Final Report Summary - UBICODE (Decoding the ubiquitin code) Cellular homeostasis necessitates spatio-temporal regulation of many cellular processes. In order to achieve this, post translational modifications (e.g. phosphorylation, lipidation, ubiquitination, etc.) are installed determining the cellular function, localization and fate of a protein at a given time at a given place in the cell. One such post translational modification is Ubiquitin, that not only marks proteins for proteasomal degradation but that also regulates many other cellular processes Unlike phosphorylation, ubiquitination is a highly complex posttranslational modification as its conjugation and deconjugation are regulated by dedicated enzymes. Moreover, ubiquitin can modify itself resulting in eight distinct Ub-chain linkages specifically modulating a cellular processes. Together with the interaction of ubiquitin and poly-ubiquitin to Ub-binding domains, biological processes are intricately controlled.One of the outstanding challenges in the ubiquitination field is the access to specific reagents required to study ubiquitin-related processes. Therefore we developed chemical tools to access the Ub-linkage specificity of deubiquitinating enzymes as well as methods to study the recognition of ubiquitin and ubiquitin chains by ubiquitin-binding domains. In the past, Ub-chains have been generated enzymatically, which requires the appropriate enzyme combinations. Although a variety of Ub-chains have been made biochemically, these laborious approaches often suffer from low yields. One major advantage of chemical synthesis is that any desired modification, e.g. mutations, fluorescent tags, affinity handles, reactive groups, crosslinkers etc. can be incorporated. We developed chemical methodologies to synthesize an array of tools based on different linkages types in high yields. This ERC grant has supported the development of chemical methods to construct Ubiquitin linkages and various reagents based on such linkages that have been used to study the Ub-linkage specificity of both Ub-binding domains deubiquitinating enzymes (DUBs). For example, the deubuiquitinase Cezanne/OTU7B has been shown to specifically recoginze and process K11-diUB chains using aforementioned tools. Another example illustrating how the chemical di-Ub reagents have furthered our understanding of ubiquitin biology is a study uncovering the Ub-linkage preference of two proteases expressed by human corona viruses (SARS and MERS), providing a starting point for developing therapeutic molecules. These findings exemplify the potential of these novel reagents in different contexts. These reagents have significantly contributed to our understanding of ubiquitin-biology and helped to elucidate the structure and biological function of deubiquitinating enzymes. Given the complexity of ubiquitin biology, these reagents, will undoubtedly uncover new aspects that may propel the development of therapeutic molecules. In addition to obtaining fundamental knowledge about Ub biology, this project has added value to peptide chemistry by introducing novel chemical technologies for broad use.
