Regulation of Peripheral EC Cytoskeletal Remodeling, Gap Closure and Barrier Restoration by nmMLCK/MYLK and Cortactin/CTTN

摘要： 项目1系统生物学研究强调了两个关键的肺细胞骨架效应蛋白/基因为中心 为了解决血管炎症、内皮细胞(EC)通透性和多器官衰竭 ARDS死亡率与ARDS血管内型的关系。我们已经令人信服地展示了多功能 非肌肉肌球蛋白轻链激酶亚型(Mylk)及其细胞骨架结合伙伴皮质酮(CTTN)， 是炎症诱导的血管通透性、白细胞转运和血管的主要调节因子。 对呼吸机产生的机械应力的反应。此外，编码nmMLCK(Myk)和nmMLCK(Myk)的基因 皮质蛋白(CTTN)含有基因变异，可增加脓毒症/创伤诱发ARDS和ARDS的风险 黑人的死亡率。与PPG主题目标同步，项目#1旨在转化机械性的见解 将nmMLCK和Cortactin的结构和功能转化为新的、有效的治疗机会，以减少ARDS 死亡率。SA#1将通过以下方式探索非肌肉Mylk和CTTN启动子的遗传/表观遗传调控：i) ROS调节(或感应)转录因子(缺氧诱导因子HIF-1a/HIF-2a和NRF2)，II) Mylk/CTTN启动子SNPs和由III)Mylk/CTTN启动子DNA甲基化(核心B)。SA#2将详细介绍EC 酪氨酸对S1PR1和TLR4受体激活引起的屏障反应的影响 NmMLCK1、nmMLCK2(促炎的Mylk剪接变异体)和Cortactin的磷酸化； Mylk/CTTN编码SNP(在黑人中过度表达)。SA#3将从功能上描述 新的nmMLCK结合蛋白(丙酮酸激酶M2，kindlin-2)和皮质蛋白结合蛋白(DOCK1/ELMO1) 在S1PR1/TLR4介导的EC细胞骨架动力学和屏障调节中。丙酮酸激酶M2(PKM2)，a 糖酵解和炎症的中央调节因子，选择性地结合nmMLCK1 IgGCAM3结构域 影响EC细胞骨架驱动的屏障修复。焦点黏附(FA)调节蛋白kindlin2是一种 Project#3目标基因，最近被确定为nmMLCK结合伙伴，可能对连接 细胞骨架与整合素介导的细胞-细胞外基质局部黏附和信号转导。DOCK1和ELMO1是关键RAC GTP酶和细胞骨架调节蛋白以及新的皮质肌动蛋白结合蛋白。SA#2和SA#3研究 利用核心C/D蛋白质组和生物物理成像方式(超分辨率，AFM)来定义蛋白质 S1PR1/TLR4介导的EC空间特异性细胞骨架重塑、间隙形成/关闭和 片状脂血症的形成和屏障调节。SA#4利用已建立的Core C大鼠和猪ARDS/VILI 评估外层含有S1PR1激动剂Tysiponate的新型促进屏障脂质体的模型 表面，涂有辛伐他汀(nmMLCK拮抗剂)、必新(NRF2激动剂、Mylk拮抗剂)或PIK (nmMLCK多肽抑制剂)。因此，通过利用与每个PPG项目和核心的集成交互， 项目1‘S系统生物学方法将阐明nmMLCK和Cortactin在ARDS和VILI中的作用 病理生物学。这些研究将加强治疗靶向，以恢复EC屏障的完整性，并增加 对ARDS健康差异的基因洞察。

ABSTRACT: Project #1 system biology studies have highlighted 2 critical lung cytoskeletal effector proteins/genes as central to addressing vascular inflammation, endothelial cell (EC) permeability and the multi-organ failure critical to ARDS mortality and the ARDS vascular endotype. We have convincingly demonstrated the multi-functional non-muscle myosin light chain kinase isoform (MYLK) and its cytoskeletal-binding partner, cortactin (CTTN), are primary regulators of inflammation-induced vascular permeability, leukocyte trafficking, and vascular responses to ventilator-derived mechanical stress. Furthermore, the genes encoding nmMLCK (MYLK) and cortactin (CTTN) harbor genetic variants that confer increased risk of sepsis/trauma-induced ARDS and ARDS mortality in Blacks. In sync with PPG thematic goals, Project #1 is designed to translate mechanistic insights into nmMLCK and cortactin structure and function into novel, effective therapeutic opportunities to reduce ARDS mortality. SA #1 will explore genetic/epigenetic regulation of the non-muscle MYLK and CTTN promoters by: i) ROS–regulated (or sensing) transcription factors (hypoxia-induced factors HIF-1a/HIF-2a, and NRF2), ii) MYLK/CTTN promoter SNPs, and by iii) MYLK/CTTN promoter DNA methylation (Core B). SA #2 will detail EC barrier-responses elicited by S1PR1 and TLR4 receptor activation that are influenced by tyrosine phosphorylation of nmMLCK1, nmMLCK2 (the pro-inflammatory MYLK splice variant) and cortactin; and by MYLK/CTTN coding SNPs (over-represented in Blacks). SA #3 will functionally characterize the involvement of novel nmMLCK-binding proteins (pyruvate kinase M2, kindlin-2) and cortactin-binding proteins (DOCK1/ELMO1) in S1PR1/TLR4-mediated EC cytoskeletal dynamics and barrier regulation. Pyruvate kinase M2 (PKM2), a central regulator of glycolysis and inflammation, selectively binds the nmMLCK1 IgGCAM3 domain to potentially influence EC cytoskeletal-driven barrier restoration. The focal adhesion (FA) regulatory protein, kindlin2, is a Project #3 target gene, and was recently identified as a nmMLCK binding partner likely crucial for linking the cytoskeleton to integrin-mediated cell-ECM focal adhesion and signaling. DOCK1 and ELMO1 are key Rac GTPase and cytoskeletal regulatory proteins and novel cortactin-binding proteins. SA #2 and SA #3 studies utilize Core C/D proteomic and biophysical imaging modalities (super resolution, AFM) to define protein interactions in S1PR1/TLR4-mediated EC spatially-specific cytoskeletal remodeling, gap formation/closure and lamellipodia formation and barrier regulation. SA #4 utilize established Core C rat and porcine ARDS/VILI models to assess a novel barrier-promoting liposome bearing the S1PR1 agonist, Tysiponate, on its outer surface, encargoed with simvastatin (nmMLCK antagonist), bixin (NRF2 agonist, MYLK antagonist), or PIK (nmMLCK peptide inhibitor). Thus, by leveraging the integrated interactions with each PPG Project and Core, Project #1's system biology approaches will clarify the contributions of nmMLCK and cortactin to ARDS and VILI pathobiology. These studies will enhance therapeutic targeting to restore EC barrier integrity and increase genetic insights into ARDS health disparities.