Bioinformatic exploration of RIO protein kinases of parasitic and free-living nematodes
Graphical abstract
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
References (79)
- et al.
RIO1, an extraordinary novel protein kinase
FEBS Lett.
(2002) - et al.
Insights into the immuno-molecular biology of Angiostrongylus vasorum through transcriptomics – prospects for new interventions
Biotechnol. Adv.
(2013) - et al.
The many faces of the helix-turn-helix domain: transcription regulation and beyond
FEMS Microbiol. Rev.
(2005) - et al.
Atypical (RIO) protein kinases from Haemonchus contortus – promise as new targets for nematocidal drugs
Biotechnol. Adv.
(2011) - et al.
Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20 S pre-rRNA in Saccharomyces cerevisiae
J. Biol. Chem.
(2003) - et al.
Testing the efficacy of RNA interference in Haemonchus contortus
Int. J. Parasitol.
(2006) - et al.
Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake
J. Biol. Chem.
(2008) Genetic analysis of pathways to Parkinson disease
Neuron
(2010)- et al.
Rescuing the bottom billion through control of neglected tropical diseases
Lancet
(2009) - et al.
The drugs we have and the drugs we need against major helminth infections
Adv. Parasitol.
(2010)
Crystal structure of A. fulgidus Rio2 defines a new family of serine protein kinases
Structure
A family portrait of the RIO kinases
J. Biol. Chem.
The RIO kinases: an atypical protein kinase family required for ribosome biogenesis and cell cycle progression
Biochim. Biophys. Acta
Evolution of protein-coding genes in Drosophila
Trends Genet.
An eye on RNAi in nematode parasites
Trends Parasitol.
Compactness of human housekeeping genes: selection for economy or genomic design?
Trends Genet.
Crystal structures of c-Src reveal features of its autoinhibitory mechanism
Mol. Cell
Interaction of tomato mosaic virus movement protein with tobacco RIO kinase
Mol. Cells
Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita
Nat. Biotechnol.
Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes
Nature
Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic
Nat. Cell Biol.
Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors
Biochem. J.
A molecular evolutionary framework for the phylum Nematoda
Nature
Genome sequence of the nematode C. elegans: a platform for investigating biology
Science
Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition
PLoS Biol.
Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity
BMC Dev. Biol.
Three independent determinants of protein evolutionary rate
J. Mol. Evol.
Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages
Proc. Natl. Acad. Sci. U.S.A.
Kinase drug discovery – what’s next in the field?
ACS Chem. Biol.
WebLogo: a sequence logo generator
Genome Res.
Genomics of Loa loa, a Wolbachia-free filarial parasite of humans
Nature
The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism
Nat. Genet.
MUSCLE: multiple sequence alignment with high accuracy and high throughput
Nucleic Acids Res.
Atypical protein kinases of the RIO family in archaea
Biochem. Soc. Trans.
ATPase-dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit
Nat. Struct. Mol. Biol.
Functional genomic analysis of C. elegans chromosome I by systematic RNA interference
Nature
Draft genome of the filarial nematode parasite Brugia malayi
Science
Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking
EMBO J.
The developmental transcriptome of Drosophila melanogaster
Nature
Cited by (11)
RIOK-2 protein is essential for egg hatching in a common parasitic nematode
2020, International Journal for ParasitologyCitation Excerpt :In the free-living nematode Caenorhabditis elegans, RIOK-2 is expressed in the pharynx (Mendes et al., 2015), and the genome-wide RNA interference screen indicates that silencing of riok-2 causes embryonic lethality and sterility (Sonnichsen et al., 2005). RIOK-2 has also been identified in several parasitic nematodes including Ascaris suum, Brugia malayi, Haemonchus contortus and Strongyloides stercoralis (Breugelmans et al., 2014; Lei et al., 2017). In the latter species, Ss-riok-2 is transcribed during all developmental stages, with a peak abundance in the free-living and parasitic stages of female worms (Lei et al., 2017).
Functional genomic exploration reveals that Ss-RIOK-1 is essential for the development and survival of Strongyloides stercoralis larvae
2017, International Journal for ParasitologyCitation Excerpt :In the context of this experiment, however, only one larva from the group expressing the mutant fusion protein (transformed with pRP8) alone progressed to the iL3 stage, and two were in the process of moulting. Based on deep bioinformatic analyses of the genomic and transcriptomic datasets from some parasitic nematode species (Campbell et al., 2011; Breugelmans et al., 2014), the RIO atypical kinase, RIOK-1, is considered to be a potential target for new drugs to control parasitic nematode diseases which affect one-quarter of the world's population (Miguel and Kremer, 2004). However, little was known about the degree to which RIOK-1 participates in regulating the development of parasitic nematodes prior to this investigation.
Advances in kinome research of parasitic worms - implications for fundamental research and applied biotechnological outcomes
2018, Biotechnology AdvancesCitation Excerpt :However, the latter study did not identify or classify divergent PKLs/aPKs, such as RIO kinases. Investigating these divergent sequences in parasites is warranted, given that they might represent parasite-specific drug targets or could provide important clues regarding the unique biology of a parasite (Breugelmans et al., 2014; Campbell et al., 2011). In addition to the S. mansoni kinome, draft kinomes for two parasitic nematodes (Loa loa and Wuchereria bancrofti) have been defined (Goldberg et al., 2013) employing the automated classification method in Kinannote and subsequent refinement based on sequence similarity with the kinases of D. discoideum, S. cerevisiae, D. melanogaster and H. sapiens, and orthology with C. elegans kinase sequences.
Genome-wide identification and characterization of the RIO atypical kinase family in plants
2018, Genes and Genomics