The generation of 02- by phagocytic cells requires the assembly of a membrane-bound complex known as the NADPH oxidase, which is composed of an integral plasma membrane flavocytochrome b and four cytosolic proteins (p40 phox, p47 phox, p67 phox, and Rac2). Although progress has been made, there is limited information about how each of these proteins regulate formation of the active NADPH oxidase complex and, similarly, what turns off the system. In the previous grant period, we identified a number of protein-protein interactions among the NADPH oxidase protein components and investigated how these interactions related to assembly of the active 02- generating system. In this renewal application, we propose studies to investigate how NADPH oxidase assembly is regulated. Based on studies performed under the previous grant period, studies by others on the phagocyte NADPH oxidase, and preliminary work described here, we hypothesize that neutrophil capacity for oxidase assembly and oxidase assembly per se are regulated at several different levels, including transcription and biosynthesis of the phox proteins, molecular assembly of the active system, and lateral organization in plasma membrane domains. Consequently, appropriate NADPH oxidase activity results from integration of these multiple levels of regulation. Because of the complex nature of the oxidase, we propose to address this hypothesis by focusing this project primarily on the role of the cytosolic phox factors in oxidase regulation. Specifically, this proposal describes strategies for: 1) characterizing the promoter for p67 phox and determining what transcription factors regulate p67 phox transcription; 2) characterizing cytosolic phox protein gene expression; 3) analyzing biosynthesis of the cytosolic phox proteins to determine relative stability of these proteins and kinetics of protein turnover; 4) identifying and characterizing flavocytochrome b domains that bind p67 phox and Rac; and 5) develop cell lines expressing GFP-phox fusion proteins for use analysis of the intermolecular interactions between cytosolic oxidase proteins and the oxidase complex in vivo. The results of these studies will further our understanding of how distinct levels of regulatory input are integrated to facilitate functional assembly and activation of the phagocyte NADPH oxidase.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042426-07
Application #
6732610
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Gretz, Elizabeth
Project Start
1996-09-30
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
7
Fiscal Year
2004
Total Cost
$337,099
Indirect Cost
Name
Montana State University Bozeman
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
625447982
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
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
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

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