Cells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell... Show moreCells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell nucleus and can be transcribed and translated into proteins, which play vital roles in numerous cellular processes. The cell relies on modifying existing proteins to carry out essential functions. These modifications can involve the conjugation of small molecules such as Ubiquitin (Ub) or Small Ubiquitin-like Modifiers (SUMOs), leading to protein degradation, conformational changes or intracellular relocation of critical proteins. The conjugation of these small molecules involves a well-orchestrated sequence of enzymatic activities performed by dedicated enzymes: E1 (activating), E2 (conjugating) and E3 (ligase). Among these, the E3 ligase enzymes hold significant importance as they confer substrate specificity.In this thesis, we have developed an advanced Mass-Spectrometry technology called TULIP2 (Targets for Ubiquitin Ligases Identified by Proteomics 2), which facilitates the identification of Ubiquitination targets for specific E3 ligases of interest. Using this technology, we have investigated the BRCA1-BARD1 E3 ligase and explore the in vivo role of the E2 UBE2D3. Furthermore, we have adapted the TULIP2 technology to create the SUMO Activated Target Traps (SATTs), enabling the identification of an E3-specific SUMO proteome. Show less
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
This work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full... Show moreThis work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full-length proteins that are able to recognize them in a linkage-specific manner. Chapter 2 describes the development of a high-throughput (HT) fluorescence polarization assay used to discover alpha-helical ubiquitin binding domains. This leads to validation of known interacting binding domains and the discovery of a novel set of K6-linkage specific UBDs. In Chapter 3, all eight diubiquitin proteins are prepared using click chemistry that renders the diubiquitin proteins immune to proteolysis by deubiquitinases (DUBs). This allowed for pulldown efforts in different cell lysates and identification of novel linkage-specific interacting proteins using tandem mass spectrometry. The procedure of this effort is outlined in Chapter 4. The study in Chapter 5 details the interaction of one of the major hits found in Chapter 3: the linkage-specific interaction of K27 diubiquitin and UCHL3. The development of a panel of specific assay reagents combined with X-ray crystallography and kinetic modelling lead to a proposed model in which the consequences of the interaction between K27Ub2 and UCHL3 are explained. In Chapter 6 future directions are offered. Show less
In this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO... Show moreIn this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO modifications with examples of substrates where the precise site of modification is important, and substrates where the modification site seems interchangeable.We show that the deubiquitinating enzymes regulate a separate subset of the ubiquitinome than the proteasome. The PARylating enzyme PARP1 show increased activity after ubiquitination, which is regulated by deubiquitinating enzymes.Furthermore, we developed an in-vivo tool to study SUMO dependent interaction using proximity labeling which can be used for microscopy or identification by mass spectrometry.Finally, we investigate binders of Ubiquitin and SUMO chains with specific linkages and elucidate a preference for some chain linkages based on biological pathway of the binder. We also explore possible binding domains and predict the structure of some chain binding complexes using protein docking. Show less
