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

? 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.