This project examines the molecular basis for assembly of NADPH oxidase, a system responsible for generation of reactive microbicidal oxidants which can cause tissue damage at sites of inflammation. This enzyme is comprised of both membrane-bound and cytosolic components that assemble into a membrane-bound complex during oxidase activation. The applicant describes a variety of strategies for exploring the central hypothesis proposing that a series of specific protein-protein interactions occurs between oxidase components and that flavocytochrome b558 acts as a final docking site where the active enzyme assembles. Proposed experimental approaches include: 1) determination of primary sequences in cytosolic oxidase components that bind to cytochrome b558 by phage display and two-hybrid screening protocols, photoaffinity labelling and site-directed mutagenesis; 2) mapping of complementary interacting sites on the cytochrome by similar approaches; 3) studies on interactions of the GTP-binding oxidase component, Rac, with other cytosolic components and the role of the guanine nucleotide exchange inhibitor, GDI; 4) mapping of sites of interactions between the cytosolic components; 5) structural studies on oxidase proteins bound to synthetic peptides, as models of the complexes formed with other oxidase components; 6) attempts to crystallize the cytosolic components p47- and p67-phox, which would be followed by x-ray diffraction studies. These studies would provide the basis for a better understanding of the structure of the active enzyme and regulation of its assembly and may eventually lead to development of therapeutic strategies aimed at preventing tissue damage associated with overproduction of oxidants in inflammatory diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042426-05
Application #
6171693
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Gretz, Elizabeth
Project Start
1996-09-30
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
5
Fiscal Year
2000
Total Cost
$181,064
Indirect Cost
Name
Montana State University Bozeman
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
City
Bozeman
State
MT
Country
United States
Zip Code
59717
Lu, Quansheng; Yang, Yu; Villar, Van Anthony et al. (2013) D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1. Hypertens Res 36:684-90
Montezano, Augusto C; Burger, Dylan; Paravicini, Tamara M et al. (2010) Nicotinamide adenine dinucleotide phosphate reduced oxidase 5 (Nox5) regulation by angiotensin II and endothelin-1 is mediated via calcium/calmodulin-dependent, rac-1-independent pathways in human endothelial cells. Circ Res 106:1363-73
Raad, Houssam; Paclet, Marie-Hélène; Boussetta, Tarek et al. (2009) Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox. FASEB J 23:1011-22
Nemali, Sailasree; Siemsen, Daniel W; Nelson, Laura K et al. (2008) Molecular analysis of the bovine anaphylatoxin C5a receptor. J Leukoc Biol 84:537-49
Mofarrahi, Mahroo; Brandes, Ralf P; Gorlach, Agnes et al. (2008) Regulation of proliferation of skeletal muscle precursor cells by NADPH oxidase. Antioxid Redox Signal 10:559-74
Ammons, Mary Cloud B; Siemsen, Daniel W; Nelson-Overton, Laura K et al. (2007) Binding of pleomorphic adenoma gene-like 2 to the tumor necrosis factor (TNF)-alpha-responsive region of the NCF2 promoter regulates p67(phox) expression and NADPH oxidase activity. J Biol Chem 282:17941-52
Kawahara, By Tsukasa; Quinn, Mark T; Lambeth, J David (2007) Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol Biol 7:109
Schepetkin, Igor A; Kirpotina, Liliya N; Khlebnikov, Andrei I et al. (2007) High-throughput screening for small-molecule activators of neutrophils: identification of novel N-formyl peptide receptor agonists. Mol Pharmacol 71:1061-74
Siemsen, Daniel W; Schepetkin, Igor A; Kirpotina, Liliya N et al. (2007) Neutrophil isolation from nonhuman species. Methods Mol Biol 412:21-34
Gauss, Katherine A; Nelson-Overton, Laura K; Siemsen, Daniel W et al. (2007) Role of NF-kappaB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-alpha. J Leukoc Biol 82:729-41

Showing the most recent 10 out of 49 publications