Project Outline

Redox balance is a central aspect of cellular function that regulates cell behaviour across many organisms from microbes to mammals. Cells maintain a balance between oxidizing species (such as hydrogen peroxide or superoxide) and antioxidants; changing the balance in either direction affects macromolecular interactions, gene expression, cell differentiation, proliferation or death. Inflammation and mitochondrial dysfunction can lead to oxidative stress through the release of reactive oxygen species. These responses are important in ageing and metabolic dysfunctions, as well as diseases. Many proteins are “redox-sensitive”: i.e. their functions and activity are affected by oxidative modification of the protein.

Phosphatase and tensin homologue (PTEN) is a tumour suppressor and dual specificity phopsphatase. Through its phospholipid phosphatase activity it antagonizes the PI3K/Akt pathway, altering metabolic balance, limiting cell proliferation, and preventing the blocking of apoptosis. As a protein phosphatase, it dephosphorylates proteins involved in cell motility and migration, also counteracting oncogenic changes. PTEN is known to be redox sensitive, and my group demonstrated that oxidation and modification by lipid oxidation products alters both its activity and its protein interactome. We have also been investigating redox-induced changes in subcellular localization, which are likely to control cell behaviour. However, questions remain over the types of modifications that can cause these changes, the mechanisms involved, and their downstream effects on cell behaviour.

The overall aim of the project is to understand the effects of redox-dependent post-translational modifications on PTEN cell signalling processes that underlie ageing, inflammation. Building on our previous research, we will use a broad, in vitro interactomics approach to determine the effect of oxidative and nitrosative stresses on PTEN and analyse its protein-protein interactions. The interactions will be validated in cellulo along with functional tests to confirm their effects on cell differentiation and proliferation. This work will be carried out in cells expressing native PTEN and PTEN containing mutations in the active site and resolving cysteines, to test the effect of blocking the redox response. The redox-dependent networks will be modelled for different cellular conditions with pathway mapping. In parallel, high resolution microscopy will be used to monitor the effects on subcellular localization and correlate with the interactome and cell function data. This will generate novel information on how the dual activities of PTEN function together in physiological and pathophysiological situations, as well as building a paradigm for integrated redox regulation at the whole-cell level.

References

Approaches to Investigating the Protein Interactome of PTEN. Smith SL, Pitt AR, Spickett CM. J Proteome Res. 2021; 20(1):60-77.

Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells. Sousa BC, Ahmed T, Dann WL, Ashman J, Guy A, Durand T, Pitt AR, Spickett CM. Free Radic Biol Med. 2019 144:223-233.

Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins. Verrastro I, Tveen-Jensen K, Woscholski R, Spickett CM, Pitt AR. Free Radic Biol Med. (2016) Jan 90: 24-34

Techniques

• Mammalian cell culture and bacterial culture.
• Biochemical techniques: cell culture, enzymatic assays, protein modification, enrichment and characterization, and molecular biology (mutagenesis and generation of fusion proteins).
• Analytical techniques: liquid chromatography and mass spectrometry (LC-MSMS), PAGE and western blotting.
• Computational techniques: quantitative data analysis, protein identification (proteomics, Mascot), lipid and oxidized lipid identification (Progenesis and other software), pathway mapping