Identification Of Interacting Proteins Of Retinoid-related Orphan Nuclear Receptor Gamma In Hepg2Cells

Background and aimsThe retinoid-related orphan nuclear receptor gamma (RORγ) is highly expressed inliver, adipose tissue, skeletal muscle and thymus, and plays critical roles in regulation ofdevelopment, immune function and metabolism. It is believed that cellular functions arenearly always the result of the coordinated action of several proteins in macromolecularassemblies and pathways. Protein complexes are highly ordered, dynamic structurestranslating biological information into function. Protein complex composition varies withtime and space to adapt to changing cellular requirements, and the analysis ofprotein-complex composition is considered an important step in the genotype-to-phenotypeintegration process. Therefore, we could infer that RORγ protein had its interacting partnersto form a functional protein complex to regulate the downstream biological process. Thoughseveral proteins have been linked to interplay with RORγ, they are not identified in a singleexperiment, indicating the limitations of the previous methods. Thus, to further investigatethe interacting proteins of RORγ, especially in a complex format that involves in RORγtranscriptional regulation, the tandem affinity purification (TAP) strategy was used tocapture the endogenous RORγ complex and the interplaying proteins of RORγ weredissected further in stably-transfected HepG2cells.Methods1. Cloning of RORγ gene from cultured HepG2cellsTotal RNA was extracted from HepG2cells, converted to complementary DNA(cDNA), and used as template to amplify the RORγ gene with RORγ-specific primers. PCRparameters were set as follows:30cycles of98oC for15sec,68oC for10sec,72oC for90sec. The PCR was then subcloned into the pMD19-T vector and verified bydouble-digestion with EcoR I and Hind III restriction enzymes and sequencing. 2. Construction of the pCeMM RORγ-CTAP(SG) plasmidThe RORγ cDNA was amplified from the pMD19-T RORγ plasmid by using restrictionenzymes specific primers. The amplified product was cloned into the EcoR I and Pme I sitesof pCeMM CTAP(SG) to construct the pCeMM RORγ-CTAP(SG) plasmid. To obtain astable transfected cell line selected by puromycin, the RORγ-CTAP(SG) or CTAP(SG)fragments were then cloned into the Xho I and EcoR I sites of pMSCVpuro vector. Theresultant plasmids were designated as pMSCVpuro RORγ-CTAP(SG) and pMSCVpuroCTAP(SG). All constructs were confirmed by sequencing.3. Cell culture, siRNA and transfectionHepG2cell line was cultured in the complete Dulbecco's modified Eagle's medium.The siRNA duplexes targeting RORγ, RIP140and HSP90corresponding to the respectivecoding regions were designed and synthesized by Invitrogen. Transfections were performedusing Lipofectamine2000(Invitrogen, USA) according to the manufacturer's instructions.For transient transfection, cells were collected24hr later. For stable transfection, cells wereselected36hr later by addition of1.5μg/ml puromycin to the complete culture medium,followed by refreshing the selective complete culture medium every two to three days.4. Western-blot analysisEqual volumes with equal protein concentration of samples were separated by4-12%NuPAGE gel electrophoresis (Invitrogen), then transferred onto polyvinylidene fluoridemembranes (PVDF) for immunoblotting. After blocking, the membranes were incubatedwith one of the following primary antibodies: anti-RORγ, SBP, protein G, RIP14, RFXDC1,HSP90, ZNF21, DOC2, Sp5, EMG1or Tubulin antibody. After1hr incubation at roomtemperature with the appropriate horseradish peroxidase (HRP)-conjugated secondaryantibody, specific protein bands on the membranes were visualized by the Super SignalWest Femto kit (Pierce Chemical, USA) according to the manufacturer's instructions.5. Immunofluorescence stainingpMSCVpuro RORγ-CTAP(SG) and pMSCVpuro CTAP(SG) transfected HepG2cellswere cultured on coated cover slides in culture medium. Cells were fixed, permeabilized,blocked, and incubated with mouse anti-SBP tag monoclonal antibody overnight at4°C.After washing, the cells were incubated for1hr at room temperature in the presence of goatanti-mouse CY3secondary antibody, followed by counterstaining with the nuclear probe DAPI and observation under fluorescence microscope.6. Tandem affinity purificationThe lysates of HepG2(5×108) cells were cleared by ultracentrifugation and thesupernatant was incubated with200μl IgG beads at4°C for4hr. After washing, the boundproteins were subjected to TEV-protease cleavage. The TEV-protease cleavage product wasincubated at4°C for2hr with100μl Streptavidin beads (Ultralink ImmobilizedStreptavidin Plus; Pierce). The