Grewal, J. S. et al. (2016) PNT1 is a C11 cysteine peptidase essential for replication of the trypanosome kinetoplast. Journal of Biological Chemistry, 291, pp. 9492-9500. (doi: 10.1074/jbc.M116.714972) (PMID:26940875) (PMCID:PMC4850289)
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Abstract
The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (H99 and C136), and an Asp (D134) in the potential S1 binding site. Immunofluorescence and cryo-electron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the parasite's mitochondrial genome (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Wilkes, Dr Jonathan and Coombs, Professor Graham and McLuskey, Dr Karen and Burchmore, Dr Richard and Brown, Miss Elaine and Lemgruber Soares, Dr Leandro and Myburgh, Dr Elmarie and Grewal, Dr Jaspreet Singh and Mottram, Professor Jeremy |
Authors: | Grewal, J. S., McLuskey, K., Das, D., Myburgh, E., Wilkes, J., Brown, E., Lemgruber, L., Gould, M. K., Burchmore, R. J., Coombs, G. H., Schnaufer, A., and Mottram, J. C. |
College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
Journal Name: | Journal of Biological Chemistry |
Publisher: | American Society for Biochemistry and Molecular Biology, Inc. |
ISSN: | 0021-9258 |
ISSN (Online): | 1083-351X |
Published Online: | 03 March 2016 |
Copyright Holders: | Copyright © 2016 The Authors |
First Published: | First published in Journal of Biological Chemistry 291:9492-9500 |
Publisher Policy: | Reproduced under a Creative Commons License |
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