Protein ubiquitination emerges as a key regulatory posttranslational modification, which is utilised both in degradative processes regulating the half-life of target proteins, as well as in signal transduction where it serves non-degradative functions. During ubiquitination, the small protein ubiquitin is attached covalently to target proteins. Ubiquitin can be ubiquitinated itself, generating ubiquitin polymers. The versatility of the ubiquitin system is achieved by the ability of ubiquitin to form at least eight types of polymers, and proteomic analysis has shown that all different ubiquitin linkages are present in cells. To date, functional and structural data is available for only three types of polyubiquitin chains. These are the well-studied Lys48-linked polyubiquitin chain, that targets modified proteins for proteasomal degradation, as well as Lys63-linked polyubiquitin that are implicated in cell signalling processes. Independent functions for the remaining chain types are emerging. We aim to generate and study the remaining ubiquitin chain types, and understand their roles in the cell. For this, we need to study ubiquitin chain assembly systems. This work has led us to determine the structure of the E3 ubiquitin ligase Parkin, explaining many mutations of individuals with early-onset Parkinson’s disease, shedding new light on the role of ubiquitin in neurodegenerative disorders. Moreover, we have identified E2 and E3 enzymes to generate Lys6- and Lys11-linked polyubiquitin chains. Availability of novel chain linkages is important to understand the molecular basis for specificity in the ubiquitin system, especially specificity of deubiquitinases and ubiquitin binding domains. Deubiquitinating enzymes hydrolyse ubiquitin chains, and are therefore key regulators of the ubiquitin system. Currently, limited information is available as how this important class of enzymes is regulated. We perform structural and functional studies on deubiquitinases of the Ovarian Tumour family, to understand how they affect cell signalling pathways at the molecular level.