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ABSTRACT: Signaling pathways controlled by reversible protein
phosphorylation (catalyzed by kinases and phosphatases)
in the malaria parasite Plasmodium are of
great interest, for both increased understanding of
parasite biology and identification of novel drug
targets. Here, we report a functional analysis in Plasmodium
of an ancient bacterial Shewanella-like
protein phosphatase (SHLP1) found only in bacteria,
fungi, protists, and plants. SHLP1 is abundant in
asexual blood stages and expressed at all stages
of the parasite life cycle. shlp1 deletion results in
a reduction in ookinete (zygote) development, microneme
formation, and complete ablation of oocyst
formation, thereby blocking parasite transmission.
This defect is carried by the female gamete and can
be rescued by direct injection of mutant ookinetes
into the mosquito hemocoel, where oocysts develop.
This study emphasizes the varied functions of SHLP1
in Plasmodium ookinete biology and suggests that it
could be a novel drug target for blocking parasite
transmission.
Cell Reports. 02/2013;
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[show abstract]
[hide abstract]
ABSTRACT: Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.
Cell reports. 02/2013;
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David S Guttery,
Benoit Poulin, David J P Ferguson,
Balázs Szöőr,
Bill Wickstead,
Paula L Carroll,
Chandra Ramakrishnan,
Declan Brady,
Eva-Maria Patzewitz,
Ursula Straschil,
Lev Solyakov,
Judith L Green,
Robert E Sinden,
Andrew B Tobin,
Anthony A Holder,
Rita Tewari
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ABSTRACT: Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl(-) mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.
PLoS Pathogens 09/2012; 8(9):e1002948. · 9.13 Impact Factor
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ABSTRACT: For patients diagnosed with advanced renal cell carcinoma (RCC), there are few therapeutic options. Radiation therapy is predominantly used to treat metastasis and has not proven effective in the adjuvant setting for renal cancer. Furthermore, RCC is resistant to standard cytotoxic chemotherapies. Targeted anti-angiogenics are the standard of care for RCC but are not curative. Newer agents, such as mTOR inhibitors and others that induce autophagy, have shown great promise for treating RCC. Here, we investigate the potential use of the small molecule STF-62247 to modulate radiation.
Using RCC cell lines, we evaluate sensitivity to radiation in addition to agents that induce autophagic cell death by clonogenic survival assays. Furthermore, these were also tested under physiological oxygen levels.
STF-62247 specifically induces autophagic cell death in cells that have lost VHL, an essential mutation in the development of RCC. Treatment with STF-62247 did not alter cell cycle progression but when combined with radiation increased cell killing under oxic and hypoxic/physiological conditions.
This study highlights the possibility of combining targeted therapeutics such as STF-62247 or temsirolimus with radiation to reduce the reliance on partial or full nephrectomy and improve patient prognosis.
Radiotherapy and Oncology 04/2012; 103(3):388-93. · 5.58 Impact Factor
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David S Guttery, David J P Ferguson,
Benoit Poulin,
Zhengyao Xu,
Ursula Straschil,
Onny Klop,
Lev Solyakov,
Sara M Sandrini,
Declan Brady,
Conrad A Nieduszynski,
Chris J Janse,
Anthony A Holder,
Andrew B Tobin,
Rita Tewari
PLoS Pathogens 03/2012; 8(3). · 9.13 Impact Factor
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David S Guttery, David J P Ferguson,
Benoit Poulin,
Zhengyao Xu,
Ursula Straschil,
Onny Klop,
Lev Solyakov,
Sara M Sandrini,
Declan Brady,
Conrad A Nieduszynski,
Chris J Janse,
Anthony A Holder,
Andrew B Tobin,
Rita Tewari
[show abstract]
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ABSTRACT: Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.
PLoS Pathogens 02/2012; 8(2):e1002554. · 9.13 Impact Factor
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Ursula Straschil,
Arthur M Talman, David J P Ferguson,
Karen A Bunting,
Zhengyao Xu,
Elizabeth Bailes,
Robert E Sinden,
Anthony A Holder,
Elizabeth F Smith,
Juliet C Coates,
Rita Tewari
[show abstract]
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ABSTRACT: Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.
PLoS ONE 01/2010; 5(9):e12901. · 4.09 Impact Factor
