“The development of PROTACs (proteolysis-targeting chimeras) have heralded a new era of therapeutics. These drugs work by co-opting existing cellular machinery to selectively target proteins for degradation, making many proteins that have long been viewed as undruggable now realistic therapeutic candidates (e.g. bromodomain containing proteins in cancer). PROTAC design requires a structural understanding of ligand binding to the protein target of interest and developing a linker to recruit an E3 ligase (e.g. VHL or CRBN) to facilitate rapid ubiquitination and proteasome-mediated degradation of the target. However, factors that determine how rapidly a ubiquitinated PROTAC target is cleared is not known. This is important, as not all proteins that are ubiquitinated are degraded rapidly, and whether this depends on the E3 ligase or other factors is not clear. This project will use a functional genomics approach to determine the key factors that facilitate degradation of existing PROTACs and uncover which other cellular components are important for the efficient depletion of these drug targets. Specifically, the project will involve our expertise in genome-wide CRISPR/Cas9 mutagenesis to screen for genes that control the degradation of the dTAG-chimera with VHL or CRBN ligands, as well as PROTACs systems targeting bromodomain proteins (e.g MZ1). Secondary validation screens with sub-pooled libraries will determine the key genes involved, and the latter part of the project will explore how these genes promote efficient protein degradation.

The student will gain a thorough training in functional genomics, cell biology and biochemistry, with direct relevance to drug development. They will also benefit from dedicated bioinformatics training in our group. Relevant literature regarding functional genomics can be found in our prior publications (e.g. Ortmann et al, Nature Genetics 2021, Bailey et at, Nature Communications 2020, and Burr et al, Cell Metabolism, 2016).”

“We exploit a range of high-throughput genetic screening techniques to uncover novel pathways regulated by the ubiquitin-proteasome system. Using a combination of microarray-based oligonucleotide synthesis, lentiviral expression screens, CRISPR/Cas9-mediated mutagenesis and next-generation sequencing, we aim to (1) identify substrates of E3 ubiquitin ligases, (2) characterise the molecular features (‘degrons’) that enable selective substrate recognition, and (3) explore how these processes are corrupted in the context of disease.

Angelman Syndrome is a devastating neurological condition caused by loss of the E3 ubiquitin ligase UBE3A. However, our understanding of how UBE3A loss affects neuronal function remains very limited, chiefly because its relevant substrates remain ill-defined. Determining the substrate repertoire of UBE3A is therefore critical both to understanding the pathophysiology of the disease and, crucially, to delineate the optimal targets downstream of UBE3A for therapeutic intervention.

The goal of this project is to overcome this bottleneck by exploiting an unbiased, global screening approach to identify and characterise UBE3A substrates. You will use a lentiviral expression library encoding ~15,000 barcoded human proteins (‘the human ORFeome’) fused to GFP to perform proteome-wide stability profiling by FACS and next-generation sequencing. By comparing wild-type cells versus cells lacking or overexpressing UBE3A, these screens will identify candidate UBE3A substrates. Biochemical characterisation of candidate substrates will then be performed in neuronal cell culture systems to explore their functional roles. Ultimately, our aim will be to identify substrates whose forced overexpression recapitulate the phenotypes observed in the absence of functional UBE3A; pharmacological inhibition of these substrates would then be a candidate therapeutic strategy for the treatment of Angelman Syndrome.”