The Biological Role of Redox Signalling by the Tumour Suppressor PTEN

Abstract

Phosphatase and tensin homologue (PTEN) is an antagonist of the Akt pathway through its phosphatase activity against phosphatidylinositol-3,4,5-phosphate (PIP3). The Akt pathway modulates numerous metabolic and cell survival processes. PTEN is redox sensitive and oxidation by reactive oxygen species (ROS) causes catalytic inactivation and changes to its interactome. ROS can oxidise phospholipids resulting in lipid oxidation products, such as acrolein and 4-hydroxynonenal (4-HNE). Whilst the effect of 4-HNE on PTEN has previously been studied, there is limited data for acrolein. The research presented in this thesis aimed to further characterise the effect of lipoxidation on PTEN by investigating the effect of acrolein on PTEN’s activity, structure and interactions using a range of proteomic techniques. In the first chapter, recombinant human PTEN-V5-His was overexpressed in Escherichia coli and purified using immobilised metal affinity chromatography. In the second chapter, PTEN-V5-His was treated in vitro with acrolein, from 0.2:1 to 20:1 acrolein:PTEN. Phosphatase activity and SDS-PAGE analysis showed dose-dependent inactivation and aggregation. Analysis using tandem mass spectrometry showed a greater susceptibility of cysteine residues to modification, with lysine modifications detected at higher acrolein concentrations. The third chapter involved affinity pull down with PTEN-V5-His, where acrolein treatment was performed prior to challenge with HCT-116 cell lysates. Proteomic analysis of the captured proteins identified changes in the interactome between untreated and acrolein-treated PTEN. HECTD1, an E3 ubiquitin ligase, was validated in vitro as a novel interactor of PTEN that showed increased binding on acrolein treatment of PTEN. The final chapter characterised the in cellulo effect of acrolein treatment, where a dose- and time-dependent loss of cell viability was identified for HCT-116 cells. On treatment with sublethal acrolein concentrations, the levels of active phosphoAkt increased whereas inactive Akt remained constant. Taken together, these results demonstrate that acrolein causes a functional change in the activity, structure and interactions of PTEN.