# Involvement of Non-Muscle Myosin Light Chain Kinase Nitration in Molecular Regulation of Inflammation-Induced Endothelial Cell Barrier Dysfunction

## TL;DR

The study shows that nitration of a specific site on the nmMLCK protein disrupts blood vessel integrity during inflammation, offering a new therapeutic target.

## Contribution

The discovery that Y1410 nitration of nmMLCK is a key driver of vascular barrier failure in inflammation.

## Key findings

- Nitration of nmMLCK at Y1410 is a critical post-translational modification that disrupts vascular barrier integrity.
- The Y1410A mutant of nmMLCK preserves Kindlin-2 binding and prevents barrier dysfunction.
- LPS-induced peroxynitrite promotes nmMLCK nitration, contributing to endothelial cell permeability.

## Abstract

What are the main findings?
The nmMLCK isoform is a critical regulator of vascular barrier integrity.We demonstrate that site-specific nitration of nmMLCK at Y1410—induced by LPS—is a key driver of vascular barrier failure.

The nmMLCK isoform is a critical regulator of vascular barrier integrity.

We demonstrate that site-specific nitration of nmMLCK at Y1410—induced by LPS—is a key driver of vascular barrier failure.

What are the implications of main findings?
Targeting Y1410-specific nmMLCK nitration provides vascular-selective protection and represents a novel therapeutic strategy to limit inflammation-induced vascular permeability.Therapeutic strategies that simultaneously inhibit nmMLCK activity and restore redox homeostasis may represent a rational approach to preserving vascular integrity in the setting of proinflammatory challenges.

Targeting Y1410-specific nmMLCK nitration provides vascular-selective protection and represents a novel therapeutic strategy to limit inflammation-induced vascular permeability.

Therapeutic strategies that simultaneously inhibit nmMLCK activity and restore redox homeostasis may represent a rational approach to preserving vascular integrity in the setting of proinflammatory challenges.

Endothelial cell (EC) barrier integrity is tightly regulated by the activity of the non-muscle myosin light chain kinase (nmMLCK) under diverse pathological inflammatory conditions (pneumonia, sepsis) and exposure to mechanical stress. Inflammatory stimuli, including lipopolysaccharide (LPS), cytokines, and damage-associated molecular patterns (DAMPs), increase EC permeability through nmMLCK-dependent EC paracellular gap formation. However, the exact mechanisms by which nmMLCK regulates vascular barrier dysfunction in acute lung injury (ALI) remain incompletely understood. We hypothesized that inflammation-induced ROS results in the peroxynitrite-mediated nitration of nmMLCK that contributes to EC barrier disruption. Human lung EC exposure to either the peroxynitrite donor, SIN-1, or to LPS, triggered significant nmMLCK nitration, which was abolished by the oxidant scavenger, MnTMPyP. Mass spectrometry of SIN-1-treated nmMLCK identified multiple nitrated tyrosines. Nitration of Y1410 proved a critical PTM as site-directed substitution with alanine (Y1410A) abolished both SIN-1- and LPS-induced nmMLCK nitration. nmMLCK nitration disrupts wild-type nmMLCK interaction with Kindlin-2, a cytoskeletal regulator of vascular barrier stability, whereas EC transfected with the Y1410A nmMLCK mutant exhibited preserved Kindlin-2 binding, reflected by alterations in trans-EC electrical resistance (TEER). Consistent with these observations, LPS-challenged murine lungs displayed enhanced nmMLCK nitration and diminished nmMLCK-Kindlin-2 association. Functionally, SIN-1 markedly impaired EC barrier integrity (TEER), which was not observed in ECs expressing the Y1410A mutant. Together, these findings suggest that nmMLCK nitration at Y1410 is a critical molecular mechanism contributing to vascular leakage, highlighting this modification as a potential therapeutic target to reduce inflammation-induced vascular permeability. Given nmMLCK’s established role in barrier regulation, we hypothesized that LPS-induced peroxynitrite formation may promote the nitration of nmMLCK tyrosine residues: a PTM that potentially contribute to nmMLCK’s regulation of EC barrier integrity.