Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA*

Background: The activation of RhoA is a critical event in acute lung injury (ALI), but the role of nitration in this process is unresolved. Results: The nitration of RhoA at Tyr34 produced GEF-like conformational changes that stimulate RhoA by decreasing GDP binding. Conclusion: We have identified a new mechanism of RhoA activation. Significance: Preventing RhoA nitration may be useful for the management of ALI. 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.

[1]  Richard E. White,et al.  Agonist of growth hormone-releasing hormone reduces pneumolysin-induced pulmonary permeability edema , 2012, Proceedings of the National Academy of Sciences.

[2]  Lung,et al.  Randomized, placebo-controlled clinical trial of an aerosolized β₂-agonist for treatment of acute lung injury. , 2011, American journal of respiratory and critical care medicine.

[3]  L. de Noronha,et al.  Increased Calcification and Protein Nitration in Arteries of Chronic Kidney Disease Patients , 2011, Blood Purification.

[4]  Yunchao Su,et al.  Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis. , 2011, Free radical biology & medicine.

[5]  Arthur S Slutsky,et al.  An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. , 2011, American journal of respiratory cell and molecular biology.

[6]  E. Lemichez,et al.  Direct modifications of Rho proteins: deconstructing GTPase regulation , 2010, Biology of the cell.

[7]  J. Catravas,et al.  Harvesting, identification and barrier function of human lung microvascular endothelial cells. , 2010, Vascular pharmacology.

[8]  J. Catravas,et al.  Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine. , 2010, Vascular pharmacology.

[9]  S. Yuan,et al.  Endothelial contractile cytoskeleton and microvascular permeability. , 2009, Cell health and cytoskeleton.

[10]  G. Martin,et al.  Recent trends in acute lung injury mortality: 1996–2005* , 2009, Critical care medicine.

[11]  Lisa N Kinch,et al.  AMPylation of Rho GTPases by Vibrio VopS Disrupts Effector Binding and Downstream Signaling , 2009, Science.

[12]  C. Irvin,et al.  Inhibition of Arginase Activity Enhances Inflammation in Mice with Allergic Airway Disease, in Association with Increases in Protein S-Nitrosylation and Tyrosine Nitration1 , 2008, The Journal of Immunology.

[13]  J. Jung,et al.  Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Augments Acute Lung Injury via Its Neutrophil Priming Effects , 2008, Journal of Korean medical science.

[14]  Catalin Vasilescu,et al.  Endotoxemia and endotoxin tolerance in patients with ARDS , 2008, Langenbeck's Archives of Surgery.

[15]  Thomas F. Rau,et al.  Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension. , 2008, American journal of physiology. Lung cellular and molecular physiology.

[16]  S. Black,et al.  Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[17]  M. Revel,et al.  Comparison of two fluid-management strategies in acute lung injury, H.P. Wiedemann, A.P. Wheeler, G.R. Bernard, B.T. Thompson, D. Hayden, B. deBoisblanc, A.F Jr. Connors, R.D. Hite, A.L. Harabin, in: N Engl J Med, 354. (2006), 2564 , 2007 .

[18]  S. Campbell,et al.  Redox regulation of RhoA. , 2006, Biochemistry.

[19]  R. Sladek,et al.  Lipopolysaccharide and TNF-α Produce Very Similar Changes in Gene Expression in Human Endothelial Cells , 2006, Journal of Vascular Research.

[20]  A. O’Brien,et al.  A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp. , 2006, Molecular microbiology.

[21]  D. Silverman,et al.  Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation. , 2006, Free radical biology & medicine.

[22]  R. Wysolmerski,et al.  Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation* , 2005, Journal of Biological Chemistry.

[23]  S. Campbell,et al.  Mechanism of Redox-mediated Guanine Nucleotide Exchange on Redox-active Rho GTPases* , 2005, Journal of Biological Chemistry.

