Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of RhoA

J Biol Chem. 2014 Feb 21;289(8):4710-22. doi: 10.1074/jbc.M114.547596. Epub 2014 Jan 7.


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.

Keywords: Enzymology; Lung Injury; Post-translational Modification; Protein Structure; Recombinant Protein Expression; Redox Regulation; Superoxide Ion.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acute Lung Injury / enzymology
  • Acute Lung Injury / pathology
  • Acute Lung Injury / physiopathology
  • Amino Acid Sequence
  • Animals
  • Bronchoalveolar Lavage Fluid
  • Cytokines / metabolism
  • Disease Models, Animal
  • Endothelial Cells / enzymology
  • Endothelial Cells / pathology
  • Enzyme Activation
  • Humans
  • Lipopolysaccharides
  • Lung Injury / chemically induced
  • Lung Injury / enzymology*
  • Lung Injury / pathology*
  • Lung Injury / physiopathology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microvessels / pathology
  • Molecular Sequence Data
  • Nitric Oxide Synthase Type III / metabolism
  • Nitrosation
  • Peptides / metabolism
  • Protective Agents / metabolism
  • Tyrosine / metabolism
  • rhoA GTP-Binding Protein / chemistry
  • rhoA GTP-Binding Protein / metabolism*


  • Cytokines
  • Lipopolysaccharides
  • Peptides
  • Protective Agents
  • Tyrosine
  • Nitric Oxide Synthase Type III
  • rhoA GTP-Binding Protein