些多态性与严重哮喘的易感性高度相关。非肌肉亚型nmMLCK是一种重要的细胞骨架效应物，其调节EC肌动蛋白细胞骨架参与血管屏障破坏、屏障恢复、肺部炎症细胞运输和对机械拉伸的血管反应。在水肿剂之后，MLCK使Ser 19和Thr 18上的MLCs磷酸化，产生屏障破坏细胞质应力纤维、空间定位的肌动球蛋白收缩和细胞旁间隙。与此相反，EC屏障保护激动剂诱导MLCK快速易位到板脂膜突起（关闭细胞旁间隙和恢复屏障完整性）和皮质肌动蛋白网络（以增强连接复合物的连接和增加屏障性能）。全长nmMLCK 1（及其选择性剪接变体nmMLCK 2）靶向特定细胞位点的机制完全未知。此外，哮喘相关的nmMLCK编码SNP（Pro21 His，Pro 147 Ser，Val 261 Ala）对MLCK结构/功能的影响同样未知。我们推测，位点特异性nmMLCK调控涉及翻译后修饰（PTM），并导致变异和SNP特异性MLCK活动。Specific Aim（SA）#1将在居住在纽约哈莱姆的非裔美国人哮喘患者中进行病例对照研究，以验证我们在芝加哥和巴巴多斯哮喘队列中的早期观察结果。具体目标#2研究将利用激酶和肌动蛋白聚合试验、GFP/YFP-MLCK融合蛋白和细胞骨架结合试验表征nmMLCK（nmMLCK 1、nmMLCK 2、MLCK编码SNP）。SA #3将利用质谱、磷酸肽图谱、GFP-MLCK融合蛋白和结合伴侣试验，检查激酶介导的PTM（Src、Abl和PKA）对位点特异性MLCK应答（nmMLCK 1、nmMLCK 2、nmMLCK-SNP）的影响。SA #4将利用可用的新型基因工程小鼠进一步确定nmMLCK（SNP）在肺部炎症损伤中的体内位点特异性参与。我们相信这些综合转化研究将导致对哮喘病理生物学的机理性见解和新的减轻水肿疗法的开发。 公共卫生相关性：哮喘是一种影响超过2000万美国人的疾病，具有不可接受的发病率和死亡率，特别是在代表性不足的少数民族人群中，如非洲裔美国人。我们的建议将提供线索的参与和重要性的细胞骨架在哮喘的发展和严重程度。

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.