final bound protein samples were eluted and run on a Readygel (Invitrogen, USA), and detected by silver-staining.7. Mass spectrometryProtein bands were excised from the SDS-PAGE gel and digested by trypsin (Promega,USA). Peptides were dissolved in0.1%formic acid and analyzed by HPLC-IonTrap-Chip-MS/MS (Agilent6300Series, USA). The mobile phase consisted of solvents A(water with0.1%formic acid) and solvents B (90%ACN,10%water with0.1%formicacid). After being treated with solvents A, the column was developed with a biphasicgradient of solvent B from5-15%over2min, followed by increases from15-40%over15min, up to70%over25min, and up to90%over35min. The nano-pump flow rate wasfixed at0.3μl/min, and the capillary-pump flow rate was fixed at3μl/min. The total analysistime was35min. Peptide and protein identifications were run automatically using theaccompanying Spectrum Mill software from Agilent (RevA.03.03.078).8. ImmunoprecipitationCells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50mM Tris-HCl,pH7.4,1%NP-40,0.1%sodium deoxycholate,150mM NaCl,1mM EDTA,1×CompleteProtease Inhibitor Cocktail tablet) and clarified lysates were precleared for1hr at4°C withProtein A/G Plus Agarose (Santa Cruz). Precleared lysates (1mg) were then incubatedovernight at4°C with4μg of control mouse IgG (Santa Cruz) and mouse anti-human RORγantibody (Abcam, USA); the following day, a2hr incubation was carried out with ProteinA/G Plus beads and four washes with RIPA buffer. Proteins remaining bound to the beadswere then analyzed by Western blot.9. Quantitative real-time PCRTotal RNA of HepG2cells was extracted by Trizol Reagent. The quantity of total RNAwas measured by NanoDrop (Agilent Technologies) and500ng of total RNA was used to synthesize cDNA with Reverse Transcription kit (TaKaRa, Japan) according to themanufacturer's instructions. GAPDH was used as endogenous control. Using specificprimers for CYP2C8(F5'-AGATCAGAATTTTCTCACCC-3'; R5'-AACTTCGTGTAAGAGCAACA-3'), PCR was carried out in25μl of total volume with0.5mM primers via aSyBr Green kit (TaKaRa) for40cycles on Rotor-Gene6000. We used the2-ΔΔCT methodfor quantifying expression relative to the GAPDH housekeeping control.Results1. Construct of pMSCVpuro RORγ-CTAP(SG) and nuclear localization ofRORγ-CTAP(SG) gene in stably-transfected HepG2cells1.1We cloned the RORγ cDNA from total mRNA of HepG2cells using a highfidelity DNA polymerase and constructed RORγ-CTAP (SG) fusion genes by cleavage andligation of the RORγ gene into the pCeMM CTAP(SG) vector.1.2The vector pCeMM RORγ-CTAP(SG) had no selectable antibiotics gene tofacilitate a stable transfected cell line, so we subcloned RORγ-CTAP(SG) or CTAP(SG)fragments into the pMSCVpuro vector to obtain the puromycin-resistant recombinantplasmid pMSCVpuro RORγ-CTAP(SG) or control pMSCVpuro CTAP(SG).1.3After the successful construction of pMSCVpuro RORγ-CTAP(SG), the plasmidspMSCVpuro RORγ-CTAP(SG) and pMSCVpuro CTAP(SG) were transiently transfectedinto HepG2cells and analyzed by Western Blot to determine whether RORγ were expressedcorrectly in HepG2cells. Our results demonstrated that the RORγ-CTAP(SG) fusion genewas able to be expressed in the pMSCVpuro RORγ-CTAP(SG)-transfected cells with theexpected molecular size (79kDa), as detected by anti-RORγ antibody. However, no suchband was observed in HepG2pMSCVpuro CTAP(SG)-transfected HepG2cells. Theendogenous RORγ protein expression was detected in all experimental cells (58kDa). Whenusing anti-PortG, RORγ-CTAP(SG) fusion protein and CTAP(SG)(21kDa) was able to bedetected in the corresponding plasmids-transfected cells, and not in HepG2cells. Similarresults were observed when using SBP tag as the detecting ligand. The tubulin protein wasdetected as an internal control to reflect nuclear protein expression, and was found to beexpressed constitutively in all experimental cells.1.4RORγ exhibits a typical nuclear receptor domain structure by which it specificallybinds to ROREs in the nucleus to regulate gene transcription. Fusion of CTAP(SG) tags into the C-terminus of the RORγ gene might have affected the nuclear localization of RORγ inHepG2cells. Therefore, we checked whether the RORγ-CTAP(SG) fusion protein was stilllocalized intranuclearly in the transfected HepG2cells by detecting the SBP tag inimmunofluorescence assays. As expected, the RORγ-CTAP(SG) fusion protein showedexclusive nuclear localization that completely overlapped with DAPI staining; whereas, thenon-fused CTAP(SG) tags were present in both nucleus and cytoplasm. The above resultsfurther support the expected ability of this system to capture endogenous RORγ proteincomplexes in HepG2cells.2. Isolation and identification