Developing mass spectrometry-based methods for protein-protein interaction analysis across the human cell division cycle

The temporal regulation of protein-protein interactions is a key feature of the cell division cycle. For example, progression from a quiescent (G0) to a proliferation-committed (G1) state is controlled by the interactions of a key protein called retinoblastoma (Rb). In quiescent cells, hypophosphorylated Rb interacts with and inhibits E2F transcription factors, which control the expression of cell cycle genes (e.g., cyclin E). Upon mitogenic stimulus, phosphorylation of Rb disrupts the interactions between Rb and E2F, thereby relieving E2F inhibition and promoting cell cycle gene expression. In human the regulation is more complex in that there are several E2F and Rb isoforms that are encoded by separate genes. Recruitment to a genomic locus can be either activating or repressing depending on the isoform. The roles of E2F and Rb has been studied extensively in the context of their dysregulation in diseases such as cancer. Less is known about their roles in specific physiological contexts such as T cell activation, where cells undergo a major change in their proliferative capacity and size.

Advances in mass spectrometry-based proteomics enables comprehensive, quantitative analysis of protein abundance and protein modification by PTMs like phosphorylation. Analysis of protein-protein interactions in a comprehensive fashion remains challenging. Progress has been made using protein correlation profiling (PCP), which combines separations of native protein complexes with MS detection. However, a drawback to PCP is that direct evidence for protein-protein interaction is lacking. More recently, researchers have demonstrated that cleavable crosslinkers, such as DSSO, enable the analysis of thousands of crosslinked peptides from a complex cellular extract.

The PhD project will examine the role of protein-protein interactions in the regulation of the G0 to G1 transition. The project will involve ‘broad’ and ‘deep’ approaches. The student will apply PCP/chemical crosslinking-based approaches to obtain an unbiased, comprehensive analysis of protein-protein interactions in CD8+ T lymphocytes. In parallel, the student will analyse the post-translational modification and interaction partners of Rb and how they differ upon T cell antigen stimulation. Candidate genes from these datasets will be identified and the functional roles investigated using genome editing. The student will learn bioinformatic tools to interrogate the datasets and perform meta-analyses with publicly available resources, such as gene ontology and other databases.

Training
The PhD research project will provide expert training opportunities in human cell culture, cell line engineering, flow cytometry, protein biochemistry, native mass spectrometry, mass spectrometry-based proteomics, chemical crosslinking, and the handling and bioinformatic analysis of large-scale proteomic data.

Ly lab website: https://dynamic-proteomes.squarespace.com
Clarke lab website: https://www.clarkelab.co.uk