Understanding substrate recruitment by BTB-Kelch family E3 ligases

Thesis

Over 600 E3 ligases are identified that covalently modify substrates with ubiquitin to control their degradation, trafficking or activity. Many are linked to human diseases and represent an important class of drug targets. An understanding of their substrate recruitment mechanisms is necessary to elucidate their functions and to enable drug design. BTB-Kelch proteins are the substrate adaptors for a large family of Cullin3-dependent E3 ligases that remain incompletely characterised. Recurrent mutations in the Kelch domain of KBTBD4 act as an oncogenic driver of medulloblastoma. I identified specific substrates of KBTBD4 mutants using mass spectrometry-based proteomics. These data suggest a novel gain-of-function mechanism for these tumour mutations. For other targets with known substrates, I performed SPOTs peptide arrays and biophysical analyses to map their binding motifs (degrons) and was able to solve the crystal structures for the substrate complexes of the E3 ligases KLHL20, KLHL12 and KLHL3 with substrates DAPK1, DVL1 and WNK3, respectively. The mapped ‘LPDLV’ motif in DAPK1 and ‘PGGPP’ motif in DVL were observed to insert into the central pockets of the Kelch domains to form hydrophobic interactions, whereas the WNK3 interface was more polar with WNK3 Thr541 forming a potential phosphorylation site to regulate salt homeostasis.

I further used the DAPK1 degron peptide to develop an Alphascreen assay for compound screening and identified the first low micromolar inhibitors of KLHL20 with potential application in cancer, as well as Alzheimer’s disease. I also established a panel of 17 human Kelch proteins for future work leading to a new structure of KLHL17 as well as a selectivity panel that revealed the high selectivity of known inhibitors of KEAP1. These results confirm the diverse structural characteristics of the Kelch domains observed from my work. Overall, these features help to explain how E3 substrate adaptors can perform their diverse roles in protein regulation.

Authors: Chen, Z

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford