PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.
PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.
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DOI：
10.1074/jbc.m116.714972
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发表时间：
2016-04-29
期刊：
影响因子：
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通讯作者：
Mottram JC
中科院分区：
文献类型：
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作者：
Grewal JS;McLuskey K;Das D;Myburgh E;Wilkes J;Brown E;Lemgruber L;Gould MK;Burchmore RJ;Coombs GH;Schnaufer A;Mottram JC
最近已经确定了来自肠道细菌Parabacteroides merdae的C11肽酶PmC 11的结构，使得能够在寄生原生动物布氏锥虫中鉴定和表征C11直向同源物PNT 1。系统发育分析确定PmC 11直系同源物在细菌，古细菌，Chromerids，球虫，动质体，后者是最分歧的。PNT 1与梭菌蛋白酶和PmC 11的一级序列比对揭示了特征性His-Cys催化二联体（His 99和Cys 136）和潜在S1结合位点中的Asp（Asp 134）的位置。免疫荧光和冷冻电子显微镜显示，PNT 1定位于动质体，一个细胞器，含有线粒体基因组的寄生虫（kDNA），与积累的蛋白质或附近的对跖点。在T.在体外培养和小鼠体内，布氏血流形式都是致命的，并且诱导的群体积累了缺乏动基体的细胞。相反，PNT 1的过表达导致细胞具有错位的动质体。在kDNA非依赖性细胞系中PNT 1的RNAi消耗导致动质体损失，但仍存活，表明PNT 1仅为动质体维持所需。重新编码的野生型PNT 1等位基因的表达，但不是一个活性位点突变体恢复寄生虫的生存能力后，在体外和体内诱导证实，PNT 1的肽酶活性是必不可少的寄生虫生存。这些数据提供的证据表明，PNT 1是一种半胱氨酸肽酶，所需的专门维护锥虫动基体。
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 (His99 and Cys136), and an Asp (Asp134) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (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.