TY - JOUR
T1 - PNT1 is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.
AU - Grewal, Jaspreet
AU - McLuskey, Karen
AU - Das, Debanu
AU - Myburgh, Elmarie
AU - Wilkes, Jonathan
AU - Brown, Elaine
AU - Lemgruber, Leandro
AU - Gould, Matthew K.
AU - Burchmore, Richard J.
AU - Coombs, Graham H.
AU - Schnaufer, Achim
AU - Mottram, Jeremy Charles
N1 - © 2016, by The American Society for Biochemistry and Molecular Biology, Inc. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details.
PY - 2016/4/29
Y1 - 2016/4/29
N2 - 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.
AB - 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.
U2 - 10.1074/jbc.M116.714972
DO - 10.1074/jbc.M116.714972
M3 - Article
SN - 0021-9258
VL - 291
SP - 9492
EP - 9500
JO - The Journal of biological chemistry
JF - The Journal of biological chemistry
IS - 18
ER -