RIOK-2 protein is essential for egg hatching in a common parasitic nematode
Introduction
Members of the protein kinase superfamily regulate signalling pathways through phosphorylation and thereby influence various cellular functions including metabolism, proliferation, migration, apoptosis, and differentiation (Manning et al., 2002). Based on functional motifs, kinases can be divided into two broad groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs) (Hanks et al., 1988, Hanks and Hunter, 1995, Manning et al., 2002). The latter group comprises 13 families including the RIOK family (Manning et al., 2002, LaRonde-LeBlanc and Wlodawer, 2005b), which has four known members: RIOK-1, RIOK-2, RIOK-3 and RIOK-B (LaRonde-LeBlanc and Wlodawer, 2005a, b). The three-dimensional crystal structures of RIOK-2 from Chaetomium thermophilum (Ct-RIOK-2), Archaeoglobus fulgidus (Af-RIOK-2) and Homo sapiens (Hs-RIOK-2) have demonstrated that they contain a trimmed version of the canonical eukaryotic protein kinase domain (termed RIOK) and an N-terminal winged helix-turn-helix (wHTH) domain which is frequently found in DNA-binding proteins and transcription factors (Gajiwala et al., 2000, Wolberger and Campbell, 2000, LaRonde-LeBlanc and Wlodawer, 2004, Ferreira-Cerca et al., 2012, Wang et al., 2019). The RIOK-2 structure also revealed a conserved disordered domain that may be involved in substrate binding (LaRonde-LeBlanc et al., 2005). Further studies showed that Af-RIOK-2 exhibits a remarkable ATPase activity capable of phosphorylating itself and myelin basic protein as a substrate (LaRonde-LeBlanc and Wlodawer, 2004).
RIOK-2 is suggested to regulate eukaryotic ribosome synthesis, which is a complex process requiring over 350 assembly factors located in the nucleolus, nucleoplasm, and cytoplasm (Gerhardy et al., 2014, Nerurkar et al., 2015). As an indispensable protein, RIOK-2 governs ribosome maturation in many ways (Schafer et al., 2003, Ferreira-Cerca et al., 2012). The catalytic activity of RIOK-2 is necessary for its release from the pre-40S particles and thereby affects the binding and dissociation of assembly factors to ribosomal subunits (Zemp et al., 2009, Ferreira-Cerca et al., 2012). The depletion of RIOK-2 results in the accumulation of 20S pre-rRNA, indicating that it is critical for D-site cleavage in the cytoplasm (Geerlings et al., 2003, Vanrobays et al., 2003). The structural analysis of ribosomes and their binding factors has also revealed that RIOK-2 binds to the A and P sites of the 40S pre-rRNA via its wHTH and two-lobed kinase domains to suppress the erroneous initiation of the protein translation (Strunk et al., 2011, Heuer et al., 2017). Not least, RIOK-2 exerts a regulatory effect on mitosis and affects tumorigenesis in glioblastoma cells (Liu et al., 2011, Mohamed et al., 2018).
Although RIOK-2 proteins of fungal and mammalian cells have been explored widely, mechanisms and functions of RIOK-2 homologs occurring in nematodes are poorly understood. 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). Ss-RIOK-2 is expressed mainly in the intestine of L1 stages (Lei et al., 2017), albeit no functional studies have been conducted in parasitic nematodes as yet, which was the primary focus of the work described herein.
Section snippets
Ethics statement
The UPD strain of S. stercoralis was maintained in steroid-treated Beagle dogs, in compliance with a protocol approved by the Committee on the Ethics of Animal Experiments of Hubei Province, China (Permit No. SYXK-2015-0029). Dogs were fed on standard laboratory chow, and kept at a constant temperature of 26 °C. All animal procedures complied with guidelines proposed by the Administration of Affairs Concerning Experimental Animals of PR China.
Parasite culture
Strongyloides stercoralis was propagated in Beagles
Aspartate 228 in the kinase motif of Ss-RIOK-2 is required for its activity
As reported earlier (Lei et al., 2017), the primary structure of RIOK-2 revealed two distinct domains with a linker region in between (Fig. 1A). The N-terminal region comprised the well-defined and conserved ATP-binding and kinase domains, whereas the C-terminal region harboured a metal-binding motif. Following in silico analysis, we tested the kinase activity of purified wild-type protein followed by the role of conserved Asp228 located in the kinase motifs (Fig. 1A). The wild-type and D228A
Discussion
This study evaluated the function and physiological importance of Ss-RIOK-2 in a clinically-relevant worm, S. stercoralis. Our data show that Ss-RIOK-2 is a catalytically competent kinase with conserved signature residues including Asp228 and Lys123 in its kinase and ATP-binding domains, respectively. Ectopic expression of mutated variants exerts a dominant negative effect, enabling us to examine the biological significance of Ss-RIOK-2 in transgenic worms. Similar to the wild-type isoform, Ss-
Acknowledgments
The authors acknowledge James B. Lok (University of Pennsylvania, USA) for sharing protocols and helpful discussion, and Robin B. Gasser (The University of Melbourne, Australia) for valuable feedback. This study was supported by “Fundamental Research Funds for the Central Universities”, China (Program 2662015PY180).
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