The production of oxygen radicals by human blood neutrophils is critical for the success of host defense, but also may be detrimental to the host in certain inflammatory processes, i.e. in the adult respiratory distress syndrome, arthritis, etc. Understanding the regulation of oxygen radical production is the long-term goal of this project and is important for developing methods of controlling various disease states. The enzyme system in neutrophils responsible for the conversion of oxygen to toxic products appears to be an activatable membrane-bound NADPH oxidase, and its mechanism of activation is not yet known. A variety of stimulating agents (i.e. phagocytic particles, chemoattractants, calcium ionophores, and phorbol esters) activate NADPH oxidase. Previous data suggest that the pathways leading to NADPH oxidase activation are complex, with both stimulus-specific and shared components, and that the shared pathway consists of at least two separately regulated steps. Recent observations have implicated protein kinase C as having a major role in receptor-mediated cellular regulation. Several events are triggered by stimulation of neutrophils that could cause activation of protein kinase C, including an increase in calcium concentration, the release of diacylglycerol, and the release of arachidonate. In addition, protein kinase C appears to be the receptor for phorbol esters, which are potent stimulants for neutrophils.
Specific Aim 1 of this project will test the hypothesis that protein kinase C is the central, shared intermediate in the pathway of NADPH oxidase activation. Preliminary data indicate that stimulation of neutrophils increases protein kinase C activity in a particulate fraction of the cell and that the increase correlates with oxidase activation. Further experiments to test the correlation between the two events will be performed. The principal investigator has recently shown that arachidonate activates NADPH oxidase in subcellular fractions from unstimulated human neutrophils and that separate, inactive fractions can be reconstituted to form an active system. This cell-free activation system will be utilized in Specific Aim 2 to determine specific biochemical mechanisms (i.e. phosphorylation of proteins, membrane phospholipid changes, membrane biophysical changes) which correlate with NADPH oxidase activation and to identify required co-factors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022564-05
Application #
3133826
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1985-07-01
Project End
1990-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
Sergeant, Susan; McPhail, Linda C (2014) Measurement of phospholipid metabolism in intact neutrophils. Methods Mol Biol 1124:89-105
Davis, Nicole Y; McPhail, Linda C; Horita, David A (2012) The NOXO1ýý PX domain preferentially targets PtdIns(4,5)P2 and PtdIns(3,4,5)P3. J Mol Biol 417:440-53
Davis, Nicole Y; McPhail, Linda C; Horita, David A (2011) Backbone 1H, 15N, and 13C resonance assignments for the NOXO1? PX domain. Biomol NMR Assign 5:139-41
Lewis, Eric M; Sergeant, Susan; Ledford, Bill et al. (2010) Phosphorylation of p22phox on threonine 147 enhances NADPH oxidase activity by promoting p47phox binding. J Biol Chem 285:2959-67
(2009) Effect of splice-site mutations in X-linked chronic granulomatous disease. Clin Immunol 130:234
Shen, Kai; Sergeant, Susan; Hantgan, Roy R et al. (2008) Mutations in the PX-SH3A linker of p47phox decouple PI(3,4)P2 binding from NADPH oxidase activation. Biochemistry 47:8855-65
Lewis, Eric M; Singla, Manav; Sergeant, Susan et al. (2008) X-linked chronic granulomatous disease secondary to skewed X chromosome inactivation in a female with a novel CYBB mutation and late presentation. Clin Immunol 129:372-80
Chial, Heidi J; Wu, Ruping; Ustach, Carolyn V et al. (2008) Membrane targeting by APPL1 and APPL2: dynamic scaffolds that oligomerize and bind phosphoinositides. Traffic 9:215-29
Sergeant, Susan; McPhail, Linda C (2007) Measurement of phospholipid metabolism in intact neutrophils. Methods Mol Biol 412:69-83
Taylor, Ross M; Lord, Connie I; Riesselman, Marcia H et al. (2007) Characterization of surface structure and p47phox SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b. Biochemistry 46:14291-304

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