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，与严重哮喘的易感性高度相关。非肌肉异构体nmMLCK是一种关键的细胞骨架效应因子，它调节EC肌动蛋白细胞骨架参与血管屏障的破坏、屏障的修复、肺炎性细胞的运输和血管对机械拉伸的反应。在水肿剂之后，MLCK将Ser19和Thr18上的MLCs磷酸化，产生屏障破坏的细胞质应激纤维、空间定位的肌球蛋白收缩和细胞旁间隙。相反，EC屏障保护激动剂诱导MLCK快速移位到板脂膜突起(关闭细胞旁间隙和恢复屏障完整性)和皮质肌动蛋白网络(加强与连接复合体的连接并增加屏障特性)。全长nmMLCK1(及其选择性剪接变异体nmMLCK2)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SNPs(Pro21His、Pro147Ser、Val261Ala)对MLCK结构/功能的影响也同样未知。我们假设，位点特异性的nmMLCK调控涉及翻译后修饰(PTM)，并导致变体和SNP特异性的MLCK活性。特殊目的(SA)#1将在居住在纽约州哈莱姆区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。特定目的#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鉴定nmMLCK(nmMLCK1、nmMLCK2、MLCK编码的SNPs)。SA#3将利用质谱学、磷酸肽图谱、GFP-MLCK融合蛋白和结合伙伴分析来研究激酶介导的PTMS(Src、Ab1和PKA)对位点特异性MLCK反应(nmMLCK1、nmMLCK2、nmMLCK-SNPs)的影响。SA#4将利用现有的和新型的基因工程小鼠来进一步确定nmMLCK(SNPs)在肺部炎症损伤中的体内特异性参与。我们相信，这些整合的翻译研究将导致对哮喘病理生物学的机械性见解，并开发新的消肿疗法。 公共卫生相关性：哮喘是一种影响2000多万美国人的疾病，其发病率和死亡率令人无法接受，特别是在非裔美国人等代表性不足的少数群体中。我们的建议将为细胞骨架在哮喘的发展和严重程度中的参与及其重要性提供线索。

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.