The ubiquitination enzymes of Leishmania mexicana

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. Although 20 deubiquitinating enzymes (DUBs) have been partially characterised in Leishmania mexicana, little is known about the role of E1 ubiquitin-activating (E1), E2 ubiquitin-conjugating (E2) and E3 ubiquitin ligase (E3) enzymes in this parasite. Using bioinformatic methods, 2 E1, 13 E2 and 79 E3 genes were identified in the L. mexicana genome. Subsequently, bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed that the E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for successful promastigote to amastigote differentiation 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, revealed a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a was found to load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. UBC2 was also shown to cooperate with human E3s RNF8 and BIRC2 in vitro to form non-K63-linked polyubiquitin chains, but association of UBC2 with UEV1 inhibits this ability. Using affinity purification proteomics, 3 putative UBC2:E3 pairs and potential substrates of UBC2 and UEV1, which include proteins associated with cellular respiration, intracellular transport and pH regulation, were identified. Therefore, the essential requirement for UBC2 and UEV1 in promastigote to amastigote differentiation could be explained by roles for UBC2-UEV1-mediated ubiquitination in regulating the changes in metabolism and protein trafficking that occur during this transition.