Giardia lamblia Nek1 and Nek2 kinases affect mitosis and excystation

Smith, Andrew; Lauwaet, Tineke; Davids, Barbara J.; Gillin, Frances D.

doi:10.1016/j.ijpara.2012.03.001

Cited by 16 publications

(17 citation statements)

References 47 publications

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“…Since Nek1 orthologs have been associated with defects in mitotic progression (e.g. Chen et al, 2002;O'Regan et al, 2007;Smith et al, 2012;Woodbury and Morgan, 2007), we used MORN1 as a marker for the spindle, spindle poles and basal complex (the growing end of the daughter bud's cytoskeleton) to monitor the progression of mitosis and cytokinesis (Gubbels et al, 2006;Hu et al, 2006). As shown in the pre-mitotic panel of Fig.…”

Section: Resultsmentioning

confidence: 99%

The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

Chen

Gubbels

2013

Journal of Cell Science

101

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SummaryThe pathology and severity of toxoplasmosis results from the rapid replication cycle of the apicomplexan parasite Toxoplasma gondii. The tachyzoites divide asexually through endodyogeny, wherein two daughter cells bud inside the mother cell. Before mitosis is completed, the daughter buds form around the duplicated centrosomes and subsequently elongate to serve as the scaffold for organellogenesis and organelle partitioning. The molecular control mechanism of this process is poorly understood. Here, we characterized a T. gondii NIMA-related kinase (Nek) ortholog that was identified in a chemical mutagenesis screen. A temperaturesensitive mutant, V-A15, possesses a Cys316Arg mutation in TgNek1 (a novel mutant allele in Neks), which is responsible for growth defects at the restrictive temperature. Phenotypic analysis of V-A15 indicated that TgNek1 is essential for centrosome splitting, proper formation of daughter cells and faithful segregation of genetic material. In vitro kinase assays showed that the mutation abolishes the kinase activity of TgNek1. TgNek1 is recruited to the centrosome prior to its duplication and localizes on the duplicated centrosomes facing the spindle poles in a cell-cycle-dependent manner. Mutational analysis of the activation loop suggests that localization and activity are spatio-temporally regulated by differential phosphorylation. Collectively, our results identified a novel temperature-sensitive allele for a Nek kinase and highlight its essential function in centrosome splitting in Toxoplasma. Moreover, these results conclusively show for the first time that Toxoplasma bud assembly is facilitated by the centrosome because defective centrosome splitting results in single daughter cell budding.

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Section: Resultsmentioning

confidence: 99%

The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

Chen

Gubbels

2013

Journal of Cell Science

101

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“…G. lamblia has fewer components of DNA synthesis, transcription, and RNA processing [1], [9]. However, massive gene expansion happened in the Nek kinase protein family [10], possibly due to the requirement of the Nek kinases for flagellar function and cell motility. Many aspects of giardial transcription are unusual.…”

Section: Introductionmentioning

confidence: 99%

DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

Lin

Weng

et al. 2013

PLoS Negl Trop Dis

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The protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.

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“…Previous attempts to use RNAi for gene knockdown have been unsuccessful and gene knockouts in Giardia are yet to be accomplished due to the tetraploid nature of trophozoites [22]. Therefore, we chose to target the remaining kinases with anti-sense translation blocking morpholino oligomers.…”

Section: Resultsmentioning

confidence: 99%

“…In other eukaryotes, NEK kinases are involved in cell cycle control [20,21]. Two Giardia NEK kinases have been shown to be active in mitosis and excystation [22]. It is worth noting that two trypanosomal NEK kinases, one of them coincidentally possessing a small gatekeeper residue, have been suggested as drug targets in Trypanosoma brucei [23].…”

Section: Introductionmentioning

confidence: 99%

Identification and Validation of Small-Gatekeeper Kinases as Drug Targets in Giardia lamblia

Hennessey

Smith

et al. 2016

PLoS Negl Trop Dis

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Giardiasis is widely acknowledged to be a neglected disease in need of new therapeutics to address toxicity and resistance issues associated with the limited available treatment options. We examined seven protein kinases in the Giardia lamblia genome that are predicted to share an unusual structural feature in their active site. This feature, an expanded active site pocket resulting from an atypically small gatekeeper residue, confers sensitivity to “bumped” kinase inhibitors (BKIs), a class of compounds that has previously shown good pharmacological properties and minimal toxicity. An initial phenotypic screen for biological activity using a subset of an in-house BKI library found that 5 of the 36 compounds tested reduced trophozoite growth by at least 50% at a concentration of 5 μM. The cellular localization and the relative expression levels of the seven protein kinases of interest were determined after endogenously tagging the kinases. Essentiality of these kinases for parasite growth and infectivity were evaluated genetically using morpholino knockdown of protein expression to establish those that could be attractive targets for drug design. Two of the kinases were critical for trophozoite growth and attachment. Therefore, recombinant enzymes were expressed, purified and screened against a BKI library of >400 compounds in thermal stability assays in order to identify high affinity compounds. Compounds with substantial thermal stabilization effects on recombinant protein were shown to have good inhibition of cell growth in wild-type G. lamblia and metronidazole-resistant strains of G. lamblia. Our data suggest that BKIs are a promising starting point for the development of new anti-giardiasis therapeutics that do not overlap in mechanism with current drugs.

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Giardia lamblia Nek1 and Nek2 kinases affect mitosis and excystation

Cited by 16 publications

References 47 publications

The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

Identification and Validation of Small-Gatekeeper Kinases as Drug Targets in Giardia lamblia

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