Elsevier

Biotechnology Advances

Volume 29, Issue 3, May–June 2011, Pages 338-350
Biotechnology Advances

Research review paper
Atypical (RIO) protein kinases from Haemonchus contortus — Promise as new targets for nematocidal drugs

https://doi.org/10.1016/j.biotechadv.2011.01.006Get rights and content

Abstract

Almost nothing is known about atypical kinases in multicellular organisms, including parasites. Supported by information and data available for the free-living nematode, Caenorhabditis elegans, and other eukaryotes, the present article describes three RIO kinase genes, riok-1, riok-2 and riok-3, from Haemonchus contortus, one of the most important parasitic nematodes of small ruminants. Analyses of these genes and their products predict that they each play critical roles in the developmental pathways of parasitic nematodes. The findings of this review indicate prospects for functional studies of these genes in C. elegans (as a surrogate) and opportunities for the design of a novel class of nematode-specific inhibitors of RIO kinases. The latter aspect is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock.

Introduction

Protein kinases are a group of enzymes that are crucial for the regulation of a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, and thus play functional roles in reversible protein phosphorylation (Hanks et al., 1988). Based on their structure, protein kinases can be classified into eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs) (Manning et al., 2002). The aPKs have kinase activity and share limited sequence similarity to the majority of eukaryotic protein kinases. Of the 518 kinases known to be encoded in the human genome, for instance, 40 are aPKs. These molecules have been classified into 13 families or groups, one of which represents the RIO kinases (designated here as RIOKs) (Manning et al., 2002). These kinases are considered essential for life, but almost nothing is known about them for multicellular organisms (metazoans), including nematodes (LaRonde-LeBlanc and Wlodawer, 2005a, LaRonde-LeBlanc and Wlodawer, 2005b).

Although the primary structures (amino acid sequences) of RIOKs are divergent from those of other protein kinase families, their conformation is similar to those of known canonical protein kinases. In yeast (Saccharomyces cerevisiae), RIOK-1 has been shown to have protein kinase activity in vitro, which is dependent upon amino acid residues recognised as being essential for kinase function (Angermayr et al., 2002). Other research has demonstrated an important role for RIOK-1 in cell-cycle progression (G1 to S transition), the regulation of the onset of anaphase and mitotic chromosome stability (Angermayr et al., 2002) as well as the processing of 20S precursor ribosomal RNA (rRNA) to the 18S species (Vanrobays et al., 2001). RIOK-2 of S. cerevisiae is also required for 20S rRNA processing. However, in contrast to RIOK-1, RIOK-2 appears to be localised predominantly to the nucleus (Vanrobays et al., 2001). Although RIOK-1 and RIOK-2 are known to be associated with the 20S precursor rRNA in yeast, the biological activities of both kinases do not overlap (Vanrobays et al., 2001, Geerlings et al., 2003). Recently, RIOK-2 has also been identified as an essential, late-acting 40S ribosome synthesis factor (Granneman et al., 2010). Interestingly, an RNA interference (RNAi) screen of human kinase genes identified that the knock-down of riok-1 and riok-2 decreased cell viability and accelerated epithelial cell migration, respectively, whereas no effect was detected for riok-3 which is specific to metazoans (Simpson et al., 2008). A recent report (Kimmelman et al., 2008) suggests that human RIOK-3 might be involved in human tumour cell motility and invasion, possibly through the modulation of the Rho family of GTPases. Taken together, this information indicates that RIOKs are involved in diverse and crucial biological processes in eukaryotes, but their precise roles remain to be elucidated in multicellular organisms.

RIOKs are encoded in the genome of the best-characterised metazoan, the free-living nematode Caenorhabditis elegans (see Manning, 2005). RNAi, which decreases messenger RNA (mRNA) levels of the targeted C. elegans gene, has been shown to affect predominantly embryonic and larval growth and/or development (Fraser et al., 2000, Ashrafi et al., 2003, Simmer et al., 2003, Rual et al., 2004, Sonnichsen et al., 2005). In spite of the functional importance of this molecule, there is no published information on aPKs for any related, parasitic nematodes, with the exception of Trichostrongylus vitrinus (order Strongylida) (see Hu et al., 2008). In the present article, we elucidate the full-length complementary cDNAs and genes of three RIOKs from Haemonchus contortus (an economically important blood-feeding strongylid nematode of small ruminants) and compare them with related molecules encoded in other organisms, infer the three-dimensional structures of RIOKs by comparison with known crystal structures of homologues, and assess the potential of these kinases as novel drug targets in parasitic helminths.

