Back to Search
Start Over
Essential roles for deubiquitination in Leishmania life cycle progression
- Source :
- PLoS Pathogens, Vol 16, Iss 6, p e1008455 (2020), PLoS Pathogens
- Publication Year :
- 2020
- Publisher :
- Public Library of Science (PLoS), 2020.
-
Abstract
- 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. Activity-based 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.<br />Author summary Leishmania parasites 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 in Leishmania are 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 deubiquitination 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 in Leishmania in vivo infection and further validates DUBs as potential drug targets in this parasite.
- Subjects :
- Life Cycles
Leishmania mexicana
Protozoan Proteins
Artificial Gene Amplification and Extension
Protozoology
Polymerase Chain Reaction
Biochemistry
Mice
Ubiquitin
Medicine and Health Sciences
Post-Translational Modification
Biology (General)
Nuclear protein
Protozoans
Leishmania
Mice, Inbred BALB C
0303 health sciences
Deubiquitinating Enzymes
biology
Cell Cycle
030302 biochemistry & molecular biology
Eukaryota
Proteases
Cell cycle
Enzymes
3. Good health
Cell biology
Chromatin
RNA splicing
Protozoan Life Cycles
Female
Intracellular
Research Article
Amastigotes
Deubiquitination
QH301-705.5
Parasitic Life Cycles
Immunology
Research and Analysis Methods
Microbiology
03 medical and health sciences
Virology
Parasitic Diseases
Genetics
Animals
Molecular Biology Techniques
Amastigote
Molecular Biology
030304 developmental biology
Parasitic life cycles
Promastigotes
Organisms
Ubiquitination
Biology and Life Sciences
Proteins
RC581-607
Parasitic Protozoans
Proteasome
Enzymology
biology.protein
Parasitology
Immunologic diseases. Allergy
Gene Deletion
Developmental Biology
Subjects
Details
- ISSN :
- 15537374 and 15537366
- Volume :
- 16
- Database :
- OpenAIRE
- Journal :
- PLOS Pathogens
- Accession number :
- edsair.doi.dedup.....64f48156a81a3b5127a298dc3d18f706