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

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

"Involvement of Non-Muscle Myosin Light Chain Kinase Nitration in Molecular Regulation of Inflammation-Induced Endothelial Cell Barrier Dysfunction" has serious concerns for the publication. The major challenge is the similarity report which is quit high. The improvements can be done as per the below comments.

1. Please check whether the text for line 16 to 25 is at right place? I think it may be after references. However, check it as per the journal guidelines.
2.  Pay attentions on i-thenticate report that is quit high and more than 36 %
3. Line 50-53 does not have any sense. It is vogue. Revise it. 
4. The authors should clarify the rationale for focusing on nmMLCK over other endothelial permeability regulators.
5. Briefly strengthen the mechanistic and translational significance of the choice of nmMLCK.
6. Conclusions are missing for this study.
7. Buffer purity should be mentioned clearly.
8. Please explain transendothelial electrical resistance (TEER) measurements and their roles.
9. The significance of phosphorylates myosin light chains (MLC) is missing.
10. Elaborate the oxidant scavenger.
11. Is there any limitation of this study? please mention.

Comments on the Quality of English Language

Too many unnecessary comas and superscripts exist. Improve the grammar and language of the manuscript. Also reduce similarity. 

Author Response

1. Please check whether the text for line 16 to 25 is at right place? I think it may be after references. However, check it as per the journal guidelines.

Response: We thank you for this observation and after reviewing the journal’s formatting guidelines we have now relocated the specified text to the appropriate section following the References.

2. Pay attentions on i-thenticate report that is quit high and more than 36 %.

Response: We acknowledge the high similarity index and have taken steps to address it. We have  now thoroughly revised the manuscript to paraphrase technical descriptions and have significantly reduced non-essential self-citations. We believe the revised manuscript will now show a substantially lower similarity score.

3. Line 50-53 does not have any sense. It is vogue. Revise it. 

Response: We apologize for the lack of clarity in the original text. We have rewritten this section to be more precise. We have revised as follows: These results suggest that nmMLCK nitration at Y1410 is a critical molecular mechanism contributing to vascular barrier leakage, highlighting this modification as a potential therapeutic target for ALI.

 

4. The authors should clarify the rationale for focusing on nmMLCK over other endothelial permeability regulators.

5. Briefly strengthen the mechanistic and translational significance of the choice of nmMLCK

Response: We agree with the reviewer’s suggestions and have now expanded the Introduction in the revised manuscript to better justify our focus on nmMLCK. We have revised as follows: We primarily focused on nmMLCK as: 1) MYLK was identified as an ARDS candidate gene in our human genetic studies and associated with both risk and severity of ARDS, 2) nmMLCK is an essential driver of endothelial cell actomyosin contraction and formation of paracellular gaps that produce vascular permeability, and 3) the unique N-terminal domain of the non-muscle MLCK isoform—specifically the Y1410 residue—provides a site for specific nitration that is not present in smooth muscle MLCK, offering a highly specific molecular mechanism for vascular-specific barrier disruption.

6. Conclusions are missing for this study.

Response: We apologize for this omission and have have now added a Conclusion section as follows: Collectively, our data establish nmMLCK nitration at Y1410 as a key post-translational modification driving LPS-induced vascular barrier dysfunction and loss of barrier integrity. This work underscores the critical role of oxidative signaling in endothelial cell mechanics anddynamic control of vascular integrity. Our results highlight the Y1410-nmMLCK axis as a viable therapeutic target for the treatment of inflammatory lung diseases, such as acute lung injury.

7. Buffer purity should be mentioned clearly.

Response: We have clarified the buffer purity in the revised manuscript. All buffers were prepared using analytical-grade reagents and diluted with ultra-pure deionized water (18.2 MΩ·cm) obtained from a Milli-Q water purification system. Commercially available pre-mixed buffers were sourced from Sigma-Aldrich and Thermo Fisher Scientific to ensure maximum consistency and purity.

