Regulation of nonmuscle myosin light chain kinase structure and function of ARDS

 描述（由申请人提供）：我们已经扩展了我们先前的研究，证明肌球蛋白轻链激酶（nmMLCK，1914 aa，基因代码-MYLK）的非肌肉亚型在以下方面是一种重要的多功能细胞骨架效应物：i）肺内皮细胞（EC）屏障破坏和屏障恢复过程; ii）肺炎症细胞运输; ii）肺血管对机械应力的反应。这些事件中的每一个对于急性呼吸窘迫综合征（ARDS）和呼吸机诱导的肺损伤（VILI）的病理生物学都是至关重要的。我们之前进行了MYLK测序，并鉴定了编码和非编码MYLK单核苷酸多态性（SNP），这些多态性有助于ARDS易感性和ARDS结局。此外，许多ARDS单核苷酸多态性在非洲裔（AD）个体中严重过度表达，这是一个存在ARDS生存率降低风险的人群。我们将讨论29个精心挑选的MYLK SNP的功能，这些SNP可能参与nmMLCK在ARDS和VILI的发展以及与EC屏障恢复相关的恢复阶段的双重EC屏障调节作用。涉及调节nmMLCK表达的机制知之甚少。SA #1将基于我们最近发表的研究来表征5'启动子和3' UTR MYLK SNP对nmMLCK表达的遗传/表观遗传调控的影响。继续我们的MYLK的结构/功能的询问，我们最近采用RNA测序分析，并证实了我们发表的研究，人肺EC表现出一个独特的1845 aa nmMLCK剪接变异体，nmMLCK 2，由外显子11的剪接缺失产生的大量表达。由于我们的数据表明肺EC暴露于炎性激动剂和过度机械应力选择性地增加nmMLCK 2表达，SA #2将检查MYLK SNP和特异性剪接因子对nmMLCK mRNA剪接变体产生的调节的影响。SA #3将扩展我们先前关于翻译后修饰（PTM）的调控影响的研究，例如位于剪接出的外显子11中的Y 464和Y 471的磷酸化。SA #3将进一步评价PTM和N末端编码SNP对nmMLCK 1和nmMLCK 2空间定向激酶活性、结构/功能关系和EC屏障反应（外周细胞骨架重塑、板状伪足形成、细胞旁间隙调节）的影响。最后， SA #4将验证选定的nmMLCK编码SNP和PTM的体内作用，并将nmMLCK拮抗作用定义为临床前ARDS和VILI模型中的潜在新型疗法。这些高度整合的系统生物学方法将使我们能够阐明肺EC细胞骨架变体、PTM和SNP对ARDS和VILI病理学的贡献，增强肺血管屏障失调的治疗靶向，并增加对ARDS健康差异的遗传基础的了解。

 DESCRIPTION (provided by applicant): We have extended our prior studies demonstrating the non-muscle isoform of myosin light chain kinase (nmMLCK, 1914 aa, gene code-MYLK) as an essential, multi-functional cytoskeletal effector in: i) lung endo- thelial cell (EC) barrier-disruptive and barrier-restorative processes; ii) in lung inflammatory cell trafficking; and in, ii) lung vascular responses to mechanical stress. Each of these events is critical to the pathobiology of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We previously per- formed MYLK sequencing and identified coding and non-coding MYLK single nucleotide polymorphisms (SNPs) that contribute to ARDS susceptibility and to ARDS outcomes. In addition, many ARDS SNPs were profoundly over-represented in individuals of African descent (AD), a population at risk for reduced survival in ARDS. We will address the functionality of 29 carefully-selected MYLK SNPs that are potentially involved in the dual EC barrier-regulatory roles of nmMLCK both in the development of ARDS and VILI and in the recovery phase associated with EC barrier restoration. The mechanisms involved in regulation of nmMLCK expression are very poorly understood. SA #1 will build upon our recently published studies to characterize the effects of 5' promoter and 3' UTR MYLK SNPs on genetic/epigenetic regulation of nmMLCK expression. Continuing our structure/function interrogation of MYLK, we recently employed RNA sequencing analysis and corroborated our published studies that human lung EC exhibit substantial expression of a unique 1845 aa nmMLCK splice variant, nmMLCK2, generated by a splicing deletion of exon11. As our data indicate that lung EC exposure to inflammatory agonists and excessive mechanical stress selectively increases nmMLCK2 expression, SA #2 will examine the influence of MYLK SNPs and specific splicing factors on the regulation of nmMLCK mRNA splice variant generation. SA #3 will extend our prior studies on the regulatory influence of post-translational modifications (PTMs) such as phosphorylation of Y464 and Y471 located in the spliced out exon 11. SA #3 will further evaluate the effects of PTMs and N-terminal coding SNPs on nmMLCK1 and nmMLCK2 spatially- directed kinase activities, structure/function relationships, and EC barrier responses (peripheral cytoskeletal remodeling, lamellipodia formation, paracellular gap regulation). Finally, SA #4 will validate the in vivo effects of selected nmMLCK coding SNPs and PTMs as well as define nmMLCK antagonism as a potentially novel therapy in preclinical ARDS and VILI models. These highly integrated system biology approaches will allow us to clarify the contributions of lung EC cytoskeletal variants, PTMs and SNPs to ARDS and VILI pathobiology, enhance therapeutic targeting of lung vascular barrier dysregulation, and increase knowledge of the genetic basis for ARDS health disparities.