Bioinformatic exploration of RIO protein kinases of parasitic and free-living nematodes

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Highlights

Abstract

Despite right open reading frame kinases (RIOKs) being essential for life, their functions, substrates and cellular pathways remain enigmatic. In the present study, gene structures were characterised for 26 RIOKs from draft genomes of parasitic and free-living nematodes. RNA-seq transcription profiles of riok genes were investigated for selected parasitic nematodes and showed that these kinases are transcribed in developmental stages that infect their mammalian host. Three-dimensional structural models of Caenorhabditis elegans RIOKs were predicted, and elucidated functional domains and conserved regions in nematode homologs. These findings provide prospects for functional studies of riok genes in C. elegans, and an opportunity for the design and validation of nematode-specific inhibitors of these enzymes in socioeconomic parasitic worms.

Introduction

Parasitic diseases continue to be a substantial burden on human and animal health worldwide. This burden is compounded by food and water shortages as well as poor sanitation (Hotez et al., 2009). The World Health Organization (WHO) estimates that more than 1 billion people are currently affected by parasitic nematodes and that the impact of these worms surpasses diabetes or lung cancer in disability adjusted life years (DALYs) (http://www.who.int/en/). Considering the limited number of approved anthelmintic drugs for widespread use in humans or other animals, current treatment programs to control these nematodes can lead to drug resistance (Osei-Atweneboana et al., 2011), requiring continued efforts to search for new interventions. The traditional approach of using high-throughput screening for drug discovery is costly and has yielded only a small number of anthelmintic drugs, with no novel classes approved for use in humans in decades (Keiser and Utzinger, 2010). An alternative approach is to understand the molecular and biochemical pathways in parasitic nematodes to guide future discovery efforts.

Major advances in high-throughput sequencing have led to a substantial expansion in the availability of genomic and transcriptomic data for parasitic nematodes in recent years. To date, the draft genomes of Trichinella spiralis (clade I) (Blaxter et al., 1998); Ascaris suum, Brugia malayi, Loa loa (clade III); Strongyloides ratti, Bursaphelenchus xylophilus, Meloidogyne hapla, Panagrellus redivivus (clade IV); Haemonchus contortus and Pristionchus pacificus (clade V) have been published (Ghedin et al., 2007, Abad et al., 2008, Dieterich et al., 2008, Opperman et al., 2008, Jex et al., 2011, Kikuchi et al., 2011, Mitreva et al., 2011, Desjardins et al., 2013, Laing et al., 2013, Schwarz et al., 2013, Srinivasan et al., 2013). In addition to the extensive genomic research linked to species of Caenorhabditis (e.g., Caenorhabditis elegans Sequencing Consortium, 1998, Gupta and Sternberg, 2003), these draft genomes represent a massive resource for the scientific community working on fundamental and applied aspects of parasitic worms.

The benefits of these resources can now be realised through a collective effort of mining and curating smaller datasets, such as particular gene families involved in essential biological processes. Protein kinases (PKs) are one of the largest gene families of metazoans and regulate a wide range of cellular processes including cell-cycle progression, transcription, DNA replication and metabolic processes (Vanrobays et al., 2001, LaRonde-LeBlanc et al., 2005, LaRonde-LeBlanc and Wlodawer, 2005a, Granneman et al., 2010, Campbell et al., 2011, Widmann et al., 2012, Read et al., 2013). PKs can activate/inactivate target proteins by catalysing the transfer of phosphate groups to specific residues (i.e. His/Asp, Ser/Thr/Tyr and Arg) on their target proteins and thus play a regulatory role in nearly all cell signalling pathways (Hanks et al., 1988). Presently, PKs can be divided into eukaryotic protein (ePKs), protein kinase-like (PKL) and atypical protein (aPK) kinases. For instance, of more than 500 human PKs, less than 10% are PKL proteins, many of which were previously classified as aPKs. The PKL kinases represent 19 families, one being the right open reading-frame kinases (RIOKs) (Kannan et al., 2007, Manning et al., 2011).

