Abstract
The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.
Highlights
The responses of pulmonary endothelial cells (ECs) to external stimuli, including bioactive agonists and mechanical stress, largely proceed through the spatially-targeted rearrangement of the dynamic, functionally complex actin cytoskeleton [1, 2]
We initially chose a limited number of such proteins for nuclear magnetic resonance (NMR) studies based on our previous studies that P147 SNP variant was more highly associated with acute lung injury (ALI) and asthma as well as on the assumption that 147P/S mutation may be directly involved in changing posttranslational modification
Using NMR and homology modeling, we have defined the potential influence of two MYLK coding SNPs, P147S, on the structure and function of non-muscle isoform of myosin light chain kinase (nmMLCK), as impacting phosphorylation and proteinprotein interaction
Summary
The responses of pulmonary endothelial cells (ECs) to external stimuli, including bioactive agonists and mechanical stress, largely proceed through the spatially-targeted rearrangement of the dynamic, functionally complex actin cytoskeleton [1, 2]. NmMLCK is essential to lung vascular barrier protection and recovery induced by barrier-enhancing or barrier-restoring agonists such as hepatocyte growth factor [13] and sphingosine 1-phosphate (S1P) [14] These agonists dramatically drive rapid and spatially-distinct increases in actin polymerization and MLC phosphorylation that are confined to lamellipodial membrane protrusions and the cortical actin ring, resulting in closure of inflammation-mediated paracellular gaps thereby restoring barrier integrity [13,14,15]. This dual role for nmMLCK in critical lung processes highlights the need to understand spatiotemporal regulatory mechanisms for this key multi-functional enzyme
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