 8. Please explain transendothelial electrical resistance (TEER) measurements and their roles.

Response: We have addressed the reviewer's request for a clearer description of TEER and its functional role by revising our this portion of the METHOD section: Endothelial barrier integrity was assessed by monitoring Transendothelial Electrical Resistance (TEER) using an Electric Cell-substrate Impedance Sensing (ECIS) system (Applied Biophysics, Troy, NY) as we have reported previously (8, 50) . HPAEC cells expressing WT-nmMLCK1 or the Y1410A mutant were seeded onto gold-plated microelectrodes in 8W10E+ arrays and grown to confluence. TEER was utilized as a high-resolution, real-time metric of paracellular permeability, reflecting the ionic conductance across the endothelial monolayer as regulated by junctional complex stability and actomyosin-driven contractile tension. Upon establishment of a stable baseline, cells were challenged with the peroxynitrite donor SIN-1. As previously described [58], Sphingosine-1-phosphate (S1P) and thrombin (Sigma-Aldrich, St. Louis, MO) were employed as positive controls to simulate barrier enhancement and disruption, respectively (50). Resistance was sampled at a frequency of 4000 Hz, enabling precise kinetic characterization of the onset of barrier dysfunction, the magnitude of paracellular gap formation, and the subsequent rate of barrier restoration.

 

9. The significance of phosphorylates myosin light chains (MLC) is missing.

Response: We have updated the manuscript to clarify that MLC2 phosphorylation at Ser19/Thr18 is the primary downstream event triggered by nmMLCK activation, which leads to actomyosin contraction and paracellular gap formation. To support this, we included new Western blot data (Fig. 5F&G) demonstrating that SIN-1 and LPS exposures, which promote nmMLCK nitration at Y1410, significantly increase p-MLC2 levels. Furthermore, we show that these increases are attenuated by the peroxynitrite scavenger MnTmPyP, further linking nitration-mediated nmMLCK activation to downstream MLC2 phosphorylation.

10. Elaborate the oxidant scavenger.

Response: To determine the specific role of reactive nitrogen species (RNS) in nmMLCK modification, we utilized MnTmPyP as a pharmacological scavenger. As a cell-permeable superoxide dismutase (SOD) mimetic and a potent peroxynitrite (ONOO-) decomposition catalyst, MnTmPyP effectively prevents the accumulation of nitrating radicals. By employing this scavenger, we were able to demonstrate that blocking oxidative/nitrative stress prevents Y1410 nitration, thereby preserving the nmMLCK-actomyosin signaling axis and maintaining barrier function. We have added these details to the Results sections (lines 200-221) to better define the scavenger's mechanism of action.

 

11. Is there any limitation of this study? please mention.

Response: In the revised manuscript, we have now noted several limitations of the current study (Lines 295–302). While we have successfully demonstrated the functional importance of Y1410 nitration in vitro and in vivo, further investigation is required to determine if additional nitrated residues cooperate with Y1410 to modulate nmMLCK activity. Furthermore, it remains to be clarified whether Y1410 modification directly alters nmMLCK kinase activity or primarily influences its scaffolding interactions. Finally, extending these findings to clinical cohorts will be essential to establish the translational relevance of nmMLCK nitration in patients with human acute lung injury (ALI) and related inflammatory syndromes.

 

12. Comments on the Quality of English Language. Too many unnecessary comas and superscripts exist. Improve the grammar and language of the manuscript. Also reduce similarity. 

Response: The manuscript has been thoroughly revised and edited for clarity, technical accuracy, and flow. We have addressed all grammatical errors and refined the scientific terminology throughout the text.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is of interest; however, several shortcomings need to be addressed to enhance its scientific impact.

1. The manuscript convincingly identifies Y1410 as a critical nitration site; however, mass spectrometry revealed multiple nitrated tyrosines on nmMLCK. The authors should provide a more apparent justification for why Y1410 is functionally dominant rather than cooperative with other nitration sites. Additional discussion or experimental evidence (e.g., combinatorial mutants or phospho- or kinase-activity assays) would strengthen the conclusion that Y1410 nitration alone is sufficient to drive barrier dysfunction.

