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2种关键的肺细胞骨架效应蛋白/基因作为核心 解决血管炎症、内皮细胞（EC）渗透性和多器官衰竭， ARDS死亡率与ARDS血管内皮型我们已经令人信服地证明了多功能 非肌肉肌球蛋白轻链激酶同种型（MYLK）及其细胞内结合配偶体，corneum蛋白（CTTN）， 是炎症诱导的血管通透性、白细胞运输和血管内皮细胞增殖的主要调节因子。 对呼吸机产生的机械应力的反应。此外，编码nmMLCK（MYLK）和 皮质激素（CTTN）具有增加脓毒症/创伤诱导的ARDS和ARDS风险的遗传变异 黑人的死亡率与PPG主题目标同步，项目#1旨在将机械见解 将nmMLCK和coronin结构和功能转化为新的、有效的治疗机会，以减少ARDS mortality. 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： ROS调节（或传感）转录因子（缺氧诱导因子HIF-1 a/HIF-2 a和NRF 2），ii） MYLK/CTTN启动子SNP，和iii）MYLK/CTTN启动子DNA甲基化（核心B）。SA #2将详细说明EC 受酪氨酸影响的S1 PR 1和TLR 4受体激活引起的屏障反应 nmMLCK 1、nmMLCK 2（促炎性MYLK剪接变体）和corpine的磷酸化;以及 MYLK/CTTN编码SNP（在黑人中过度表达）。SA #3将在功能上描述以下人员的参与 新的nmMLCK结合蛋白（丙酮酸激酶M2，kindlin-2）和皮质素结合蛋白（DOCK 1/ELMO 1） 在S1 PR 1/TLR 4介导的EC细胞骨架动力学和屏障调节中。丙酮酸激酶M2（PKM 2），a 糖酵解和炎症的中枢调节因子，选择性结合nmMLCK 1 IgGCAM 3结构域， 影响EC细胞壁驱动屏障恢复。黏着斑（FA）调节蛋白Kindlin 2是一种免疫调节蛋白。 项目#3靶基因，最近被鉴定为nmMLCK结合伴侣，可能对连接 细胞骨架与整合素介导的细胞-ECM粘着斑和信号传导。DOCK 1和ELMO 1是关键Rac GT3和细胞骨架调节蛋白和新型皮质素结合蛋白。SA #2和SA #3研究 利用Core C/D蛋白质组学和生物物理成像模式（超分辨率，AFM）来定义蛋白质 S1 PR 1/TLR 4介导的EC空间特异性细胞骨架重塑、间隙形成/闭合和 板状伪足形成和屏障调节。SA #4使用已建立的Core C大鼠和猪ARDS/VILI 模型，以评估一种新型的屏障促进脂质体，其外部携带S1 PR 1激动剂Tysiponate， 表面，负载辛伐他汀（nmMLCK拮抗剂）、bixin（NRF 2激动剂、MYLK拮抗剂）或PIK （nmMLCK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的系统生物学方法将阐明nmMLCK和coronin对ARDS和VILI的作用 病理生物学这些研究将增强治疗靶向，以恢复EC屏障的完整性，并增加 对ARDS健康差异的遗传学见解。

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.