Ubiquitination is best known as a signal for proteasome-mediated protein degradation. Recent studies have uncovered new functions of ubiquitin and ubiquitin-like proteins. These functions signaling roles in DNA repair, immune responses and regulation of membrane dynamics.
Our lab aims to elucidate the function of ubiquitination and deubiquitination in these pathways. We have an interest in conjugating enzymes (E1-E2-E3’s) and deubiquitinating enzymes (DUBs) as therapeutic targets in diseases such as cancer and neurodegenerative diseases. Approaches include finding inhibitors of specific DUBs using state of the art high-throughput screening. It is our ultimate goal to validate specific targets with such small-molecule inhibitors of specific DUBs in cellular and animal models.
The ubiquitin (de)conjugation machinery is fiercely investigated the past two decades and for instance the basic dogma underlining the joint activity of E1, E2 and E3 enzymes to ligate ubiquitin to a target protein is widely accepted. Many of the intricacies of these systems however remain open for discussion and preparing new tools using a chemical approach to study ligase and hydrolase systems are essential for a better understanding. An example of understudied enzymatic activities is the cross-talk between ubiquitination and ADP-ribosylation. Bacterial effector proteins, such as the class of Legionella SidE enzymes, catalyze the covalent ADPribosylation of ubiquitin on Arg42, eventually leading to the phosphoribosyl ubiquitination of serine containing target proteins. As the matter-of-fact the interplay between ADPribosylation and ubiquitination is more prevalent in nature, in processes such as bacterial infection but also during DNA-damage responses in mammals, and we focus on studying such systems using a chemical biology approach.
Ubiquitination most commonly leads to destruction of the modified protein, but can also change its activation, interactions or localization. These modifications are controlled by a complex enzymatic system, with more than 700 players in the ubiquitin network, and disruption within these systems could lead to diseases such as cancer and neurodegeneration. Detailed insights into the mechanism behind this is however yet to be obtained, thus slowing the development of targeted medicines for treatment of these diseases.
Much of our work aims to unravel the workings of these systems. Utilizing both activity-based probes as well as biochemistry and cell biology techniques, we are currently seeking to unravel the role of ubiquitination and the proteasome in Huntington’s disease, a dominantly inherited neurodegenerative disease. Moreover, we have a research interest in E3 ligases, the enzymes that attach ubiquitin to a substrate. Our research pursues the development of platform technologies to gain understanding on the biological and mechanistic information of E3 ligases, while capitalizing on insights to design strategies aimed at targeting specific E3 ligases. Together, this offers opportunities to exploit the ubiquitin system not only by small molecules but also by targeted protein degradation approaches as PROTACs. The success of the PROTAC strategy in targeting diverse pathological proteins relies, however, on the number and specificity of E3 ligases that can be recruited for this purpose. An exciting possibility to explore is the recruitment of E3 ligases with disease-specific expression or that are cell- or tissue- specific. This knowledge is exactly what is aimed for in my lab, along with the means (assays, reagents, probes) to reveal such knowledge.
Based on the total chemical synthesis of ubiquitin, we developed a fluorescence polarization assay to assess ubiquitinated peptide selectivity of DUBs which turned out to be crucial in the elucidation of Ub linkage specificities of OTU DUBs. This technique was further extended to Ub-like proteins to assess bacterial effector proteases and the ISG15-specific protease USP18. This work laid the basis for the development of full-length diUb FRET probes with which the Ub linkage specificity of DUBs can be quantified. Such different assaying techniques are now further used to develop small-molecule cell permeable DUB inhibitors and activity-based probes.