Structural And Functional Characterisation Of RIOK-2 Protein Kinase Encoding Gene Of Strongyloides Stercoralis

Parasitic nematodes not only cause serious economic losses to the livestock industry,but also can infect people and seriously affect human health.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).Widespread and uncontrolled use of anthelmintic drugs in human or other animal parasitic disease control has led to serious drug resistance problem worldwide.The development of new anthelmintic drugs and vaccines requires in-depth study of the functions of the important genes controlling parasitic nematodes’ development.Protein kinases play an important role in regulating important biological processes and have been reported as drug targets for the treatment of diseases,but few studies have been done in parasites.The atypical serine protein kinase RIOK-2 is essential for ribosome biogenesis and the cell-cycle progression in yeast and humans.The present research choose RIOK-2 protein kinase and using the zoonotic parasitic nematode Strongyloides stercoralis to study the characterization and functions of Ss-RIOK-2 in the development of this parasite.Meanwhile,transgenic technology and RNA interference technology were used to analyze the functions of Ce-RIOK-2 in the free-living nematode Caenorhabditis elegans.(1)The gene characterization of Ss-riok-2The c DNA of Ss-riok-2 is 1572 bp in length,encoding a predicted protein of 523 amino acids containing a conserved N-terminal winged helix(w HTH)domain and a RIO kinase domain.A three-dimensional structure(3D)model of ATP bound and an unbound forms of Ss-RIOK-2 were generated using the Chaetomium thermophilum RIOK-2 protein kinase(Ct-RIOK-2)crystal structure 4GYG as a template employing Modeller program.Structural characterization identified some critical sites for ATP binding and metal binding.These newly binding sites potentially give opportunity to design a specific inhibitor against the Ss-RIOK-2 protein kinase and so may contribute to drug discovery.The full length g DNA of Ss-riok-2 is 1620 bp.There is one intron in the coding sequence,which is 48 bp in length.The putative promoter of Ss-riok-2 is 1308 bp in length and contains a number of conserved elements.(2)The expression and transcriptional profiles of Ss-RIOK-2The recombinant protein of Ss-RIOK-2 was expressed in E.coli,and the kinase enzyme showed that Ss-RIOK-2 has the capable of auto-phosphorylation.RNAseq revealed that Ss-riok-2 is transcribed throughout all developmental stages with the highest transcript abundance in free-living females and parasitic females.Patterns of transcriptional reporter expression in transgenic S.stercoralis larvae indicated that Ss-RIOK-2 is expressed in intestinal cells.(3)The function of Ce-RIOK-2The putative promoter of Ce-riok-2,a RIOK-2 protein kinase encoding gene of the free-living nematode C.elegans,drives GFP expression in intestinal cells of C.elegans.In addition,the anatomic expression pattern was weaker in the dauer larvae.Furthermore,silencing of Ce-riok-2 by RNA interference up-regulates Ce-vit-2 expression and causes arrested development,sterility and protruding vulva phenotype in a wild-type background.This study provides the first insight into the functions of RIOK-2 in the parasite nematode S.stercoralis and the free-living nematode C.elegans,which demonstrates significant advancement in molecular and cellular biological study of S.stercoralis and of parasitic nematodes generally,and provides a foundation for further functional genomic studies.