title: Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of RhoA.
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 Tyr(34) in RhoA. Tyr(34) 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 Tyr(34) 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 Tyr(34) 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.
authors.affiliation: From the Program in Pulmonary Vascular Disease, Vascular Biology Center and.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Rafikov
authors.forename: Christiana
authors.initials: C
authors.lastname: Dimitropoulou
authors.forename: Saurabh
authors.initials: S
authors.lastname: Aggarwal
authors.forename: Archana
authors.initials: A
authors.lastname: Kangath
authors.forename: Christine
authors.initials: C
authors.lastname: Gross
authors.forename: Daniel
authors.initials: D
authors.lastname: Pardo
authors.forename: Shruti
authors.initials: S
authors.lastname: Sharma
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Jezierska-Drutel
authors.forename: Vijay
authors.initials: V
authors.lastname: Patel
authors.forename: Connie
authors.initials: C
authors.lastname: Snead
authors.forename: Rudolf
authors.initials: R
authors.lastname: Lucas
authors.forename: Alexander
authors.initials: A
authors.lastname: Verin
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: John D
authors.initials: JD
authors.lastname: Catravas
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
mesh_terms: D055371:Acute Lung Injury / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D000595:Amino Acid Sequence; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000201:enzymology / Q000473:pathology; D004789:Enzyme Activation; D006801:Humans; D008070:Lipopolysaccharides; D055370:Lung Injury / Q000139:chemically induced / Q000201:enzymology* / Q000473:pathology* / Q000503:physiopathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000473:pathology; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015538:Nitrosation; D010455:Peptides / Q000378:metabolism; D020011:Protective Agents / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D010455:Peptides; D020011:Protective Agents; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D020742:rhoA GTP-Binding Protein
keywords: Enzymology; Lung Injury; Post-translational Modification; Protein Structure; Recombinant Protein Expression; Redox Regulation; Superoxide Ion