Screening And Identification Of A Novel HTERT-interacting Protein

Background and goals:There is a close relationship between telomeres-telomerase and normal cellproliferation, differentiation and age-related diseases. Telomeres, the complex protein-DNAstructures at the end of linear chromosomes, play an important role in maintaining genomicstability and integrity. Telomeres shorten proportionally with the cell division andproliferation in normal human cells, which might eventually limit cell replication, resultingin replicative senescence. Telomerase, known as a unique cellular ribonucleoprotein (RNP)enzyme in tissue progenitor cells and cancer cells, synthesizes and elongates the telomericDNA sequences when its activity is alleviated. The telomerase activity decides themaintenance of telomeres’ length and thereby controls the replicative capacity of cells. Atpresent telomerase has shed light on a broad spectrum of research fields including normalageing, various forms of cancer, stem cells and age-related disease pathology such as heartdisease and diabetes. These crucial roles for telomerase highlight the need for betterunderstanding on the mechanisms of human telomerase structure and function and how thisenzyme contributes to human disease.The regulation of telomerase activity is fairly complicated. Human telomerase reversetranscriptase (hTERT) is not only the catalytic component of human telomerase, but alsothe rate-limiting determinant of telomerase activity. The regulation of telomerase activityoccurs at various levels, including transcription, mRNA splicing, maturation andmodifications of hTERT, assembly of the functional ribonucleoprotein telomeraseholoenzyme, transportation to functionally relevant nuclear compartments, and access ofthe enzyme to its substrates. All of these biology events need the protein-proteininteractions between hTERT and its interactive proteins. The precise actions of hTERT andits partners in the multistep processes of telomerase assembly and their potential functions in regulating telomerase activity and in the accessibility of telomerase to telomeres remainto be determined. The mass spectrometry technology has developed quickly in these yearswith the advantages of high accuracy and speed. For these reasons theCo-immunoprecipitation combined with mass spectrometry-based proteomics technologyemerging as a powerful approach has been widely used to search for new protein-proteininteractions physiologically in recent years. In this study, protein complex of hTERT werepurified and identified by Co-immunoprecipitation (Co-IP) combinded High PressureLiquid Chromatography, Chip Hermeticity In Plastics, Mass Spectrum (HPLC-CHIP-MS-MS)and a novel hTERT interacting partner called Protocadherin10(PCDH10) was discovered.Primary research has also focused on the effect of the interaction between PCDH10andhTERT on telomerase activity and biological behavior of tumor cell such as tumor cellproliferation, adhesion, migration and invasion. Our study confirmed that this approach ishigh-performance and accurate.Methods and Results：1. Screening of hTERT interacting proteins using Co-IP technology and massspectrometric analysis1.1Separation and identification of hTERT immunoprecipitate complexThe fresh normal testis tissues used in this study were obtained from the operationpatients undergoing cancer of prostate in the department of urology in Southwest Hospitaland identified by pathology. Through Co-IP Kit, the total protein was obtained by using IPLysis/Wash Buffer and the hTERT immunoprecipitate complex was purified via Co-IP withspecific hTERT antibody. Western Blotting analysis suggested hTERT exsited in theimmunoprecipitate complex, and simultaneously the complex was separated by SDS-PAGEand stained by coomassie brillian blue. Followed by in-gel digestion of the target specificband, the peptide was analyzed through HPLC-CHIP-MS-MS. The peptide massfingerprints were searched by Swiss-Prot protein database and PCDH10was obtained as anovel hTERT interacting protein under the valid model.1.2Identification of interaction between hTERT and PCDH10by Co-IPThe total proteins of testis tissue were immunoprecipitated with anti-hTERT and detected with anti-PCDH10, and were immunoprecipitated with anti-PCDH10and detectedwith anti-hTERT separately. PCDH10was detected by anti-PCDH10in theimmunoprecipitates precipitated with anti-hTERT and hTERT was detected by anti-hTERT in the immunoprecipitates precipitated with anti-PCDH10. The results confirmedthe interaction between hTERT and PCDH10, which certified the validity of the MS result.2. The regulation role of the interaction between PCDH10and hTERT on telomerasefunctions2.1Construction of recombinant adenovirus encoding human PCDH10geneThe full length of the coding region of PCDH10was amplified by RT-PCR from testistissue and inserted into the MCS KpnI and XbaI of adenovirus shuttle vectorpAdTrack-CMV by T4ligase to generate the recombined shuttle plasmid. Linearized byPmeI, the shuttle plasmid pAdTrack-CMV-PCDH10and adenovirus genome-containingpAdEasy-1plasmid underwent homologous recombination into E.coli BJ5183cells, and therecombinant adenovirus plasmid pAdEasy-PCDH10was constructed successfully. At lastthe recombinant adenovirus was packed and propagated after