Regulation of Lung Barrier Function by Endothelial Cell (EC) MLCK

项目1的研究表明，编码多功能细胞骨架蛋白肌球蛋白的基因 轻链激酶(MLCK)编码的多态与易感性密切相关 致急性肺损伤(ALI)。非肌肉异构体nmMLCK是一种关键的细胞骨架效应器，它调节 EC肌动蛋白细胞骨架参与肺血管屏障破坏、屏障修复 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使Ser19和Thr18上的MLCs磷酸化，产生破坏细胞质屏障的应激纤维， 空间定位的肌动球蛋白收缩和细胞旁间隙。相比之下，EC屏障保护性激动剂 诱导MLCK快速移位到板脂膜突起(以关闭细胞旁间隙 并恢复屏障的完整性)和皮质肌动蛋白网络(以加强与连接复合体和 增加阻隔性能)。全长nmMLCKI(和它的五个交替)的机制 剪接变异体)针对特定的细胞位点是完全未知的。此外，ALI相关的影响 MLCK结构/功能上的nmMLCK编码SNP(Pro21His、Pro147Ser、Val261Ala)类似 未知。我们假设特定位点的nmMLCK调控涉及翻译后修饰 (PTMS)，并导致变异和SNP特异性MLCK活性。特定目标(SA)#1将进行研究 利用激酶鉴定nmMLCK(nmMLCKI、nmMLCK剪接变异体、MLCK编码SNPs) 肌动蛋白聚合实验、GFP/YFP-MLCK融合蛋白和细胞骨架结合实验。SA#2 将研究激酶介导的PTMS(Src、Abl、ERK和PKA)对位点特异性MLCK的影响 利用质谱学的反应(nmMLCKI，nmMLCK-变体，nmMLCK-SNPs)，磷酸肽 定位、GFP-MLCK融合蛋白和结合伙伴分析。SA#3将审查MLCK对 肌动蛋白聚合和EC片层黏附重塑(对细胞旁缝隙关闭至关重要) 用GFP-MLCK-和巴西林融合蛋白耦合原子力显微镜。SA#4将利用 可用的和新的基因工程小鼠进一步确定nmMLCK剪接变异体在体内的作用 (+/-)SNPs与肺炎性损伤这些翻译研究整合到我们的整个PPG和Lead 对EC屏障调节和新型消肿疗法的发展有机械性的见解。

Project #1 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 acute lung injury (ALI). 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 nmMLCKI (and its five alternatively spliced variants) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of ALIassociated 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 studies to characterize nmMLCK (nmMLCKI, nmMLCK splice variants, MLCK-coding SNPs) utilizing kinase and actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #2 will examine the influence of kinase-mediated PTMs (Src, Abl, ERK and PKA) on site-specific MLCK responses (nmMLCKI, nmMLCK-variants, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptide mapping, GFP-MLCK fusion proteins, and binding partner assays. SA #3 will examine MLCK regulation of actin polymeriza-tion and focal adhesion remodeling in EC lamellipodia (critical to paracellular gap closure) using GFP-MLCK- and paxillin fusion proteins coupled to atomic force microscopy. SA #4 will utilize available and novel genetically-engineered mice to further define the in vivo role of nmMLCK splice variants (+/- SNPs) in lung inflammatory injury. These translational studies integrate across our entire PPG and lead to mechanistic insights into EC barrier regulation and the development of novel edema-reducing therapies.