A Multimodal Mass Spectrometry Platform to Investigate the Structural and Functional Mechanisms of Cysteine Redox Modifications

Lead Supervisor Dr David Clarke, University of Edinburgh, School of Chemistry
Co-Supervisor Prof Ted Hupp, Institution and School: Edinburgh Cancer Research, University of Edinburgh

Project Outline: The intracellular redox environment is a highly compartmentalised and regulated state function which varies considerably in differing subcellular location and during cell cycle, cell differentiation, and cell death. Dysregulation of cellular redox potential, particularly to pro-oxidative states, is implicated in the initiation and proliferation of several disease states (particularly in age-related diseases such as cardiovascular disease, neurodegenerative disease and cancer). On a molecular level, flux in cellular redox potential can directly influence protein structure by altering the oxidation state of redox-sensitive cysteine (Cys) residues. There is an emerging realisation that in many proteins, these reversible redox modifications (RMs) act as molecular switches to regulate function or activity. Therefore, investigating the molecular details and the structural/functional consequences of RMs in susceptible proteins is crucial for successfully understanding the role by which redox environment can regulate specific individual proteins and biochemical pathways.

This project will build upon recent work investigating oxidation pathways in p53 (Clarke and Hupp groups). The p53 transcription factor is a key regulator of cell cycle which safeguards cells from growing unchecked by mediating the cellular response to oncogenic environmental signals. Deactivation of p53 by mutation allows cells to evade apoptosis and tumours to progress; and p53 is one of the most frequently mutated genes in human cancers. Deactivating mutations usually result in a single amino acid substitution in the protein’s DNA binding domain. There is now growing evidence that p53 activity is influenced by its redox environment and that the Cys-rich p53 DNA binding-domain is susceptible to RM. We have recently used a MS-based approach to delineate the molecular mechanisms of two different oxidation pathways in wild-type p53 (1). In addition, recent reports have highlighted several electrophilic molecules which can reactivate oncogenic p53 mutants – restoring antitumour activity. We have demonstrated that the action of this class of electrophiles (all Michael acceptors, e.g. MIRA-1) is mediated by ligand binding to specific Cys in the core domain of p53 (2). We have also outlined new methodology, Mass Spectrometry Redox Profiling (MSRP), to quantitate specific RMs based on their redox midpoint potential (3).
Aim: During this project, the molecular details of redox-cysteine modifications in wild-type p53 will be delineated using top-down mass spectrometry and quantified using our MSRP methodology. In vitro assays will be performed in order to determine their functional role. The structural effects of specific modifications will be assessed using native MS and ion mobility MS. Following this, these same techniques will be applied to the analysis of chemical modifications of oncogenic mutant forms of p53.

Thus, this study will shed light of the chemical and structural details of cysteine redox regulation in p53 and the phenomenon of mutant p53 reactivation by Cys modification.

Training: You will learn skills in molecular biology, protein chemistry; and apply these techniques to the production and characterisation of recombinant proteins. This project will utilise a comprehensive range of state-of-the-art MS-based techniques for protein analysis. You will receive training and extensive hands on experience in - top-down fragmentation, native MS, and ion mobility MS.

Applicants should possess a recent first class or 2(i) in Chemistry, Biochemistry or closely related subject. The ideal candidate will have a strong interest in mass spectrometry, protein chemistry and structural biology; and excellent written and oral communication skills.

Previous experience in the field of mass spectrometry is highly desirable.