Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Burge, R. J., Damianou, A., Wilkinson, A. J., Rodenko, B. and Mottram, J. C. (2020) Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex. PLoS Pathogens, 16(10), e1008784. (doi: 10.1371/journal.ppat.1008784) (PMID:33108402) (PMCID:PMC7647121)

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Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Rodenko, Dr Boris and Damianou, Andreas and Mottram, Professor Jeremy
Creator Roles:
Damianou, A.Investigation, Writing – review and editing
Rodenko, B.Conceptualization, Supervision, Writing – review and editing
Mottram, J. C.Conceptualization, Funding acquisition, Supervision, Writing – review and editing
Authors: Burge, R. J., Damianou, A., Wilkinson, A. J., Rodenko, B., and Mottram, J. C.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:PLoS Pathogens
Publisher:Public Library of Science
ISSN:1553-7366
ISSN (Online):1553-7374
Published Online:27 October 2020
Copyright Holders:Copyright © 2020 Burge et al.
First Published:First published in PLoS Pathogens 16(10): e1008784
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
168381Proteolysis and life cycle progression in LeishmaniaJeremy MottramMedical Research Council (MRC)MR/K019384/1III - Parasitology