Identification And Function Of A Telomerase Reverse Transcriptase-interacting Protein In Giardia Duodenalis

G.duodenalis as a kind of zoonotic parasitesis,mainly parasitizes in the the duodenum and jejunum that causes giardiasis,with diarrhea being the major symptom.Giardia spp.are zoonotic parasites that greatly impact the health of humans and other mammals..At present,the control of giardiasis mainly depends on anti-Giardia drugs,such as metronidazole,tinidazole and albendazole.However,because a detailed understanding of the molecular biological characteristics of Giardia and its regulatory mechanisms is lacking,few ideal vaccines can be applied clinically to control giardiasis.Because Giardia can be immortalized in vitro,and immortalization mainly depends on telomerase to replicate the telomere repeat sequence and maintain its length.Therefore,it is of great practical significance to study the telomere related functions of Giardia.As a special DNA protein complex,telomere structure of Giardia exists at the end of chromosome in nucleus,and its function mainly depends on the regulation of telomerase.Telomerase of Giardia consists of telomerase RNA（TR）,telomerase reverse transcriptase（TERT）and telomerase associated protein（TAP）.Among them,tert protein mainly includes two domains: telomerase RNA binding domain（TRBD）and reverse transcriptase domain（RT）.At present,there are few reports about TRBD interacting proteins.The isolation and identification of TRBD interacting proteins of Giardia is helpful to reveal the cellular and molecular biological characteristics of Giardia,as well as the mechanism of telomerase activity and cell cycle regulation.In addition,as a model organism,Giardia is in the middle of the transition from prokaryote to eukaryote,which is of great value in evolutionary biology research,so it has become an important research object of zoonotic protozoa.However,it is still unclear whether the genes contained in the binucleate of Giardia are identical or not.It is of great significance to study the co localization of telomerase interacting proteins in Giardia.In our research,we fisrt try to identify the TRBD-associated proteins by yeast double-hybrid screening and pulldown and coimmunoprecipitation（co-IP）assays,then we explored the location of the TRBD and its associated proteins.To ensure the function of TRBD-associated proteins in regulating telomerase,the viral vector-mediated hammerhead ribozyme was used to evaluate telomere length and telomerase activity.In addition,we also intends to analyze the related proteins or factors that may affect the interacting proteins through database prediction,so as to construct a complete protein regulatory network.The result showed that After amplifying the Yeast two-hybrid Library of G.duodenalis,we screened proteins involved in regulating the TRBD in a yeast two-hybrid system.NCBI BLAST software showed that the clone sequence（candidate 325 protein）was identical to that of Zinc Finger Domain（GL50803₂0802,ZFD）.To demonstrate interaction between the TRBD and ZFD in vitro,TERT and ZFD were amplified and cloned into plasmids,respectively.His-TRBD and GST-ZFD proteins were purified,pulled down and analyzed by Western blot（WB）assay.The results show indicated that His-TRBD bound directly to GST-ZFD in vitro but not to GST alone,which was consistent with the results of the two-hybrid analysis.293 T cells were transfected with the recombinant plasmids pc DNA3.1-MycRBD/pc DNA3.1-HA-ZFD,pc DNA3.1-Myc-TRBD,and pc DNA3.1-HA-ZFD.The result shown that both TRBD and ZFD can be expressed.Co-IP shown that TRBD bound directly to ZFD in vitro.The TRBD was fused to c SNAP（p TRBD-c SNAP）,and ZFD was fused to n SNAP（p ZFD-n SNAP）after TRBD and ZFD fragments was amplified by PCR.Co-expression of p TRBD-c SNAP and p ZFD-n SNAP vectors in 293 T cells resulted in fluorescence complementation,which was also observed in the G.duodenalis nucleus.The results showed that protein protein interaction（PPI）occurred in both nuclei of G.duodenalis.Identification the interaction domain of the PPI: The ZFD protein was divided into two different domain fragments,and the specific interaction position with TRBD was analyzed.The result shown that N-terminal domain can interact with TRBD,indicating the specific interaction sites of the interacting proteins.In vitro ribozyme cleavage of ZFD m RNA: A hammerhead ribozyme targeting ZFD was designed as Ham-ZFD according to the design principle of hammerhead ribozyme.After p C631-SNAP-ZFD was mixed with p C631-SNAP-Ham-ZFD,the results confirmed 84% knockdown of ZFD.It was shown that the hammerhead ribozyme can be used in further studies.Expression of ribozyme in G.duodenalis and its effect on the proliferation of G.duodenalis: After we knocked down ZFD expression in G.duodenalis by transfecting ZFD-targeting ribozymes,the cell density on the 5th day was only approximately 41% of that of wildtype cells.These data suggested that electroporation with Ham-ZFD in G.duodenalis may reduce cell growth.Then we analyized the telomerase activity by TRAP.Result indicating that the decrease in ZFD reduced telomerase activity in G.duodenalis.Moreover,the TL was analyized by RT-PCR.It is said that the TL in the Ham-ZFD group was 0.91 ± 0.07 relative to that in the control group TL.These results suggest that ZFD may be partly involved in TL control.Analysis of physical and chemical properties of ZFD protein and identification of interaction protein: the molecular simulation analysis of ZFD showed that the formula of ZFD was C 923 H 1438 N 274 O 295 S 15,the relative molecular weight was 21.6kd,the isoelectric point was 6.70,the lipolysis index of ZFD protein was 54.61,so the protein was predicted to be hydrophilic.In addition,ZF’D structure analysis may interact with GSB₄558 protein,and the interaction between the proteins was verified by pull-down.In summary,the studies successfully screened the TERT-interacting protein ZFD.Pull-down and immunoprecipitation confirmed the PPI.By divided ZFD protein into two different domain fragments,the specific interaction position with TRBD was analyzed.In addition,through the functional analysis,the theoretical and experimental basis for the establishment and improvement of the interaction network of the telomere related proteins of G.duodenalis was established at the molecular level.The completion of the experiment has laid a good foundation for the further study of G.duodenalis specific drugs and vaccine targets.