Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA |
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Authors: | Ruslan Rafikov Christiana Dimitropoulou Saurabh Aggarwal Archana Kangath Christine Gross Daniel Pardo Shruti Sharma Agnieszka Jezierska-Drutel Vijay Patel Connie Snead Rudolf Lucas Alexander Verin David Fulton John D Catravas Stephen M Black |
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Institution: | From the ‡Program in Pulmonary Vascular Disease, Vascular Biology Center and ;the §Department of Surgery, Georgia Regents University, Augusta, Georgia 30912 |
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Abstract: | Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr34 in RhoA. Tyr34 is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr34 against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr34 nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of “shielding” peptides to prevent RhoA nitration in the management of ALI. |
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Keywords: | Lung Injury Post-translational Modification Protein Structure Recombinant Protein Expression Redox Regulation Superoxide Ion Enzymology |
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