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 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.
Co-promotor: Prof.dr. P.ten Dijke