Biochemical and structural studies of E3 ubiquitin ligase DTX3L – a protein at a crossroads of ubiquitination and ADP-ribosylation

Proteins form support structures for cells, they respond to stimuli to generate signals or catalyse biochemical reactions. Proteins even regulate their levels by modulating gene expression or through the ubiquitin-proteasome system. The presence of a chain of ubiquitin molecules in proteins directed to proteasomal degradation was the spark that initiated research on ubiquitination. Ubiquitination is a post-translational modification that results in a covalent bond between a protein and ubiquitin.
Ubiquitination is best known for the proteasome pathway, but it also has non-degradative outcomes. Protein ubiquitination is the result of an enzymatic cascade where an E1, E2 and E3 enzyme acts in concert. As E3 enzymes confer specificity to the reaction, most ubiquitination research focuses on the mechanism by which these enzymes act. Furthermore, the dysregulation of E3 ligases has been linked to cancer, Parkinson’s and Alzheimer’s diseases. As a result, there are ongoing efforts to identify inhibitors of these enzymes.
The work developed as part of this thesis focused on DTX3L, a RING-type ubiquitin E3 ligase. The roles of DTX3L in cells are still undefined, but research links it to immune response, DNA damage repair and androgen receptor signalling. Specifically for DNA repair and androgen receptor, the process is dependent on ADP-ribose binding, possible thanks to its interacting partner, PARP9. PARP9 has two macrodomains, which act as recognition motifs of ADP-ribosylation. Moreover, the complex also integrates ADP-ribosylation by possibly acting as a transferase for ubiquitin.
Our studies led to the determination of the binding affinity between DTX3L and PARP9 and the regions responsible for the interaction. We proposed that PARP9 modulates DTX3L activities. We determined the crystal structure of D2, the principal mediator of DTX3L oligomerisation, which revealed a tetrameric assembly. We also assessed the evolutionary conservation of D2 and the relevance of DTX3L oligomers. Our results indicate that the tetramer is a biologically functional unit required for the recognition of ADP-ribosylated androgen receptor. Finally, considering the correlation between DTX3L and cancer, we developed a homogeneous FRET-based assay that helps to study the activity of the ligase in real-time. The assay, compatible with high-throughput screening, facilitated the discovery of the first inhibitors of the ubiquitination activity of DTX3L.