Agonist-specific Protein Interactomes of Glucocorticoid and Androgen Receptor as Revealed by Proximity Mapping
2017; Elsevier BV; Volume: 16; Issue: 8 Linguagem: Inglês
10.1074/mcp.m117.067488
ISSN1535-9484
AutoresJoanna K. Lempiäinen, Einari A. Niskanen, Kaisa-Mari Vuoti, Riikka Lampinen, Helka Göös, Markku Varjosalo, Jorma J. Palvimo,
Tópico(s)Biotin and Related Studies
ResumoGlucocorticoid receptor (GR) and androgen receptor (AR) are steroid-inducible transcription factors (TFs). The GR and the AR are central regulators of various metabolic, homeostatic and differentiation processes and hence important therapeutic targets, especially in inflammation and prostate cancer, respectively. Hormone binding to these steroid receptors (SRs) leads to DNA binding and activation or repression of their target genes with the aid of interacting proteins, coregulators. However, protein interactomes of these important drug targets have remained poorly defined. We used proximity-dependent biotin identification to map the protein interaction landscapes of GR and AR in the presence and absence of their cognate agonist (dexamethasone, 5α-dihydrotestosterone) and antagonist (RU486, enzalutamide) in intact human cells. We reproducibly identified more than 30 proteins that interacted with the GR in an agonist-specific manner and whose interactions were significantly influenced by the DNA-binding function of the receptor. Interestingly, the agonist-dependent interactome of the GR overlapped considerably with that of the AR. In addition to known coactivators, corepressors and components of BAF (SWI/SNF) chromatin-remodeling complex, we identified a number of proteins, including lysine methyltransferases and demethylases that have not been previously linked to glucocorticoid or androgen signaling. A substantial number of these novel agonist-dependent GR/AR-interacting proteins, e.g. BCOR, IRF2BP2, RCOR1, and TLE3, have previously been implicated in transcription repression. This together with our data on the effect of BCOR, IRF2BP2, and RCOR1 on GR target gene expression suggests multifaceted functions and roles for SR coregulators. These first high confidence SR interactomes will aid in therapeutic targeting of the GR and the AR. Glucocorticoid receptor (GR) and androgen receptor (AR) are steroid-inducible transcription factors (TFs). The GR and the AR are central regulators of various metabolic, homeostatic and differentiation processes and hence important therapeutic targets, especially in inflammation and prostate cancer, respectively. Hormone binding to these steroid receptors (SRs) leads to DNA binding and activation or repression of their target genes with the aid of interacting proteins, coregulators. However, protein interactomes of these important drug targets have remained poorly defined. We used proximity-dependent biotin identification to map the protein interaction landscapes of GR and AR in the presence and absence of their cognate agonist (dexamethasone, 5α-dihydrotestosterone) and antagonist (RU486, enzalutamide) in intact human cells. We reproducibly identified more than 30 proteins that interacted with the GR in an agonist-specific manner and whose interactions were significantly influenced by the DNA-binding function of the receptor. Interestingly, the agonist-dependent interactome of the GR overlapped considerably with that of the AR. In addition to known coactivators, corepressors and components of BAF (SWI/SNF) chromatin-remodeling complex, we identified a number of proteins, including lysine methyltransferases and demethylases that have not been previously linked to glucocorticoid or androgen signaling. A substantial number of these novel agonist-dependent GR/AR-interacting proteins, e.g. BCOR, IRF2BP2, RCOR1, and TLE3, have previously been implicated in transcription repression. This together with our data on the effect of BCOR, IRF2BP2, and RCOR1 on GR target gene expression suggests multifaceted functions and roles for SR coregulators. These first high confidence SR interactomes will aid in therapeutic targeting of the GR and the AR. The glucocorticoid receptor (GR)1 and androgen receptor (AR) are hormone-activated transcription factors that belong to the steroid receptor (SR) subfamily of nuclear receptors (NRs). GR mediates the