[24]  F. Huang,et al.  Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk. , 2005, American journal of physiology. Lung cellular and molecular physiology.

[25]  A. Hopkins,et al.  RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. , 2004, American journal of physiology. Cell physiology.

[26]  S. Rounds,et al.  Barrier dysfunction and RhoA activation are blunted by homocysteine and adenosine in pulmonary endothelium. , 2004, American journal of physiology. Lung cellular and molecular physiology.

[27]  R. Kallet.  Evidence-based management of acute lung injury and acute respiratory distress syndrome. , 2004, Respiratory care.

[28]  K. Walley,et al.  Mechanisms of beta-receptor stimulation-induced improvement of acute lung injury and pulmonary edema , 2004, Critical care.

[29]  B. Graham,et al.  RhoA-Derived Peptide Dimers Share Mechanistic Properties with Other Polyanionic Inhibitors of Respiratory Syncytial Virus (RSV), Including Disruption of Viral Attachment and Dependence on RSV G , 2004, Journal of Virology.

[30]  T. Akaike,et al.  Quantitative Assessment of Protein-bound Tyrosine Nitration in Airway Secretions from Patients with Inflammatory Airway Disease , 2004, Free radical research.

[31]  R. Dietz,et al.  Hydroxymethylglutaryl Coenzyme A Reductase Inhibition Reduces Chlamydia pneumoniae‐Induced Cell Interaction and Activation , 2003, Circulation.

[32]  C. Kirkpatrick,et al.  GM-CSF expression by human lung microvascular endothelial cells: in vitro and in vivo findings. , 2002, American journal of physiology. Lung cellular and molecular physiology.

[33]  E. Klann,et al.  Peroxynitrite-induced tyrosine nitration and inhibition of protein kinase C. , 2001, Biochemical and biophysical research communications.

[34]  A. Ridley,et al.  Rho and Rac but not Cdc42 regulate endothelial cell permeability. , 2001, Journal of cell science.

[35]  S. Kuroda,et al.  An Open Conformation of Switch I Revealed by the Crystal Structure of a Mg2+-free Form of RHOA Complexed with GDP , 2000, The Journal of Biological Chemistry.

[36]  K. Scheffzek,et al.  The Rac–RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI , 2000, Nature Structural Biology.

[37]  R. Schaeffer,et al.  RhoA inactivation enhances endothelial barrier function. , 1999, American journal of physiology. Cell physiology.

[38]  M. Wysocki,et al.  Prevalence, etiologies and outcome of the acute respiratory distress syndrome among hypoxemic ventilated patients , 1999, Intensive Care Medicine.

[39]  U. Rapp,et al.  Deamidation of Cdc42 and Rac by Escherichia coliCytotoxic Necrotizing Factor 1: Activation of c-Jun N-Terminal Kinase in HeLa Cells , 1999, Infection and Immunity.

[40]  Y. Zheng,et al.  Negative Regulation of Rho Family GTPases Cdc42 and Rac2 by Homodimer Formation* , 1998, The Journal of Biological Chemistry.

[41]  M. Shirakawa,et al.  Crystal Structure of Human RhoA in a Dominantly Active Form Complexed with a GTP Analogue* , 1998, The Journal of Biological Chemistry.

[42]  D. Perl,et al.  Protein Nitration in Parkinson's Disease , 1998, Journal of neuropathology and experimental neurology.

[43]  K. Aktories,et al.  Glucosylation and ADP ribosylation of rho proteins: effects on nucleotide binding, GTPase activity, and effector coupling. , 1998, Biochemistry.

[44]  W. Minor,et al.  Crystal structure of RhoA–GDP and its functional implications , 1997, Nature Structural Biology.

[45]  Theresa M. Wizemann,et al.  Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia , 1994, Journal of leukocyte biology.

[46]  J S Beckman,et al.  Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. , 1992, Archives of biochemistry and biophysics.

[47]  Marseille,et al.  Société de Réanimation de Langue Française , 1985 .