Regulation of Asthmatic Inflammation by Non-Muscle MLCK

非肌肉 MLCK 对哮喘炎症的调节

• 批准号: 7681504
• 负责人: Joe G. N. Garcia
• 金额: $ 39.22万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681504
• 关键词: ABL1 gene; Actins; Actomyosin; Acute; Affect; African; African American; Agonist; Alternative Splicing; American; Asthma; Automobile Driving; Barbados; Binding; Binding Sites; Biological; Biological Assay; Blood Vessels; Case-Control Studies; Cell surface; Cells; Chicago; Chimeric Proteins; Chronic; Code; Complex; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Cytoskeleton; Development; Disease; Edema; Endothelial Cells; Endothelium; Environmental Risk Factor; Epithelial; European; Exons; Gene Frequency; Genes; Genetic; Genetic Determinism; Genetic Polymorphism; Genetically Engineered Mouse; Hepatocyte Growth Factor; Human; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Lead; Length; Locales; Location; Lung; Lung Inflammation; Lung diseases; MYLK gene; Maps; Mass Spectrum Analysis; Mechanics; Mediating; Membrane; Membrane Microdomains; Modeling; Morbidity - disease rate; Movement; Mus; Muscle; Myosin ATPase; Myosin Light Chain Kinase; Ovalbumin; Pathogenesis; Peptide Signal Sequences; Phenotype; Phosphopeptides; Phosphorylation; Phosphotransferases; Physiological; Population; Positioning Attribute; Post-Translational Protein Processing; Predisposition; Proline; Property; Protein Binding; Protein Isoforms; Proto-Oncogene Protein pp60 (c-src); Publishing; RNA Splicing; Regulation; Role; Sampling; Serine; Severities; Site; Stress Fibers; Stretching; Structure; Susceptibility Gene; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Translational Research; Underrepresented Minority; Variant; Work; actin kinase; airway hyperresponsiveness; airway remodeling; alveolar epithelium; c-abl Proto-Oncogenes; case control; cohort; human EMS1 protein; in vivo; insight; lung injury; mortality; novel; polymerization; protein kinase A kinase; public health relevance; response; restoration; sphingosine 1-phosphate; therapeutic target; trafficking; translational study

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.

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）的基因含有与严重哮喘易感性高度相关的编码多态性。非肌肉亚型 nmMLCK 是一种关键的细胞骨架效应子，可调节 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血管对机械拉伸的反应。在使用水肿剂后，MLCK 在 Ser19 和 Thr18 上磷酸化 MLC，产生屏障破坏的细胞质应激纤维、空间局部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快速易位至板状足膜突起（以闭合细胞旁间隙并恢复屏障完整性）和皮质肌动蛋白网络（以增强与连接复合物的连接并增加屏障特性）。全长 nmMLCK1（及其选择性剪接变体 nmMLCK2）靶向特定细胞位点的机制完全未知。此外，哮喘相关 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 nmMLCK 调控涉及翻译后修饰 (PTM)，并导致变体和 SNP 特异性 MLCK 活性。具体目标 (SA) #1 将对居住在纽约州哈莱姆区的患有哮喘的非裔美国人进行病例对照研究，以验证我们之前对芝加哥和巴巴多斯哮喘队列的观察结果。具体目标 #2 研究将利用激酶和肌动蛋白聚合测定、GFP/YFP-MLCK 融合蛋白和细胞骨架结合测定来表征 nmMLCK（nmMLCK1、nmMLCK2、MLCK 编码 SNP）。 SA #3 将利用质谱、磷酸肽图谱、GFP-MLCK 融合蛋白和结合来检查激酶介导的 PTM（Src、Abl 和 PKA）对位点特异性 MLCK 反应（nmMLCK1、nmMLCK2、nmMLCK-SNP）的影响伙伴分析。 SA #4 将利用现有的新型基因工程小鼠来进一步确定 nmMLCK (SNP) 在肺部炎症损伤中的体内特定位点参与。我们相信这些综合转化研究将带来对哮喘病理学的机制见解和新型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，其发病率和死亡率令人无法接受，特别是在非洲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和重要性提供线索。