effects of glucocorticoids in a plethora of fundamental biological processes in the human body, such as metabolism, cell proliferation, development, inflammation, and immune responses (1.Ramamoorthy S. Cidlowski J.A. Corticosteroids: Mechanisms of action in health and disease.Rheum. Dis. Clin. North Am. 2016; 42: 15-31Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 2.Vandevyver S. Dejager L. Libert C. Comprehensive overview of the structure and regulation of the glucocorticoid receptor.Endocr. Rev. 2014; 35: 671-693Crossref PubMed Scopus (157) Google Scholar, 3.Weikum E.R. Knuesel M.T. Ortlund E.A. Yamamoto K.R. Glucocorticoid receptor control of transcription: Precision and plasticity via allostery.Nat. Rev. Mol. Cell Biol. 2017; 18: 159-174Crossref PubMed Scopus (274) Google Scholar). Synthetic glucocorticoid agonists are widely used pharmaceuticals because of their potent anti-inflammatory and anti-immune effects (4.Kadmiel M. Cidlowski J.A. Glucocorticoid receptor signaling in health and disease.Trends Pharmacol. Sci. 2013; 34: 518-530Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Androgens and the AR are imperative for the development, differentiation and function of male reproductive organs and they also regulate sexually dimorphic characteristics and processes in nongenital tissues, including development of muscle strength (5.Bardin C.W. Catterall J.F. Testosterone: A major determinant of extragenital sexual dimorphism.Science. 2005; 211: 1285-1294Crossref Scopus (294) Google Scholar, 6.Gao W. Bohl C.E. Dalton J.T. Chemistry and structural biology of androgen receptor.Chem. Rev. 2015; 105: 3352-3370Crossref Scopus (399) Google Scholar). The AR is also an important drug target (7.Watson P.A. Arora V.K. Sawyers C.L. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer.Nat. Rev. Cancer. 2015; 15: 701-711Crossref PubMed Scopus (776) Google Scholar). Synthetic AR antagonists, antiandrogens, such as enzalutamide, are widely used in the treatment of metastatic prostate cancer (8.Scher H.I. Fizazi K. Saad F. Taplin M.E. Sternberg C.N. Miller K. de Wit R. Mulders P. Chi K.N. Shore N.D. Armstrong A.J. Flaig T.W. Flechon A. Mainwaring P. Fleming M. Hainsworth J.D. Hirmand M. Selby B. Seely L. de Bono J.S. AFFIRM Investigators Increased survival with enzalutamide in prostate cancer after chemotherapy.N. Engl. J. Med. 2012; 367: 1187-1197Crossref PubMed Scopus (3395) Google Scholar). The GR and the AR, like all NRs, consist of three main functional domains: The N-terminal transactivation domain (NTD), the central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD). Steroid binding to the LBD causes a conformational change, allowing the SR to homodimerize and translocate into the nucleus. In the nucleus, the SR binds to palindromic DNA motifs, steroid response elements (SREs). The SRE recognition is mediated by two zinc fingers of the DBD which is the most conserved domain among the NRs (9.Helsen C. Claessens F. Looking at nuclear receptors from a new angle.Mol. Cell. Endocrinol. 2014; 382: 97-106Crossref PubMed Scopus (102) Google Scholar). The SREs reside often on distal enhancers where the SRs cooperate with other transcription factors to activate target gene transcription, whereas direct DNA-binding of SRs seems to be less frequently involved in repression of SR target genes (10.Kassel O. Herrlich P. Crosstalk between the glucocorticoid receptor and other transcription factors: Molecular aspects.Mol. Cell. Endocrinol. 2007; 275: 13-29Crossref PubMed Scopus (222) Google Scholar). The DBDs of SRs are conserved to an extent allowing the AR and the GR but also other 3-keto SRs, mineralocorticoid receptor and progesterone receptor, to recognize the same canonical androgen/glucocorticoid/progesterone/mineralocorticoid response element (ARE/GRE/PRE/MRE), whereas estrogen receptor (ER) α and β bind to a different element (11.Cotnoir-White D. Laperriere D. Mader S. Evolution of the repertoire of nuclear receptor binding sites in genomes.Mol. Cell. Endocrinol. 