The origin of deubiquitinases – An evolutionary approach towards identification and understanding of novel DUBs and DUB-like enzymes.

• Applicant: Professor Dr. Kay Hofmann
• Subject Area: Biochemistry ; Bioinformatics and Theoretical Biology ; Structural Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 449991788

The covalent attachment of one or more ubiquitin units to proteins, known as ubiquitination, is a posttranslational modification that – besides its importance for proteostasis – also regulates a wide range of other cellular processes, including the response to intracellular pathogens. Important regulators of ubiquitination are the so-called deubiquitinases (DUBs), which remove ubiquitin from substrates or chains. Bacteria, which do not have a ubiquitin system of their own, often encode DUBs that are thought to interfere with the host defense pathways. Our preliminary work has shown that most of the eukaryotic deubiquitinase classes are related to each other and probably evolved from a ‘proto-DUB’. It appears that most – if not all – bacterial deubiquitinases have the same evolutionary origin and have probably been co-opted from host-encoded DUBs, although in most cases rapid evolution and structural rearrangements are obscuring this relationship Our project aims at understanding of what properties make a protease a DUB, and which factors determine deubiquitination specificity. A deeper understanding of these factors will enable us to identify DUBs, and possibly proteases for ubiquitin-related modifiers, from their sequences. A useful by-product will be the ability to identify DUBs in the genomes of bacteria and other pathogens that have to counteract ubiquitin-based defense mechanisms, but for which no deubiquitinases could be identified so far. Ubiquitination targets intracellular bacteria for degradation by the autophagy pathway, which is a major obstacle to intracellular survival of the pathogens. In preliminary work, we have developed a bioinformatical strategy for DUB discovery, which will allow us to identify structurally rearranged deubiquitinase families and even those with an altered active site topology. We are going to use these methods to identify DUBs in bacterial genomes of medical and/or agronomical importance, including grampositive bacteria, for which no such activity has been identified. We will also investigate two new DUB-like activities, which we have discovered recently: bacterial ‘ubiquitin C-terminal clippases’ and ‘trans-ubiquitinases’. The former class of enzymes removes ubiquitin like similar to a conventional DUB, but cleaves ubiquitin before the C-terminal GlyGly motif rather than after this motif. As a consequence, this type of deubiquitination is irreversible and over time will destroy the host ubiquitin system. At first sight, these clippases appear to be ideal for bacteria to disarm ubiqutin-based host defenses. Nevertheless, only few bacteria appear to have evolved this pathway, and we will try to find out why. The ‘trans-ubiquitinases’ do not simply cleave ubiquitin chains to its monomers, but will directly conjugate the freed ubiquitin onto another target. We predict that enzymes with this kind of activity are found in several pathogens and will investigate their roles.

• DFG Programme: Research Grants