Essential roles for deubiquitination inLeishmanialife cycle progression
Authors: Damianou Andreas , Burge Rebecca , Catta-Preta Carolina M. C. , Geoghegan Vincent , Nievas Y. Romina , Newling Katherine , Brown Elaine , Burchmore Richard , Rodenko Boris , Mottram Jeremy C. 
Published: March 2020

The parasitic protozoanLeishmaniarequires 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. Activity-based protein profiling ofL. mexicanaC12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when Bar-seq was applied to a pool of 16 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, 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 deletionin vitroandin 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 ofLeishmaniaand that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.

Author Summary

Leishmaniaparasites require a variety of protein degradation pathways to enable the parasite to transition through the various life cycle stages that occur in its insect and mammalian hosts. Several enzymes involved in protein degradation inLeishmaniaare known to be essential, including a multi-protein complex, the proteasome, but little is known about how proteins are targeted to the proteasome for degradation. Here, we analyse components of the deubiqutination pathway, including twenty cysteine peptidases (DUBs) that remove the posttranslational modifier ubiquitin from substrates tagged for proteasomal degradation. We used chemical probes to measure active enzymes in parasite lysates and genome engineering to create DUB gene deletion mutants. We identified some DUBs that are essential for parasite viability and some that are required for life cycle progression. We carried out a detailed analysis of the essential DUB2, which has broad deubiquitinase activity and is found in the nucleus. This enzyme interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. This work demonstrates the important role that DUBs play inLeishmania in vivoinfection and further validates DUBs as potential drug targets in this parasite.