pAdeasy-PCDH10wastransfected into293T cells. The high purity adenovirus was obtained after5passages andcalled Ad-PCDH10.2.2The effect of PCDH10over expression on telomerase activity of HS776T cellsHuman pancreatic cancer cells HS776T were transfected with the Ad-PCDH10andthe empty Ad vector (the untransfected cells as a control). After the transfection ofPCDH10, the mRNA and protein expression levels of PCDH10were higher compared withthe control (P<0.01). But the Real-Time PCR and Western Blotting analyses showed thatPCDH10had no effect on mRNA and protein level of the endogenous hTERT (P>0.05).hTERT was detected in the immunoprecipitates with anti-PCDH10of PCDH10-transfectedcells by Western Blotting and the telomerase activity of PCDH10-transfected cells wasdecreased obviously by TRAP analyses compared with control(P<0.01). Taken together,these data indicated that the over-expression of PCDH10could be associated physicallywith telomerase through interaction with hTERT in vivo and that this association couldinduce significant reduction in the telomerase activity. 3. The effect of PCDH10over expression on proliferation, cell cycle, migration andinvasion of HS776T cellsHuman pancreatic cancer cells HS776T were transfected with the Ad-PCDH10andthe empty Ad vector (the untransfected cells as a control).3.1The effect of PCDH10over expression on proliferationAfter the transfection of PCDH10at24h,48h,72h,96h,120h and144h, the CCK-8assay showed that there was no difference in the cell proliferation between theuntransfected cells and the transfected cells with the empty Ad vector (P>0.05). After thetransfection of PCDH10, the cell proliferation descended gradually with the increasing oftime and the relative ability of cell proliferation deceased by12.73%at96h (P<0.05),19.87%at120h (P<0.01) and30.14%at144h (P=0.000), respectively compared withcontrol.3.2The effect of PCDH10over expression on cell cycleAfter the transfection of PCDH10at72h, the flow cytometry assay (FCM) showed thecell cycle was no difference in the transfected cells with Ad-PCDH10compared with theunintransfected cells and the intransfected with the empty Ad vector (P>0.05), whichsuggested that PCDH10has no effect on the cell cycle..3.3The effect of PCDH10over expression on cell migrationAfter the transfection of PCDH10at24h and48h, the wound migration assay showedthere was no difference in the cell migration between the untransfected cells and thetransfected with the empty Ad vector (P>0.05), which suggested that the empty Ad vectorhas no effect on the cell migration. After the transfction of PCDH10, the relative ability ofcell migration deceased by30.07%at24h (P=0.000) and52.95%at96h (P=0.000)compared with control, which suggested that PCDH10could significantly inhibit the abilityof cell migration.3.4The effect of PCDH10over expression on cell invasionAfter the transfection of PCDH10at24h, the Transwell chamber assay analyses therewas no difference in the trans-membrane cell count between the untransfected cells and thetransfected with the empty Ad vector (P>0.05), which suggested that the empty Ad vector has no effect on the cell invasion. After the transfection of PCDH10, the celltrans-membrane count descended and the relative ability of cell invasion deceased by58.56%(P=0.000) compared with control, which suggested that PCDH10couldsignificantly inhibit the ability of cell invasion.Conclusions:In summary, this is the first time to screen hTERT interacting proteins in the normaltestis tissue and PCDH10was identified as a novel hTERT partner. Moreover we confirmedthere was a real interaction between PCDH10and hTERT and then further found thisinteraction was involved in the regulation process of the activity of telomerase.Up-regulated expression of PCDH10could significantly inhibit the activity of telomerase.To move forward a single step, we clarified the over-expression of PCDH10couldsignificantly inhibit the ability of proliferation, migration and invasion of HS776T cells.To our best knowledge, there is no similar report about PCDH10and telomerase yet.The tumor suppressor gene (TSG) PCDH10was first reported as a negative factor oftelomerase and these findings lay a foundation for exploring the regulatory roles of otherPCDH10-associated proteins on telomerase function, which will open a new window for theinvestigation concerning the regulatory pathways of telomerase. And further we have raised ahypothesis that the absence of PCDH10because of tumorspecific promoter methylation in multiplecarcinomas and cells resulted in a weakened interaction with hTERT, which weakens the inhibitionto the activity of telomerase by their interaction and leads to the proliferation, migration andinvation of tumor cells; on the contrary, the over-expession of PCDH10resulted in a enhancedinteraction with hTERT, which strengthens the inhibition to the activity of telomerase by theirinteraction and futher inhibited the proliferation, migration and invation of tumor cells. This may bea crucial mechanism of tumor suppression of PCDH10, which suggests PCDH10as an inhibitor oftelomerase may be a new potential targets in anti-cancer therapeutic, which could provide apivotal thread and clue for the development, prevention, diagnosis and therapy of age-relateddiseases and tissue-engineering applications.