University of Surrey

Test tubes in the lab Research in the ATI Dance Research

Protein Structure and Biochemical Characterisation of the p22phox and p47phox in NADPH Oxidase-2 Activation.

Meijles, Daniel N. (2013) Protein Structure and Biochemical Characterisation of the p22phox and p47phox in NADPH Oxidase-2 Activation. Doctoral thesis, University of Surrey (United Kingdom)..

Available under License Creative Commons Attribution Non-commercial Share Alike.

Download (9MB) | Preview


It has been well documented that a superoxide-generating NADPH oxidase 2 (Nox2) is involved in the pathogenesis of oxidative stress-related cardiovascular diseases such as hypertension and atherosclerosis. However, the structure of the Nox2 protein subunits and the regulatory mechanisms of Nox2 activation remain largely unknown. Therefore the purpose of this Ph.D. research project is to use a combination of in silico computational protein structure modelling and molecular cell biology to investigate the three-dimensional (3D) protein structural changes of the p22phox (the Nox2 partner of the cytochrome b558) and p47phox (a key regulatory subunit of the Nox2) in the phosphorylation regulation of Nox2 activation. A consensus in silico 3D protein structure model for the p22phox was generated by incorporating transmembrane-specific structural predictions with the computational modelling program molecular operating environment (MOE). The results showed that the p22phox consists of an N-terminal transmembrane domain (124 a.a.) with three membrane transecting helices, an extensive extracellular region between helices two and three, and a C-terminal cytoplasmic domain (71 a.a.). Using the p22phox structure model generated in this thesis, I examined the C242T polymorphism, which has been shown to be protective against the progression of atherosclerosis. My results showed that the C242T polymorphism, which corresponds to substitution of Histidine-72 to Tyrosine-72, is located in the extracellular loop of the p22phox, which causes significant morphological changes of the p22phox and affects its interaction with the catalytic Nox2 subunit. Transfection of p22phox plasmid baring C242T polymorphism into human endothelial cells significantly reduced TNFα (100U/ml, 30 min)-induced reactive oxygen species (ROS) production as compared to cells transfected with wild-type (WT) p22phox plasmid and vector controls. These results were further verified in p22phox-defficient HeLa cells. The process of p22phox binding to phosphorylated p47phox has been found to be a crucial step in Nox2 activation. However, it has only been suggested, but not proven that under resting conditions, the p47phox is in its auto-inhibited conformation such that the p22phox binding groove is masked by a polybasic auto-inhibitory region (AIR). Phosphorylation of the AIR exposes the p22phox binding groove and is known to initiate Nox2 activation. In order to investigate the phosphorylation-induced morphological changes in p47phox, I modelled the p47phox, based on the available crystal structures, in both resting and the activated forms by molecular dynamics. My results showed that phosphorylation of the serines-303/4, 310, 315 and 379 located at the C-terminus of p47phox moves the auto-inhibitory region away, which in turn exposes the p22phox binding groove and initiates a process essential for the activation of Nox2. In summary, my Ph.D. project combines the knowledge and techniques of bioinformatics with cellular and molecular biology. The results of the in silico models of both p22phox and p47phox can be used for further investigation of the mechanisms of Nox2 activation, and can be exploited for the design of small molecules to inhibit Nox2 activation. This thesis has also provided important insights into the mechanism of the C242T polymorphism, which explains its clinical relevance to inhibit endothelial dysfunction reported by previous clinical studies.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Meijles, Daniel N.
Date : 2013
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 2013.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:06
Last Modified : 06 May 2020 14:09

Actions (login required)

View Item View Item


Downloads per month over past year

Information about this web site

© The University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom.
+44 (0)1483 300800