2. While the data demonstrate altered protein–protein interactions (Kindlin-2 binding), it remains unclear whether Y1410 nitration directly alters nmMLCK catalytic activity. Measurement of MLC phosphorylation in WT versus Y1410A-expressing cells under SIN-1 or LPS stimulation would help distinguish whether the observed barrier effects are driven by altered kinase activity, scaffolding function, or both. The authors should experimentally address this point.

3. SIN-1 is a commonly used but indirect peroxynitrite donor that generates both superoxide and nitric oxide simultaneously. The authors should more explicitly discuss potential off-target oxidative effects of SIN-1 and clarify whether additional controls (e.g., decomposed SIN-1 or alternative RNS scavengers) were used or considered to ensure specificity of peroxynitrite-mediated nitration.

4. Many key experiments rely on the overexpression of GFP-tagged nmMLCK constructs in HEK293 or endothelial cells. The authors should discuss how overexpression levels compare with physiological nmMLCK abundance and whether similar nitration patterns are detectable for endogenous nmMLCK under inflammatory conditions.

5. Although LPS-treated mice show increased nmMLCK nitration and reduced Kindlin-2 interaction, the data remain correlative. Genetic or pharmacologic strategies that selectively block nmMLCK nitration (e.g., endothelial-specific Y1410A knock-in or antioxidant intervention linked to functional lung permeability endpoints) would significantly strengthen causal inference. The authors should experimentally address this point.

6. The manuscript refers primarily to nmMLCK1, yet the lung expresses multiple MYLK isoforms. The authors should clarify whether nmMLCK2 is also nitrated under these conditions, or whether Y1410 is unique to a specific isoform involved in endothelial barrier regulation.

7. While representative ECIS traces are shown, inclusion of summary metrics (e.g., area under the curve, peak resistance drop, or recovery kinetics) would allow for more rigorous quantitative comparison between WT and Y1410A conditions.

8. The disruption of nmMLCK–Kindlin-2 interaction is a central mechanistic claim. However, the binding interface remains undefined. At least the authors provide a brief discussion or reference to known Kindlin-2 interaction domains—or to how Y1410 nitration may sterically or electrostatically interfere with binding—this would enhance mechanistic clarity.

9. The authors appropriately acknowledge the lack of patient data as a limitation. Nevertheless, a brief discussion referencing existing evidence of protein nitration in human ARDS or sepsis samples would help contextualize the translational relevance of nmMLCK nitration.

Author Response

REVIEWER #2.

 

1. The manuscript is of interest; however, several shortcomings need to be addressed to enhance its scientific impact. The manuscript convincingly identifies Y1410 as a critical nitration site; however, mass spectrometry revealed multiple nitrated tyrosines on nmMLCK. The authors should provide a more apparent justification for why Y1410 is functionally dominant rather than cooperative with other nitration sites. Additional discussion or experimental evidence (e.g., combinatorial mutants or phospho- or kinase-activity assays) would strengthen the conclusion that Y1410 nitration alone is sufficient to drive barrier dysfunction.

Response: We thank the reviewer for this insightful comment. We agree that other sites might play secondary or cooperative roles, perhaps in modulating the kinetics of recovery or long-term protein stability. Future studies using phosphoproteomics and multi-site mutagenesis is required to determine if secondary sites play a supporting role in long-term barrier recovery versus acute injury. While our mass spectrometry analysis identified several nitrated tyrosine residues, we focused on Y1410 for the following reasons: Y1410 is located within the unique N-terminal domain of the non-muscle isoform. This region is known to be critical for nmMLCK's association with the actin cytoskeleton and its subcellular localization, which may explain why its modification has a more profound functional impact compared to other sites. Our data show that the Y1410A single mutant is sufficient to significantly prevent peroxinitrite-induced nmMLCK nitration and paracellular gap formation.