This article provides new insights into three different atypical (RIO) kinases in H. contortus, one of the economically most important parasites of livestock. Based on bioinformatic and phylogenetic analyses, these kinases are proposed to be novel drug targets. Currently, computational approaches (e.g., Krasky et al., 2007, Caffrey et al., 2009, Doyle et al., 2010) are increasingly being used to assess the potential of key genes/gene products as novel drug targets in parasitic worms. In addition, structure-based virtual screening has been proven to be useful in the identification of compounds able to inhibit the activity of molecules whose three dimensional structure had been established using homology models (reviewed by Villoutreix et al., 2007). For instance, in silico docking for ~ 200,000 compounds (i.e., ChemDiv) into the binding site of an homology model of a human BCR-ABL tyrosine kinase (which is known to play a crucial role in the pathogenesis of chronic myeloid leukaemia; Deininger et al., 2000) led to the identification of 15 compounds selected for biological testing, eight of which were demonstrated to significantly inhibit tumour cell growth (Peng et al., 2003). In another study (Vangrevelinghe et al., 2003), novel and selective inhibitors of protein casein kinase II were identified by in silico docking of an homology model of CK2 with a subset of 400,000 molecules available in the Novartis database (Vangrevelinghe et al., 2003). Although numerous examples of protein-ligand interaction studies and drug design using in silico approaches are described in the literature (cf. Villoutreix et al., 2007, Cavasotto and Phatak, 2009, Hammami and Fliss, 2010), and computational structure prediction methods are cost- and time-effective in the absence of experimental structures, the success of these approaches depends on the accuracy of the model predicted and on the sequence similarities between the protein used as a template and the homologous sequence(s) (see Cavasotto and Phatak, 2009).

Section snippets

Nucleic acids

Genomic DNA was extracted from 50 mg of pooled H. contortus using a small-scale sodium dodecyl-sulphate (SDS)/proteinase K extraction procedure, followed by purification over a mini-column (Wizard Clean-Up, Promega) (Gasser et al., 2006). The specific identity and mono-specificity of the parasite material was verified by PCR-coupled, automated sequencing of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA from genomic DNA (see Bott et al., 2009). Total RNA was extracted

Three riok genes and their inferred gene products for Haemonchus contortus

The full-length cDNAs (designated Hc-riok-1, Hc-riok-2 and Hc-riok-3) were 1842, 1590 and 1434 nucleotides (nt) in length, respectively (GenBank accession nos. HQ198854.1-HQ198857.1 and HQ207527.1-HQ207528.1.; Table 1). The transcripts representing Hc-riok-1, Hc-riok-2 and Hc-riok-3 were reproducibly detected by reverse transcription PCR in all developmental stages examined, except for the exsheathed L3 (Fig. 1, Conder and Johnson, 1998). Transcription was usually greatest in eggs, L4 female

Inference of gene function and interactions based on information available for C. elegans and other eukaryotic organisms

In C. elegans, riok-1 is involved in biological processes essential for nematode viability and fertility as well as endocytosis and fat storage (Fraser et al., 2000, Ashrafi et al., 2003, Simmer et al., 2003, Rual et al., 2004, Sonnichsen et al., 2005, Balklava et al., 2007, Ceron et al., 2007; cf. www.wormbase.org; Table 2). In contrast, knowledge of the functions of the C. elegans riok-2 and riok-3 is limited to the observation that gene perturbation by RNAi results in lethality and

Three-dimensional structural modelling

In this section, all residue numbers refer to the sequence of Hc-RIOK-1 (see Fig. 4). Topologically, the three RIOKs differ in the N- and C-terminal domains that flank the central kinase domain. RIOK-1 usually possesses an N-terminal domain of ~ 100 aa residues, with few predicted secondary structure elements. Af-RIOK-1 from A. fulgidus, the only RIOK-1 for which an experimental three-dimensional structure is available (PDB accession code 1ztf) (LaRonde-LeBlanc et al., 2005b), seems to be rather

Application of the RIOK-1 model for the prediction of drugs in silico

In a first attempt to probe the active site of RIOKs, with a view toward drug discovery, we conducted an in silico screen using the homology model of Hc-RIOK-1 employing the SPECS database. The top 12 binding compounds identified from this screen are listed in Table 3. Interestingly, four of these 12 compounds possess a carbohydrate moiety. For the compounds ranked third and tenth, a second binding mode was observed and ranked in positions 8 and 11, respectively, indicating an increased

Conclusions and future prospects

Based on modelling, structural comparison of the three RIOKs shows that the RIOK domain harbouring the catalytic site is a well-conserved fold among parasitic nematodes, in particular between H. contortus and T. vitrinus. However, despite this fold, there are several aa substitutions in functionally important, conserved secondary structure elements (Supplementary Fig. 4), whose impact can only be assessed from three-dimensional structures determined experimentally. Future structural studies

Acknowledgements

Funding from the Australian Research Council (ARC) is gratefully acknowledged (RBG). PRB is supported by funding from the National Health and Medical Research Council (NHMRC).

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