In the present study, we extend previous studies of RIOKs of selected strongylid nematodes (Hu et al., 2008, Campbell et al., 2011, Ansell et al., 2013), indicating that they (i) are likely to be crucial for development, survival and/or reproduction, (ii) are conserved among nematodes, but (iii) are divergent from related molecules in other ecdysozoans and vertebrates. Multicellular organisms studied to date all have three riok genes (riok-1, riok-2 and riok-3). Functional studies of these genes in Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (elegant worm), Drosophila melanogaster (vinegar fly) and vertebrate cell lines indicate key roles for RIOKs in ribosome maturation, cell-cycle progression and/or chromosome stability (Vanrobays et al., 2001, Angermayr and Bandlow, 2002, Geerlings et al., 2003, Ceron et al., 2007, Simpson et al., 2008, Granneman et al., 2010, Strunk et al., 2011, Widmann et al., 2012, Esser and Siebers, 2013). Recently, another study (Read et al., 2013) has indicated that D. melanogaster riok-2 (Dme-riok-2) is involved in cell cycle progression through the regulation of the Akt signalling pathway, suggesting that riok-2 might be part of a complex cell-cycle signalling system. In spite of this information, the essential roles of RIOKs have only been assumed for most organisms, including parasitic nematodes (Vanrobays et al., 2001, LaRonde-LeBlanc et al., 2005, LaRonde-LeBlanc and Wlodawer, 2005a, Granneman et al., 2010, Campbell et al., 2011, Widmann et al., 2012, Read et al., 2013). To date, structural information for the RIOK family is based on two crystal structures for Afu-RIOK-1 and Afu-RIOK-2 of Archaeoglobus fuldigus (Archaea) and on Cth-RIOK-2 of Chaetomium thermophilum (thermophilic fungus) (LaRonde-LeBlanc and Wlodawer, 2004, LaRonde-LeBlanc et al., 2005, Ferreira-Cerca et al., 2012). Remarkably, the latter, fungal kinase acts in vitro as an ATPase rather than a kinase, suggesting that RIOKs might be involved in a process that is distinct from the assumed canonical serine/threonine kinase activity (Ferreira-Cerca et al., 2012). Gaining insights into the structural characteristics and molecular functions of RIOKs, and their involvement in cellular pathways, might provide a basis for the future design of novel drugs against parasitic nematodes. As a first step, we undertook here a detailed investigation of the riok gene family in 12 nematode species representing different phylogenetic clades, and for which draft genomes and transcriptomes are presently available.

Section snippets

Gene prediction and identification

We extracted genomic, transcriptomic and protein datasets for the identification, isolation and curation of full-length riok genes from 13 nematode species from WormBase (WS238; www.wormbase.org) for C. elegans, C. briggsae, T. spiralis, L. loa, A. suum, B. xylophilus, M. hapla, Meoidogyne incognita and from other repositories for H. contortus (GenBank Assembly IDs: GCA_000469685.1 and GCA_000442195.1), P. redivivus (GenBank Assembly ID: GCA_000341325.1), Pr. pacificus (//www.pristionchus.org

Detailed characterisation of riok genes in nematodes

Based on the three characterised Cel-rioks (WBGene00019698, WBGene00013688 and WBGene00014012), we identified partial rioks in the draft genomes of 12 other nematode species for which extensive published genomic/transcriptomic data were available. After manual curation, full-length gene sequences were defined for each riok-1 to -3 for eight of the 12 nematodes (Table 1). No full-length rioks were found for the three plant-parasitic nematodes, B. xylophilus, M. hapla and M. incognita, or the

Discussion

We combined a bioinformatics approach with manual curation to provide improved insights into functional and structural aspects of the RIOK family of parasitic and free-living nematodes whose draft genomes are publicly available (Ghedin et al., 2007, Dieterich et al., 2008, Opperman et al., 2008, Jex et al., 2011, Mitreva et al., 2011, Desjardins et al., 2013, Laing et al., 2013, Schwarz et al., 2013, Srinivasan et al., 2013). Due to a lack of reliable culturing systems to propagate parasites in

Acknowledgements

This project was funded by the National Health and Medical Research Council (NHMRC) of Australia, and the Australian Research Council (ARC). This project was also supported by a Victorian Life Sciences Computation Initiative (VLSCI), Australia, grant number VR0007 on its Peak Computing Facility at the University of Melbourne, Australia, an initiative of the Victorian Government. Other support from the Australian Academy of Science, the Australian-American Fulbright Commission, Alexander von

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