 

2. While the data demonstrate altered protein–protein interactions (Kindlin-2 binding), it remains unclear whether Y1410 nitration directly alters nmMLCK catalytic activity. Measurement of MLC phosphorylation in WT versus Y1410A-expressing cells under SIN-1 or LPS stimulation would help distinguish whether the observed barrier effects are driven by altered kinase activity, scaffolding function, or both. The authors should experimentally address this point.

Response: We agree that distinguishing between nmMLCK’s catalytic activity and its scaffolding function is vital for understanding the role of Y1410 nitration. To address this, we have now performed several experiments: 1) We measured MLC2 phosphorylation (p-MLC2) in endothelial cells following treatment with LPS and SIN-1. As shown in the new Figure 5 F&G, both stimuli significantly increased p-MLC2 levels. These increases were attenuated by the peroxynitrite scavenger MnTmPyP, directly linking nitration-mediated stress to downstream kinase activation and signaling. 2) Following the reviewer’s suggestion, we compared p-MLC2 levels in cells expressing WT-nmMLCK versus the Y1410A mutant under SIN-1 or LPS stimulation. Our results showed a mild reduction in p-MLC2 levels in cells expressing the Y1410A mutant compared to WT. While the Y1410A mutant demonstrated a clear trend toward reduced kinase activity, we observed that high-level viral transduction of these constructs can sometimes mask subtle physiological nuances. However, the consistent reduction in p-MLC2 in the Y1410A mutant combined with the MnTmPyP data strongly suggests that Y1410 nitration contributes, at least in part, to the modulation of nmMLCK catalytic activity in addition to its protein-binding interactions.

 

3. SIN-1 is a commonly used but indirect peroxynitrite donor that generates both superoxide and nitric oxide simultaneously. The authors should more explicitly discuss potential off-target oxidative effects of SIN-1 and clarify whether additional controls (e.g., decomposed SIN-1 or alternative RNS scavengers) were used or considered to ensure specificity of peroxynitrite-mediated nitration.

Response: This is a excellent point. To evaluate the specific role of peroxynitrite-mediated nitration, we utilized the peroxynitrite scavenger MnTmPyP, which inhibits the formation of nitrating radicals. This enabled us to characterize the impact of site-specific nitration on nmMLCK. As a negative control, we prepared decomposed SIN-1 by incubating the compound in cell culture media at 37°C in the dark for 4 hours, allowing for complete aerobic degradation. Immunoprecipitation (IP) analysis confirmed that while fresh SIN-1 induced robust nmMLCK nitration, decomposed SIN-1 had no effect, further validating that the observed modifications are dependent on active peroxynitrite generation (Data not shown).

 

4. Many key experiments rely on the overexpression of GFP-tagged nmMLCK constructs in HEK293 or endothelial cells. The authors should discuss how overexpression levels compare with physiological nmMLCK abundance and whether similar nitration patterns are detectable for endogenous nmMLCK under inflammatory conditions.

Response: This is a very fair point, and therefore, to study nmMLCK nitration under physiological conditions, we examined endogenous levels in inflamed lung tissues derived from mice received to LPS (Figure 5A). Given the inherent challenges in detecting post-translational nitration, we performed immunoprecipitation to concentrate nmMLCK prior to analysis. This endogenous evidence was complemented by overexpression studies to provide a robust visualization of the modification.

 

5. Although LPS-treated mice show increased nmMLCK nitration and reduced Kindlin-2 interaction, the data remain correlative. Genetic or pharmacologic strategies that selectively block nmMLCK nitration (e.g., endothelial-specific Y1410A knock-in or antioxidant intervention linked to functional lung permeability endpoints) would significantly strengthen causal inference. The authors should experimentally address this point.

Response: We thank the reviewer for these helpful suggestions. Unfortunately, primary endothelial cells have a limited lifespan, limiting the generation of stable Y1410A knock-in lines. We agree that investigating whether antioxidant intervention prevents nmMLCK nitration and impacts lung permeability is a critical next step. We plan to address these mechanisms in a follow-up study.

