Thesis defense Bianca van Tol

The research described in the 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. Conjugation and removal of ubiquitin can enhance or suppress these signals and their consequences. 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, including cancer and neurological disorders. 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. The enzymatic activity and selectivity read-out is done qualitatively by SDS-PAGE analysis, or quantitively by fluorescence intensity measurements and mass spectrometry.