of RORγ protein complexes by TAP/MS2.1The acquisition of protein complexes by tandem affinity purification followed byMS analysis of the captured samples is an effective and efficient method to identifybiologically relevant macromolecular complexes. In our work, TAP was performed afterinduction of expression of RORγ-CTAP(SG) or its control CTAP(SG) tags instably-transfected HepG2cell lines. The purification yields from each step of the TAPprocedure were evaluated by Western blot analysis. Results showed that the initial totalinput contained RORγ-CTAP(SG) fusion protein. After being treated with TEV (tobaccoetch virus) protease, the RORγ-CTAP(SG) fusion protein was effectively cleaved intoRORγ-SBP, as observed in the Western blot assay of the final eluate.2.2The final eluates from pMSCVpuro RORγ-CTAP(SG)-or pMSCVpuroCTAP(SG)-transfected HepG2cells were separated by SDS-PAGE and stained with silverregents. Results demonstrated a total of eight bands that differed from the control.2.3These eight bands were excised and digested with trypsin, peptides were extracted,separated by nanoflow LC, and introduced into an ion-trap mass spectrometer. MS/MSanalysis and database searching of the sequenced peptides resulted in the identification of8proteins. Two representative MS/MS spectrum of the tryptic digest from band1and band3are the peptide sequence (K)GMSSHLNGQAR(T) and (R)TLTLVDTGIGMTK(A) whichmatched separately to RIP140and HSP90by database searching.3. Confirmation of the RORγ complex3.1There is no doubt that any interacting protein detected by the TAP method must beconfirmed by different assays. In this study, confirmation of the interaction was conductedby immunoprecipitating RORγ from total extracts, followed by Western blot assay to determine whether those newly-identified proteins were interacting with endogenous RORγprotein. Results showed that RIP140and HSP90proteins, rather than EMG1, ZNF21,DOC2A, Sp5and RFXDC1proteins, could be co-immunoprecipitated with RORγ protein,confirming the interaction among RIP140, HSP90and RORγ in the HepG2cells.3.2To determine whether RIP140and HSP90have functional relevance with RORγ,siRNA duplexes were designed for down-regulation of RIP140and HSP90. The ability ofthe siRNA duplexes to repress RIP140or HSP90protein expression was confirmed byWestern blot assay. An irrelevant siRNA was used as negative control for transfection, andtubulin was used as the internal standard for Western blot. Introduction of RORγ siRNA-2,RIP140siRNA-3and HSP90siRNA-1in HepG2cells resulted in a significant decrease ofRORγ, RIP140and HSP90protein levels, while other siRNA had less effect on the levels ofthese proteins. We then examined the effects of decreased RORγ, RIP140and HSP90protein expression via RORγ-transcribed CYP2C8gene in HepG2cells by quantitativeRT-PCR. A significant decrease in CYP2C8mRNA expression was detected afterdownregulation of RIP140or/and HSP90protein.Conclusions1. We cloned the RORγ cDNA and constructed RORγ-CTAP (SG) fusion genes bycleavage and ligation of the RORγ gene into the pCeMM CTAP(SG) vector. Then wesubcloned RORγ-CTAP(SG) or CTAP(SG) fragments into the pMSCVpuro vector to obtainthe puromycin-resistant recombinant plasmid pMSCVpuro RORγ-CTAP(SG) or controlpMSCVpuro CTAP(SG).2. We transfected the plasmids pMSCVpuro RORγ-CTAP(SG) and pMSCVpuroCTAP(SG) into HepG2cells and confirmed the expression of RORγ-CTAP(SG) andCTAP(SG) by Western Blot. We also confirmed the RORγ-CTAP(SG) fusion protein waslocalized intranuclearly in the transfected HepG2cells.3. We acquired the RORγ protein complexes by tandem affinity purification, and thenidentified eight proteins by MS/MS analysis and database searching from the silvered finalelute.4. We checked the newly identified seven RORγ-interacting proteins and found thatRIP140and HSP90proteins could be co-immunoprecipitated with RORγ protein,confirming the interaction among RIP140, HSP90and RORγ in the HepG2cells. Furthermore, a significant decrease in CYP2C8mRNA expression was detected afterdownregulation of RIP140or/and HSP90protein by using RNAi strategy.In conclusion, we have identified and verified RIP140and HSP90as the interactors ofRORγ in a complex format in HepG2cells and comfirmed that RIP140and HSP90canupregulate RORγ-mediated CYP2C8gene expression. These findings support thehypothesis that RORγ is a coregulator-dependent transcription factor and functions in acomplex. HSP90may act as a chaperone by binding to RORγ protein and helping RORγexecute its regulatory roles involved in liver metabolism and other biological events whileRIP140might function as a coactivator of RORγ in HepG2cells, which has to be clarifiedin the future.