Molecular mechanisms of the ubiquitin system

How does ubiquitin achieve specificity in regulating virtually all aspects of eukaryotic homeostasis? A key point is the
action of E3 ubiquitin ligases (E3Ls), the most diversified class of enzymes in the ubiquitin system. The pharmaceutical
industry is actively developing new drugs including PROTAC to recruit E3Ls for the on-demand proteasomal degradation of
disease-causing proteins. At least 600 E3Ls are encoded in the human genome, yet only a handful have been exploited so
far. To exploit these enzymes therapeutically, it is essential to obtain structural and mechanistic insights of how E3Ls work
and are regulated. Here, we will use protein biochemistry, cell biology and cryo-EM to study (i) the structure of E3 ligases
UPF1 and HECTD1; (ii) how their activity/specificity is regulated, and the cross-talk of the ubiquitin system with other
post-translational modifications, e.g. phosphorylation; (iii) the potential for these E3Ls as new modalities for
targeted protein degradation.
The nonsense-mediated mRNA decay (NMD) factor UPF1 comprises a CH-domain with two Ring-modules, characteristic
of Ring domain E3 ubiquitin ligases but UPF1's E3L activity has to be demonstrated yet. Human UPF1 is involved in
downregulation of MYOD, a key regulator of myogenesis, and we now will investigate whether UPF1 is the E3L regulating
MYOD levels. We further aim to identify and validate potential other substrates of the UPF1 E3L using cell biology and
proteomics. We are particularly interested to see if there is a link between UPF1 E3L activity and associated protein
degradation or ubiquitin signalling and the NMD pathway that degrades faulty mRNAs.
The HECT E3 ubiquitin ligase HECTD1 has multiple roles including in cell migration, Wnt signalling and cell division as we
have shown recently. Currently, HECTD1 is the only E3L which can assemble, on its own, branched polyubiquitin chains
which is a potent degradative signal. To understand how these complex ubiquitin signals are made and how
HECTD1 ligase activity is regulated, we will characterise HECTD1 biochemically, biophysically and structurally
using cryo-EM and/or crystallography.
The student will be trained in eukaryotic protein expression and purification, ubiquitination assays, biophysical
techniques, protein crystallography and state-of-the-art cryo-EM. An important goal will be to establish the expression
and purification of full-length E3 enzymes for activity assays, biophysical and structural characterisation. The student will
benefit from ongoing international collaborations, supportive dynamic research environments and our combined
expertise in the Bristol and Bath teams.