 

6. The manuscript refers primarily to nmMLCK1, yet the lung expresses multiple MYLK isoforms. The authors should clarify whether nmMLCK2 is also nitrated under these conditions, or whether Y1410 is unique to a specific isoform involved in endothelial barrier regulation.

Response: This is an astute observation as nmMLCK1 and nmMLCK2 are identical except for the alternative splicing of Exon11.  The Y1410 residue is not located within Exon 11 and is conserved in both isoforms. We therefore hypothesize that nmMLCK2 is also nitrated by peroxynitrite under these conditions, similar to nmMLCK1, but did not address this directly.

 

7. While representative ECIS traces are shown, inclusion of summary metrics (e.g., area under the curve, peak resistance drop, or recovery kinetics) would allow for more rigorous quantitative comparison between WT and Y1410A conditions.

Response: We agree that quantitative summary metrics provide a more rigorous comparison than representative traces alone. Following the reviewer’s suggestion, we have now calculated the Area Under the Curve (AUC) and recovery kinetics (measured as the slope of resistance increase post-insult) for both WT and Y1410A conditions. These summary data have been added as Figure 3 and confirm a statistically significant difference in barrier recovery between the two groups (p < 0.05).

 

8. The disruption of nmMLCK–Kindlin-2 interaction is a central mechanistic claim. However, the binding interface remains undefined. At least the authors provide a brief discussion or reference to known Kindlin-2 interaction domains—or to how Y1410 nitration may sterically or electrostatically interfere with binding—this would enhance mechanistic clarity.

Response: This is a very important suggestion as we agree that defining the biochemical basis for the nmMLCK–Kindlin-2 interaction is crucial. While a full structural characterization of the interface is beyond the scope of this study, we have now added a discussion (Lines 423-436) in the reviswed manuscript that addresses how Y1410 nitration likely disrupts this binding. Kindlin-2 typically interacts with proteins via its Ferm domain (specifically the F1 or F3 subdomains). Tyrosine nitration adds a bulky, electronegative nitro group to the phenolic ring. We hypothesize that Y1410 is either part of a critical hydrophobic binding pocket or that the added negative charge and steric bulk of the nitro group electrostatically repel the Kindlin-2 binding interface, thereby preventing the recruitment of Kindlin-2 to the nmMLCK complex. While the precise binding interface between nmMLCK and Kindlin-2 remains to be crystallographically defined, our results suggest that the Y1410 residue is a critical determinant of this interaction. Kindlin-2 is known to utilize its tripartite FERM domain (subdivided into F1, F2, and F3) to scaffold signaling proteins at the cell membrane. It is highly probable that Y1410 resides within a hydrophobic pocket of the Kindlin-2 F3 subdomain, a common site for integrin-associated protein binding. The addition of a nitro group at the Y1410 position introduces both steric bulk and a significant increase in electronegativity. Such modifications are known to disrupt protein-protein interfaces through electrostatic repulsion or by physically preventing the close apposition of binding surfaces, thereby explaining the loss of nmMLCK–Kindlin-2 association observed in our study.

 

9. The authors appropriately acknowledge the lack of patient data as a limitation. Nevertheless, a brief discussion referencing existing evidence of protein nitration in human ARDS or sepsis samples would help contextualize the translational relevance of nmMLCK nitration.

Response: We thank the reviewer for this suggestion to strengthen the translational context of our work. While patient samples were not directly analyzed in this study, there is substantial clinical evidence of increased protein nitration in the lungs and plasma of patients with ARDS and sepsis. We have now added a paragraph to the Discussion (Lines 434-441) with citation of studies that have identified elevated 3-nitrotyrosine levels in human bronchoalveolar lavage fluid (BALF) and lung tissues,findings supporting the clinical relevance of our proposed nmMLCK nitration mechanism.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for revision.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my comments.