Authors
A Damianou1; P H Celie3; R Burchmore2; B Rodenko4; J C Mottram1; 1 Department of Biology, University of York; 2 Institute of Infection and Inflammation, University of Glasgow; 3 Netherlands Cancer Institute; 4 UbiQ Bio BV, Amsterdam Discussion
DUBs are a class of peptidases whose function is to cleave the post-translational modifier ubiquitin from proteins or ubiquitin-conjugates. DUBs play crucial roles in many biological processes such as protein degradation and gene regulation. Leishmania also has a ubiquitin system and its genome suggest the presence of 20 DUB orthologues. Still, the identity, function and essentiality of DUBs in Leishmania remain to be revealed. In this study, a chemical proteomics approach using a fluorescent ubiquitin-based probe was used for activity-based protein profiling, revealing the presence of many active DUBs in L.mexicana. A number of stage-specific DUBs have been identified, including some that have amastigote-specific activity. The DiCRe inducible gene knockout system is being used to evaluate L.mexicana DUB1, with preliminary data suggesting that DUB1 is essential. Furthermore, active recombinant DUB1 protein has been expressed and purified using a baculovirus expression system and an HTS-compatible fluorescence polarisation assay developed based on the proteolysis of tetramethylrhodamine-labelled Lys(Ub)Gly. Our approach combines chemical and genetic screening to identify essential Leishmania DUBs as a starting point for drug discovery activities. 
