Use this URL to cite or link to this record in EThOS:
Title: The biochemical and biophysical characterisation of p47phox activation
Author: Hussain, F.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2008
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
The multi-component enzyme NADPH oxidase plays a central role in host defence against microbial infection, due to its ability to support the production of reactive oxygen species. The enzyme is dormant in resting cells where its six hetero-subunits are separated into cytoplasmic and membrane compartments. Activation and assembly of the NADPH oxidase is a complex process that is regulated, in large part, by changes in protein-protein and protein-lipid interactions. An important step in the activation process is the phosphorylation-induced translocation of the cytoplasmic P40phox-p67phox-p47phox complex to the membrane-bound heterodimeric P22phox-gp91phox complex (flavocytochrome b.sss). This interaction is prevented in the resting state due to an intramolecular interaction in p47phox that maintains the protein in an auto-inhibited conformation. Phosphorylation of eight serine residues in p47phox is essential for enzyme activation, of which five are located in the polybasic region and are known to relieve the auto-inhibition. Glutamate mutants were made to mimic phosphoserine residues, and various biochemical and biophysical techniques were used to demonstrate that phosphorylation of the remaining three serine residues, located in the C-terminal region, do not contribute to the release of auto-inhibition, and hence do not promote binding to p22phox. Instead, they weaken the interaction with p67phox. Based on these data, a new model for phosphorylation-induced changes in NADPH oxidase assembly is proposed. Binding of the polybasic region and p22phox to the tandem SH3 domains of p47phox occurs with high affinity. This affinity is mediated through the formation of a 'superSH3 domain', which is largely dependent on two structural features: the covalent SH3-SH3 linker and a 'GWW' motif located in the n-Src loops of either SH3 domain. Mutant proteins were made and quantitative binding assays were performed to demonstrate that the chemical nature of the linker is more important for the intramolecular interaction with the polybasic region in the auto-inhibited state, than for an intermolecular interaction such as that with p22phox in the active state. Furthermore, it was shown that the tryptophan residues of the GWW motif play different roles in the stabilisation of the superSH3 domain conformation. Their precise role depends on whether additional interactions between the superSH3 domain and its target can occur outside of the conserved ligand binding site, as observed in the auto-inhibited state. These data suggest that the sequence requirements for the formation of a superSH3 domain are relatively flexible, making it more likely that other proteins containing multiple SH3 domains may also form a superSH3 domain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available