RT Journal Article SR Electronic T1 Characterisation of the OTU domain deubiquitinase complement of Toxoplasma gondii JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202201710 DO 10.26508/lsa.202201710 VO 6 IS 6 A1 Mary-Louise Wilde A1 Ushma Ruparel A1 Theresa Klemm A1 V Vern Lee A1 Dale J Calleja A1 David Komander A1 Christopher J Tonkin YR 2023 UL https://www.life-science-alliance.org/content/6/6/e202201710.abstract AB The phylum Apicomplexa contains several parasitic species of medical and agricultural importance. The ubiquitination machinery remains, for the most part, uncharacterised in apicomplexan parasites, despite the important roles that it plays in eukaryotic biology. Bioinformatic analysis of the ubiquitination machinery in apicomplexan parasites revealed an expanded ovarian tumour domain–containing (OTU) deubiquitinase (DUB) family in Toxoplasma, potentially reflecting functional importance in apicomplexan parasites. This study presents comprehensive characterisation of Toxoplasma OTU DUBs. AlphaFold-guided structural analysis not only confirmed functional orthologues found across eukaryotes, but also identified apicomplexan-specific enzymes, subsequently enabling discovery of a cryptic OTU DUB in Plasmodium species. Comprehensive biochemical characterisation of 11 Toxoplasma OTU DUBs revealed activity against ubiquitin- and NEDD8-based substrates and revealed ubiquitin linkage preferences for Lys6-, Lys11-, Lys48-, and Lys63-linked chain types. We show that accessory domains in Toxoplasma OTU DUBs impose linkage preferences, and in case of apicomplexan-specific TgOTU9, we discover a cryptic ubiquitin-binding domain that is essential for TgOTU9 activity. Using the auxin-inducible degron (AID) to generate knockdown parasite lines, TgOTUD6B was found to be important for Toxoplasma growth.