The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activitybased protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.

14 The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal 15 proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle.16 The proteasome is essential for parasite proliferation, yet little is known about the requirement 17 for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based 18 protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases 19 (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic 20 promastigote to amastigote. However, when Bar-seq was applied to a pool of 16 DUB null 21 mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was 22 observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for 23 successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 10, 24 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are 25 essential for promastigote viability and the essential role of DUB2 in establishing infection was 26 demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the 2 27 nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, 28 mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-29 ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs 30 play in differentiation and intracellular survival of Leishmania and that amastigotes are 31 exquisitely sensitive to disruption of ubiquitination homeostasis. 33Author Summary 34 Leishmania parasites require a variety of protein degradation pathways to enable the parasite 35 to transition through the various life cycle stages that occur in its insect and mammalian hosts.36 Several enzymes involved in protein degradation in Leishmania are known to be essential, 37 including a multi-protein complex, the proteasome, but little is known about how proteins are 38 targeted to the proteasome for degradation. Here, we analyse components of the 39 deubiqutination pathway, including twenty cysteine peptidases (DUBs) that remove the 40 posttranslational modifier ubiquitin from substrates tagged for proteasomal degradation. We 41 used chemical probes to measure active enzymes in parasite lysates and genome engineering 42 to create DUB gene deletion mutants. We identified some DUBs that are essential for parasite 43 viability and some that are required for life cycle progression. We carried out a detailed 44 analysis of the essential DUB2, which has broad deubiquitinase activity and is found in the 45 nucleus. This enzyme interacts with nuclear proteins associated with transcription/chromatin 46 dynamics, mRNA splicing and mRNA capping. This work demonstrates the important role that 47 DUBs play in Leishmania in vivo infection and further validates DUBs as potential drug targets 48 in this parasite. 49 50 3 51 Introduction 52 Leishma...

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AbstractPost-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved interface, highlighting the importance of stable UBC2-UEV1 interaction in the function of this complex across diverse eukaryotes. Furthermore, recombinant L. mexicana E1 UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can also cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.Author summaryThe post-translational modification of proteins is key for allowing Leishmania parasites to transition between the different life cycle stages that exist in its insect vector and mammalian host. In particular, components of the ubiquitin system are important for the transformation of Leishmania from its insect (promastigote) to mammalian (amastigote) stage and normal infection in mice. However, little is known about the role of the enzymes that generate ubiquitin modifications in Leishmania. Here we characterise 28 enzymes of the ubiquitination pathway and show that many are required for life cycle progression or mouse infection by this parasite. Two proteins, UBC2 and UEV1, were selected for further study based on their importance in the promastigote to amastigote transition. We demonstrate that UBC2 and UEV1 form a heterodimer capable of carrying out ubiquitination and that the structural basis for this activity is conserved between Leishmania, Saccharomyces cerevisiae and humans. We also show that the interaction of UBC2 with UEV1 alters the nature of the ubiquitination activity performed by UBC2. Overall, we demonstrate the important role that ubiquitination enzymes play in the life cycle and infection process of Leishmania and explore the biochemistry underlying UBC2 and UEV1 function.