Primary Supervisor: Professor Corinne M. Spickett, Life & Health Sciences
Secondary supervisors: Dr Alex Cheong
PhD project title:Regulation of protein-protein interactions by cellular redox state
University of Registration: Aston University
Fundamental cell processes including signaling and the function of the cytoskeleton depend on protein-protein interactions (PPIs). Understanding the processes that alter these interactions is a key challenge in biochemistry, and it is becoming clear that the redox status of the cell can affect specific PPIs through oxidation of redox sensitive cysteines. This is particularly important in inflammatory conditions, where increased production of reactive oxygen species occurs, and in ageing as the cellular antioxidant response pathways are impaired.
Most studies have investigated specific, pairwise interactions under simple oxidative conditions induced by hydrogen peroxide treatment, which mainly oxidizes low pKa cysteine thiols. Very few studies have adopted a broader “interactomics” approach to investigating redox effect. Recently, we developed a new approach, based on proteomics and chemical biology, to demonstrate that the interactome of the dual specificity phosphatase and metabolic regulator PTEN is altered by the oxidation of its catalytic cysteine residue to form an intramolecular disulfide. We identified 86 interacting proteins, the binding of fourteen of which was shown to be affected by the thiol/disulfide status of PTEN , including proteins important in cytoskeletal rearrangement and DNA damage repair.
The redox balance in cells is also more complex than simply cysteine thiol-disulfide interconversion. Oxidative stress leads to oxidation of other residues including methionine, histidine and lysine, while tyrosine and tryptophan can be modified by nitroxidative stress, and lipids can be oxidized to generate reactive electrophilic species that also modify the structure and function of proteins . The aim of this project is to expand our novel interactomics approach to study the effects of redox-dependent post-translational modifications on cellular processes that underlie inflammation, ageing and diseases such as cancer and diabetes.
Objectives and methodology
Objective 1. To determine the effect of reactive nitrogen species and lipoxidation by short chain lipid breakdown products on PPIs in vitro.
Initially, PTEN will be used as a well-validated model. His-tagged protein will be chemically modified, immobilized on a resin and interacting proteins captured from mammalian cell lysates and characterized using proteomics techniques relying on liquid chromatography - mass spectrometry. Following this, other redox-sensitive enzymes of key cellular importance will be investigated: pyruvate kinase M2, which is known to be important in cancer and is inactivated by oxidation and lipoxidation, and the thiol-disulfide regulated central signaling phosphatase PTP1B.
Objective 2: Validation of the redox-dependent interactions in vivo. The most significant changes identified in Objective 1 will be validated in cultured cells. The human breast cancer cell line MCF7 will be grown under normal or redox stress conditions, the cells lysed, and PPIs confirmed by immunoprecipitation and western blotting. Site-directed mutagenesis followed by functional tests will be used to confirm the key residues involved in the redox-interactions in cells.
Objective 3: Modelling of redox-dependent interactions. Information from objectives 1 and 2 will used to model the redox-dependent networks of the proteins using pathway mapping, e.g. Panther.
- Approaches to Investigating the Protein Interactome of PTEN. Smith SL, Pitt AR, Spickett CM. J Proteome Res. 2021 Jan 1;20(1):60-77.
- Modification of proteins by reactive lipid oxidation products and biochemical effects of lipoxidation. Spickett CM, Pitt AR. Essays Biochem. 2020 Feb 17;64(1):19-31.
- 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 Nov 20;144:223-233.
BBSRC Strategic Research Priority: Understanding the rules of life: Structural Biology and Systems Biology
Techniques that will be undertaken during the project:
- The main biochemical techniques will be cell culture, enzymatic assays, protein modification, enrichment and characterization, and 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 Corinne Spickett, Aston University