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.
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