REGULATION OF SUPEROXIDE PRODUCTION BY HUMAN NEUTROPHILS

Summary

Principal Investigator: Ulla Knaus
Abstract: The superoxide-generating NADPH oxidase is a complex, multicomponent system that is present in a dormant form in unstimulated neutrophils, but is activated after exposure of the cells to a wide range of stimuli. Superoxide, and the reactive species derived from it, generally serve a beneficial role in killing pathogenic microbes, but can cause serious damage to normal tissues during inflammation. For example, in adult respiratory distress syndrome, pulmonary damage results from the excessive production of phagocyte oxidants, while in chronic granulomatous disease (CGD), failure to produce these molecules leads to life-threatening infections. The long-term objectives of the proposed research are: 1) to elucidate the biochemical mechanisms that regulate NADPH oxidase activity; and 2) to develop drugs capable of modulating this activity. In clinical situations where there is excessive inflammation, agents that suppress this activity may be of therapeutic use. Conversely, drugs that augment NADPH-oxidase activity in a way that improves the antimicrobial efficiency of the neutrophil could be advantageous to patients suffering from overwhelming infections. Several key methods have been developed in this laboratory to study this problem. A fully soluble cell-free system will be used to reconstitute oxidase activity from highly purified and recombinant oxidase proteins to determine how they regulate NADPH oxidase. A group of >300 CGD patients who have a variety of mutations encompassing four of the oxidase components have been characterized and are available for further studies. The cell-free system has made it possible to study the functional defects in the mutant proteins expressed by these patients. In addition, studies will be performed using site-directed mutant forms of phox proteins expressed in mammalian cell lines. Polyclonal and monoclonal antibodies will be used in surface plasmon resonance, immunoprecipitation, inhibition and topological studies of NADPH oxidase components. The following specific aims are designed to focus on several of the major conceptual and technical deficiencies that currently impede the long-range goals of this project: 1) To determine how the cytosolic factors and their cofactors regulate the activity of NADPH oxidase; 2) To study the molecular interactions of the cytosolic and membrane factors and their cofactors in unstimulated, primed and activated neutrophils; 3) To identify and characterize mutations in NADPH oxidase components to understand how they result in CGD. These studies may ultimately lead to new pharmacological methods for controlling oxygen radical-mediated tissue damage as well as an improved understanding of the pathophysiology of CGD.
Funding Period: 1986-09-01 - 2010-02-27
more information: NIH RePORT

Top Publications

  1. ncbi Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity
    Yanmin Zhu
    Herman B Wells Center for Pediatric Research, Department of Pediatrics Hematology Oncology, Microbiology Immunology, and Medical and Molecular Genetics, James Whitcomb Riley Hospital for Children, Indianapolis, Indiana 46202, USA
    J Biol Chem 281:30336-46. 2006
  2. ncbi Silencing of DUOX NADPH oxidases by promoter hypermethylation in lung cancer
    Sylvia Luxen
    Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA
    Cancer Res 68:1037-45. 2008
  3. ncbi P21-activated kinase is required for mitotic progression and regulates Plk1
    B Maroto
    Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Oncogene 27:4900-8. 2008
  4. pmc Inhibitory action of NoxA1 on dual oxidase activity in airway cells
    Sandrine Pacquelet
    Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California 92037, USA
    J Biol Chem 283:24649-58. 2008
  5. pmc Mutational analysis reveals distinct features of the Nox4-p22 phox complex
    Katharina von Löhneysen
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA
    J Biol Chem 283:35273-82. 2008
  6. pmc Lung epithelial injury by B. anthracis lethal toxin is caused by MKK-dependent loss of cytoskeletal integrity
    Mandy Lehmann
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America
    PLoS ONE 4:e4755. 2009
  7. pmc Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells
    Sylvia Luxen
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Sci 122:1238-47. 2009
  8. pmc Structural insights into Nox4 and Nox2: motifs involved in function and cellular localization
    Katharina von Löhneysen
    Department of Immunology and Microbial Science, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
    Mol Cell Biol 30:961-75. 2010

Scientific Experts

  • Ulla Knaus
  • Deborah Noack
  • Katharina von Löhneysen
  • Sylvia Luxen
  • Mandy Lehmann
  • Monika Frausto
  • Sandrine Pacquelet
  • B Maroto
  • Mary C Dinauer
  • Algirdas J Jesaitis
  • Yanmin Zhu
  • Malcolm R Wood
  • Jeffrey S Friedman
  • Malcolm Wood
  • Bruce E Torbett
  • Suzel Davanture
  • Marta Perego
  • M B Ye
  • Karine Regazzoni
  • K von Lohneysen
  • Steven A Belinsky
  • A Schnelzer
  • Amy Jo Casbon
  • Natalie Stull
  • Sally McCormick
  • Christophe C Marchal
  • William M Nauseef

Detail Information

Publications8

  1. ncbi Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity
    Yanmin Zhu
    Herman B Wells Center for Pediatric Research, Department of Pediatrics Hematology Oncology, Microbiology Immunology, and Medical and Molecular Genetics, James Whitcomb Riley Hospital for Children, Indianapolis, Indiana 46202, USA
    J Biol Chem 281:30336-46. 2006
    ....
  2. ncbi Silencing of DUOX NADPH oxidases by promoter hypermethylation in lung cancer
    Sylvia Luxen
    Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA
    Cancer Res 68:1037-45. 2008
    ..Our results suggest that an area on chromosome 15 that includes DUOX1, DUOX2, and their maturation factors is a frequent target for epigenetic silencing in lung cancer...
  3. ncbi P21-activated kinase is required for mitotic progression and regulates Plk1
    B Maroto
    Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Oncogene 27:4900-8. 2008
    ..Our results reveal a role for Pak in regulating Plk1 activity and mitotic progression, and connect Pak to the complex protein interaction network enabling cell division...
  4. pmc Inhibitory action of NoxA1 on dual oxidase activity in airway cells
    Sandrine Pacquelet
    Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California 92037, USA
    J Biol Chem 283:24649-58. 2008
    ..This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components...
  5. pmc Mutational analysis reveals distinct features of the Nox4-p22 phox complex
    Katharina von Löhneysen
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA
    J Biol Chem 283:35273-82. 2008
    ..These studies highlight the distinct interaction of the key regulatory p22(phox) subunit with Nox4, a feature which could provide the basis for selective inhibitor development...
  6. pmc Lung epithelial injury by B. anthracis lethal toxin is caused by MKK-dependent loss of cytoskeletal integrity
    Mandy Lehmann
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America
    PLoS ONE 4:e4755. 2009
    ..These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax...
  7. pmc Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells
    Sylvia Luxen
    Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Sci 122:1238-47. 2009
    ..As these locations probably define signaling specificity of Duox1 versus Duox2, these findings will facilitate monitoring Duox isoform expression in lung disease, a first step for early screening procedures and rational drug development...
  8. pmc Structural insights into Nox4 and Nox2: motifs involved in function and cellular localization
    Katharina von Löhneysen
    Department of Immunology and Microbial Science, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
    Mol Cell Biol 30:961-75. 2010
    ..In contrast, Nox4 released H(2)O(2) and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design...