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Title: Structural and biochemical studies of the components of the NADPH oxidase in human neutrophils
Author: Shao, Dongmin
ISNI:       0000 0001 3397 0183
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2003
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The NADPH oxidase generates O2- in phagocytes such as neutrophils. The system is dormant in resting cells and activated on exposure to stimuli following the assembly of the cytosolic components p40phox, p47phox, p67phox and rac2 with cytochrome b558 in the membrane. A region of homology of about 100-110 residues in length exists between the N-termini of p40phox and p47phox and has been termed the PX domain, the role of which is unknown. To further understand the role of this domain with respect to the cytosolic factor proteins p40phox and p47phox of the NADPH oxidase, we have carried out expression, purification, characterisation and crystallisation trials of the PX domain of p40phox and p47phox, and full-length p47phox. We also expressed and purified the full-length p40phox and truncated p67phox (302-460). A complex of full-length p40phox and p67phox (302- 460) has been made and the binding has been characterised. Numerous attempts to crystallise this complex have been carried out and protein crystals have been obtained. We also identified a PX domain binding partner in neutrophils using affinity column made from recombinant PX domain of p40phox. Actin was found to bind to PX domains of p40phox and p47phox. In vitro binding studies confirmed PX domains bound to the side of F-actin. Significantly, PX domains co-localised with F-actin when PX domain of p40phox and p47phox constructs were microinjected into fibroblasts nuclear, suggesting a positive role of actin filaments in the NADPH oxidase activation. Recent evidence shows that specific lipid microdomains, "rafts", are involved in signal transduction and in activation of many cells. Here we have examined the contribution of lipid rafts to NADPH oxidase activation in neutrophils. We found that the NADPH oxidase components in neutrophils associated with membrane rafts. Stimulation of neutrophils with PMA or via Fc receptors induced cytosolic components and PKC isotypes to be recruited to membrane rafts. Treatment of neutrophils with a cholesterol depletion drug-MβCD resulted in less efficient activation of the NADPH oxidase by PMA and IgG coated S. aureus, hence pointing to a function of membrane rafts as a "trap" to concentrate molecules and increasing the efficiency of cross-talking between molecules.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available