Essential roles for deubiquitination inLeishmanialife cycle progression

Abstract

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...

Leishmaniadifferentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

“…Previous work has underscored the importance of the ubiquitination system in Leishmania by demonstrating both the essentiality of the parasite proteasome and a key role for DUBs in the life cycle of this parasite (8,15). Our generation of a select ubiquitination gene null mutant library revealed that 1 out of 2 ubiquitin E1s and 4 out of 13 ubiquitin E2s could not be deleted and therefore may be essential in promastigotes.…”

“…Our generation of a select ubiquitination gene null mutant library revealed that 1 out of 2 ubiquitin E1s and 4 out of 13 ubiquitin E2s could not be deleted and therefore may be essential in promastigotes. A smaller proportion (4 out of 20) cysteine peptidase DUBs were shown to be essential in promastigotes, perhaps due to a greater degree of redundancy in DUB function (8). UBC12, a putative Nedd8 E2, also appears to be essential in promastigotes, suggesting it functions independently of the Nedd8 E1 UBA3, which is non-essential in promastigotes.…”

“…Exploring the potential interaction between UBC2-UEV1 or HECT2 and protein kinases involved in stress responses is an interesting avenue for further study. Additionally, the identification of UBC2 and UEV1 in an interactome of L. mexicana DUB2, which is able to cleave K63-linked diubiquitin in vitro, suggests a possible interplay between these proteins in the regulation of K63-linked ubiquitin chains (8).…”

“…The post-translational modifications phosphorylation and ubiquitination, for example, are thought to contribute significantly to the differentiation process (5)(6)(7)(8).…”

“…To date, two ubiquitin-activating enzymes have been described in Leishmania major (14), and at least 20 cysteine peptidase DUBs exist in Leishmania mexicana, many of which are essential for the promastigote to amastigote transition (8). The importance of the ubiquitination system in these species is further exemplified by the finding that the Leishmania proteasome is essential for parasite survival, since many forms of ubiquitin modification target proteins for proteasomal degradation (15).…”