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The role of redox biology in the functions of the tumour suppressor PTEN

Primary Supervisor: Professor Andrew Pitt, Life & Health Sciences

Secondary Supervisors: Professor Anna Hine, Professor Corinne Spickett

PhD project title: The role of redox biology in the functions of the tumour suppressor PTEN

University of Registration: Aston University

Project outline:

PTEN (Phosphatase and tensin homolog) is a redox-sensitive, dual-specificity protein phosphatase involved in regulating fundamental cellular processes, including metabolism, apoptosis, cell proliferation and survival, and is a key tumour suppressor. Loss of PTEN function is linked to a large number of cancers, for example over 70% of prostate cancers have low PTEN activity. The main function of PTEN is as a lipid phosphatase, catalyzing the dephosphorylation of phosphatidylinositol-3,4,5- triphosphte (PIP3) to phosphatidylinositol-4,5-diphosphate (PIP2), reversing the action of phosphoinositide-3-kinase (PI3K) and thus down-regulating the PI3K/Akt signaling pathway. However, PTEN has also been implicated in a number of other cellular processes, such as DNA damage repair and cytoskeletal reorganization, but the mechanisms by which it does this are unclear.

PTEN can be reversibly inactivated by oxidation of a catalytic cysteine residue to form an intramolecular disulfide, which plays an important part in the control of its enzymatic activity. Recently, using a new approach based on proteomics and chemical biology, we have demonstrated that the interaction of PTEN with other proteins, the PTEN interactome, changes depending on the redox status of PTEN. We identified 86 interacting proteins, the binding of fourteen of which was shown to be affected by the redox status of PTEN. Two of the proteins, Annexin A2 (AnxA2) and DNA Damage Binding Protein 1 (DDB1) could be related to the role of PTEN in cytoskeletal rearrangement and DNA damage repair. AnxA2 is involved in diverse cellular processes including cell motility, linkage of membrane-associated protein complexes to the actin cytoskeleton, endocytosis, and ion channel formation. DDB1 is involved in numerous processes in the cell including DNA base excision repair, DNA replication and chromatin remodeling. The binding of AnxA2 to PTEN was shown to be dependent on the redox status of PTEN, binding more strongly to the oxidized form, suggesting a role of redox regulation in DNA damage repair processes.

The project will build on 3 key areas:

i) In vitro studies of the redox interactome of PTEN to identify protein interactions that show redox sensitivity, looking at the effects of both PTEN redox status and the redox status of the cell. Using carefully characterized reduced and oxidized PTEN immobilized on a resin, interacting proteins will be captured from a cell lysate and characterized using proteomics techniques relying on liquid chromatography coupled to mass spectrometry. Lysates will be generated from human cancer cells lines stressed under redox conditions to understand the interplay between cellular oxidative stress and PTEN function.

ii) Validation of the changes identified in the PTEN redox interaction in vivo. Cells will be grown under normal and redox stress conditions, the cells lysed, and the interaction of PTEN with key proteins confirmed by western blotting. In addition proximity labelling using the BioID2 methodology (PTEN fused to a biotin ligase) will be developed to unlock more detail of the in vivo PTEN redox interactome using chemical and proteomic methods.

iii) Elucidation of the mechanistic effects that the redox changes in the PTEN interactome have on cellular processes, focusing initially on cytoskeletal rearrangement and DNA damage repair. This will be achieved by generating PTEN mutants with changes in catalytic activity, redox sensitivity or alternative binding, and probing the effect of the mutants on the functioning on the relevant cellular processes using well proven molecular markers.

References:

  1. The effect of HOCl-induced modifications on phosphatase and tensin homolog (PTEN) structure and function. Verrastro, I., Tveen Jensen, K., Spickett, C. M. & Pitt, A. R., 2018, Free Radical Research, 1-471.

  2. The effect of HOCl-induced modifications on phosphatase and tensin homolog (PTEN) structure and function. Verrastro, I., Tveen Jensen, K., Spickett, C. M. & Pitt, A. R., 2018, Free Radical Research, 1-471.

BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing: Understanding the rules of life: Structural Biology

Techniques that will be undertaken during the project:

  • The main biochemical techniques will be: cell culture, enzymatic assays, protein modification and enrichment, molecular biology (mutagenesis and generation of fusion proteins).
  • The main analytical techniques will be: liquid chromatography and mass spectrometry (LC-MSMS), PAGE and western blotting, uv-vis assays, protein purification.
  • The main computational techniques will be: quantitative data analysis, protein identification (proteomics), pathway mapping.

Contact: Professor Andrew Pitt, Aston University