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），含有编码多态性，与易感性高度相关 急性肺损伤（ALI）。非肌肉亚型nmMLCK是一种重要的细胞骨架效应因子， EC肌动蛋白细胞骨架参与血管屏障破坏、屏障恢复、肺内 炎症细胞运输和血管对机械拉伸的反应。继水肿剂， MLCK磷酸化Ser 19和Thr 18上的MLCs，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。相反，EC屏障保护激动剂 诱导MLCK快速移位至板状脂膜突起（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， 增加阻隔性能）。全长nmMLCKI（和其五个替代性地， 剪接变体）靶向特定细胞位点的情况是完全未知的。此外，ALI相关的影响 MLCK结构/功能上的nmMLCK编码SNP（Pro21 His、Pro 147 Ser、Val 261 Ala）类似地 未知我们假设nmMLCK的位点特异性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将进行研究 表征利用激酶的nmMLCK（nmMLCKI、nmMLCK剪接变体、MLCK编码SNP） 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。SA #2 将研究激酶介导的PTM（Src，Abl，ERK和PKA）对位点特异性MLCK的影响 响应（nmMLCKI，nmMLCK变体，nmMLCK-SNP），利用质谱，磷酸肽 作图、GFP-MLCK融合蛋白和结合伴侣测定。SA #3将检查MLCK调节， EC板状伪足中肌动蛋白聚合和粘着斑重塑（对细胞旁间隙闭合至关重要） 使用GFP-MLCK-和桩蛋白融合蛋白偶联到原子力显微镜。SA #4将使用 可用的和新的基因工程小鼠，以进一步确定nmMLCK剪接变体的体内作用 （+/- SNPs）在肺部炎症损伤中的作用。这些转化研究整合了我们的整个PPG和领先的 对EC屏障调节和新型减轻水肿疗法的发展的机械见解。

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.