Ubiquitination is an important posttranslational modification that plays a role in virtually all cellular pathways. Ubiquitin signals are made out of one or multiple ubiquitin proteins that are targeted towards substrates by a cascade of E1, E2 and E3 enzymes, where the E3-ligase catalyzes the transfer of the ubiquitin from the E2 onto the target. The RING-in-between-RING (RING1-IBR-RING2 / RBR) subfamily of RING E3-ligases is a novel identified class of E3-ligases that are characterized by the presence of a RBR domain. The RBRs are involved in major pathways such as the nuclear factor of kappa-B (NF-kB) pathway and play a role in the development of diseases like Parkinson disease and cancer. Therefore, a detailed understanding of the molecular mechanisms underlying their functioning is critical to understand the implication of mutations and to facilitate the development of molecular medicine. In this thesis we analyze the molecular mechanisms by which the RBR E3-ligases Triad1 and HOIP mediate the transfer of ubiquitins from the E2s onto their targets. We show that the specificity of ubiquitin chain formation by Triad1 is dependent on the E2 in the reaction, while HOIP determines it chain formation specificity by a unique region in its C-terminus. Finally, we show that HOIP functions via the combined RING/HECT-type mechanism that was identified in two other RBR E3-ligases. Therefore, we conclude that the RING/HECT-type mechanism is likely to be general for all RBRs, while the target selection and ubiquitin chain-formation specificity are regulated in different ways for the separate RBRs.

E3-ligase, HOIP, LUBAC, RING, triad1, ubiquitin
T.K. Sixma (Titia)
Erasmus University Rotterdam , Antonie van Leeuwenhoek Ziekenhuis, Amsterdam
This thesis was printed with financial support from Erasmus University Rotterdam and The Netherlands Cancer Institute.
This work was funded by the European Commission 7th Framework Programme; grant id erc/281699 - Decoding the ubiquitin code (UBICODE), This work was funded by the European Commission 7th Framework Programme; grant id erc/249997 - The balance of ubiquitin conjugation and deconjugation (UBIQUITIN BALANCE)
Erasmus MC: University Medical Center Rotterdam

Smit, J.J. (2013, May 28). Ubiquitin chain formation by RBR E3-ligases. Antonie van Leeuwenhoek Ziekenhuis, Amsterdam. Retrieved from http://hdl.handle.net/1765/40175