Assembly of the phagocyte NADPH oxidase complex: chimeric constructs derived from the cytosolic components as tools...

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Signaling

Background:

• Phagocytes utilize reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate superoxide (O2*-).
• The enzyme complex comprises membrane-bound flavocytochrome b559 (gp91phox and p22phox) and cytosolic components (p47phox, p67phox, Rac).
• Activation involves cytosolic component translocation and cytochrome b559 interaction, leading to O2*- production.

Purpose of the Study:

• To design and characterize novel activators of NADPH oxidase.
• To investigate the role of protein-protein interactions and conformational changes in NADPH oxidase activation.
• To propose a new model for the mechanism of NADPH oxidase activation.

Main Methods:

• Design and synthesis of chimeric proteins combining segments of p67phox and Rac1.
• Cell-free system assays to measure NADPH oxidase activation.
• Biophysical techniques including fluorescence resonance energy transfer (FRET), small angle X-ray scattering (SAXS), and gel filtration to study chimera conformation.

Main Results:

• A prototype chimera, p67phox(1-212)-Rac1(192), potently activated NADPH oxidase in a cell-free system.
• Prenylated chimeras activated the oxidase independently of p47phox and anionic amphiphiles.
• Intrachimeric bonds between p67phox and Rac1 moieties were essential for activation, influencing chimera conformation.

Conclusions:

• Intrachimeric bonds are critical for NADPH oxidase activation by p67phox-Rac1 chimeras.
• Conformational changes within the chimeras are directly linked to their activation capability.
• A 'propagated wave' model is proposed, where conformational changes initiated in Rac transmit through p67phox to gp91phox, activating the enzyme.