Screening And Function On The Interacting Proteins Of Telomerase RNA Binding Domain TRBD Of Giardia Duodenalis

Giardia duodenalis,referred to as Giardia,is an important zoonotic parasite which is widely parasitic in humans and mammals and causing intestinal diseases with diarrhea as the main symptom.It’s harm to human and animal health with a high infection rate.The drugs,such as metronidazole,tinidazole,were used in clinical treatment of giardia.However,anti-giardia drugs which were used frequently is prone to the risk of teratogenic and carcinogenic.The reason is that we lack the understanding of the molecular biology and cell cycle of Giardia.Therefore,it is significance to further explore the regulation mechanism of Giardia life cycle activities and find good drug targets.Giardia is an ideal eukaryotic "model organism" which is considered to be a transitional stage between prokaryotes and eukaryotes.Giardia trophozoites have two nuclei with the similar size and shape.Studies have shown that the two nuclei can synchronize DNA replication and transcription,but there is still debate about whether the two nuclei have the same genes and function.Giardia trophozoites proliferate by binary fission and can be pure cultured in vitro and subculture repeatedly,which shows the phenomenon of "immortalization".A large number of studies have shown that the cell immortalization is related to the regulation of telomeres and telomerase.Telomerase is a ribonucleic-protein complex with reverse transcriptase activity that synthesizes telomere DNA to lengthen telomere ends.It can maintain telomere length and stability,and participate in life processes such as cell death and aging.Telomerase reverse transcriptase（TERT）is a catalytic telomerase subunit containing a highly conserved domain,telomerase RNA binding domain（TRBD）,which is involved in the assembly of ribosomal protein complexes and is critical to telomerase activity.Currently,it has been reported that RFC1 and ZFD which interact with the telomerase TRBD positively regulate Giardia telomerase activity and influence proliferation in Giardia.The regulation of telomerase activity in Giardia is a complex regulatory process that may require the combined action of multiple proteins,so screening of new telomerase-related proteins provides a basis for elucidating the regulatory network of telomerase in Giardia and in-depth study on the cell cycle regulation mechanism of Giardia.Therefore,the yeast two-hybrid system was used to screen the interacting proteins of Giardia duodenalis telomerase RNA-binding domain TRBD and confirmed the interaction by Bimolecular fluorescence complementation、Co-immunoprecipitation、Pull down.We use Immunofluorescence and Duolink（?） PLA methods to determine the localization of TRBD and its interacting protein in Giardia,and explore the effect of telomerase TRBD-interacting protein on the activity of Giardia telomerase,which is in order to lay a foundation for further exploration of the regulatory mechanism of telomerase in Giardia.Screening of Giardia duodenalis TRBD-interacting proteins: The bait plasmid p GBKT7-TRBD was constructed and transformed into Y2 H Gold yeast for toxicity and self-activation detection.Then,the bait plasmid and the Giardia duodenalis yeast two-hybrid c DNA plasmid library were co-transformed into yeast cells and screened through different auxotrophic media.The results showed that the bait plasmid p GBKT7-TRBD had no toxic effect on yeast cells and could not cause self-activation.Five positive clones,which namely Histone acetyltransferase type B subunit 2,b ZIP family protein,WD-repeat protein,NHLrepeat-containing protein,and Heat shock protein 90,were preliminary screeed by yeast two-hybrid system.And Heat shock protein 90（Hsp90）,the interaction protein of TRBD,was obtained through return verification.Interaction verification between TRBD and Hsp90 protein of Giardia duodenalis: Firstly,Bimolecular fluorescent recombinant plasmids pb Jun-Hsp90 and pb Fos-TRBD were constructed and were co-transfected into HEK-293 T cells.It is showed red fluorescence in Bimolecular fluorescent recombinant,which showed the interaction between TRBD and Hsp90 in HEK-2933 T cells.At the same time,p CDNA3.1-His-TRBD and p CDNA3.1-HA-Hsp90 eukaryotic expression plasmids were constructed and co-transfected into HEK-293 T cells.The protein was collected and verified by Co-immunoprecipitation.The results showed that TRBD interacted with Hsp90 in cells.Finally,p ET-32a-TRBD and p GEX-4T-1-Hsp90 recombinant plasmids were constructed and fusion protein were inducted expression and purified.GST-Pull down results showed that TRBD interacted with Hsp90.The location of TRBD and Hsp90 protein in Giardia duodenalis: The mouse anti-TRBD and rabbit anti-Hsp90 polyclonal antibodies were prepared.The distribution of TRBD and Hsp90 in Giardia was observed by immunofluorescence,and the co-localization of the two in the binucleus of Giardia was observed by Duolink（?） PLA.The experimental results showed that TRBD existed in the nucleus and cytoplasm of Giardia,while Hsp90 was mainly distributed in the cytoplasm,and a small part is also present in the nucleus;and TRBD and Hsp90 co-localized on the binuclear of Giardia.Effects of telomerase interacting protein Hsp90 on telomerase activity in Giardia duodenalis: Firstly,the recombinant plasmid of chimeric hammerhead ribozyme mediated by Giardia duodenalis virus vector was constructed.The Real-Time PCR showed that the cutting efficiency in vitro reached 69.83%,which met the requirements of subsequent experiments.The recombinant plasmids of chimeric hammerhead ribozyme mediated by Giardia duodenalis virus vector were electro-transfected into Giardia trophozoites,respectively,Real-Time PCR and Western Blot analysis showed that the transcript content of Hsp90 in the p C631-pac-Ham-Hsp90 electro-transformation group was 46.1% of that in the control group,the expression level of Hsp90 protein decreased and the proliferation rate of Giardia slowed down;TRAP-silver staining method detected telomerase activity was also significantly reduced.The results showed that the reduction of Hsp90 expression could inhibit the growth and telomerase activity of Giardia duodenalis.In conclusion,Hsp90,the interaction protein of telomerase RNA binding domain TRBD of Giardia duodenalis was successfully screened by yeast two hybrid system,and verified the interaction between TRBD and Hsp90 by Bimolecular fluorescence recombinant,Co-immunoprecipitation and GST-Pull down.At the same time,we obversed the distribution of TRBD and Hsp90 in Giardia by Immunofluorescence and determine that they can be co-located on the binuclear by Duolink（?） PLA.Finally,the effect of Hsp90 on Giardia telomerase activity was investigated by chimeric hammerhead ribozyme mediated by Giardia duodenalis virus vector.This study preliminarily explored the regulatory effect of Hsp90 on telomerase of Giardia,the results showed that the reduction of Hsp90 expression could inhibit telomerase activity and the proliferation of Giardia.It is laid a foundation for further study on the regulatory mechanism of Giardia telomerase.