2011; 334: 76-82Crossref PubMed Scopus (47) Google Scholar). Moreover, the AR and the GR share a significant amount of chromatin binding sites (12.Sahu B. Laakso M. Pihlajamaa P. Ovaska K. Sinielnikov I. Hautaniemi S. Janne O.A. FoxA1 specifies unique androgen and glucocorticoid receptor binding events in prostate cancer cells.Cancer Res. 2013; 73: 1570-1580Crossref PubMed Scopus (163) Google Scholar, 13.Pihlajamaa P. Sahu B. Janne O.A. Determinants of receptor- and tissue-specific actions in androgen signaling.Endocr. Rev. 2015; 36: 357-384Crossref PubMed Scopus (83) Google Scholar), and the shared binding sites are associated with genes that are regulated by both androgens and glucocorticoids (12.Sahu B. Laakso M. Pihlajamaa P. Ovaska K. Sinielnikov I. Hautaniemi S. Janne O.A. FoxA1 specifies unique androgen and glucocorticoid receptor binding events in prostate cancer cells.Cancer Res. 2013; 73: 1570-1580Crossref PubMed Scopus (163) Google Scholar). However, also AR-selective binding sites not recognized by the GR exist (14.Denayer S. Helsen C. Thorrez L. Haelens A. Claessens F. The rules of DNA recognition by the androgen receptor.Mol. Endocrinol. 2010; 24: 898-913Crossref PubMed Scopus (112) Google Scholar). The NTD of SRs contains the ligand-independent transcription activation function 1 (AF1) required for the maximal transcriptional activity of the SRs, and the LBDs contain the second transcription activation function (AF2) that is ligand-dependent (15.Warnmark A. Treuter E. Wright A.P. Gustafsson J.A. Activation functions 1 and 2 of nuclear receptors: Molecular strategies for transcriptional activation.Mol. Endocrinol. 2003; 17: 1901-1909Crossref PubMed Scopus (194) Google Scholar). Transcriptional regulation by NRs requires, in addition to RNA polymerase II and general TFs, several NR-interacting proteins, coregulators. The coregulators regulate transcription through a variety of functions, such as chromatin remodeling, histone-binding, and post-translational modification of histones and other proteins (16.Millard C.J. Watson P.J. Fairall L. Schwabe J.W. An evolving understanding of nuclear receptor coregulator proteins.J. Mol. Endocrinol. 2013; 51: T23-T36Crossref PubMed Scopus (66) Google Scholar, 17.Meier K. Brehm A. Chromatin regulation: How complex does it get?.Epigenetics. 2014; 9: 1485-1495Crossref PubMed Scopus (81) Google Scholar). Initially, coactivators, such as NCOA1, NCOA2, and NCOA3 (SRC-1, SRC-2, and SRC-3), were thought to be recruited by hormone-bound nuclear receptors to enhance gene expression and corepressors, e.g. NCOR1 (N-CoR) and NCOR2 (SMRT), in turn by nonliganded or antagonist-bound nuclear receptors to repress gene expression (18.Collingwood T.N. Urnov F.D. Wolffe A.P. Nuclear receptors: Coactivators, corepressors and chromatin remodeling in the control of transcription.J. Mol. Endocrinol. 1999; 23: 255-275Crossref PubMed Scopus (268) Google Scholar, 19.Perissi V. Rosenfeld M.G. Controlling nuclear receptors: The circular logic of cofactor cycles.Nat. Rev. Mol. Cell Biol. 2005; 6: 542-554Crossref PubMed Scopus (402) Google Scholar, 20.Dasgupta S. O'Malley B.W. Transcriptional coregulators: Emerging roles of SRC family of coactivators in disease pathology.J. Mol. Endocrinol. 2014; 53: R47-R59Crossref PubMed Scopus (43) Google Scholar). Most of the NR coregulators were originally identified through genetic screens with LBDs as baits in yeast (yeast two-hybrid systems), a milieu that does not normally express any NR or homologue. The early studies focused on coregulator interactions with the AF2 domain in the NR LBD (21.Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma.Nature. 1998; 395: 137-143Crossref PubMed Scopus (1681) Google Scholar). Interactions of full-length NRs with other proteins are still relatively ill-defined and, proteomics-based NR interactomes, including those of the AR and the GR, have remained surprisingly poorly defined (22.Khorasanizadeh S. Rastinejad F. Visualizing the architectures and interactions of nuclear receptors.Endocrinology. 2016; 157: 4212-4221Crossref PubMed Scopus (25) Google Scholar). The latter may be because of transient nature of interactions and challenges in solubility and solubilization of chromatin-associated proteins when using traditional proteomic methods, such as affinity purification coupled to mass spectrometry (AP-MS) (23.Lambert J.P. Pawson T. Gingras A.C. Mapping physical interactions within chromatin by proteomic approaches.Proteomics. 2012; 12: 1609-1622Crossref PubMed Scopus (15) Google Scholar, 24.Lambert J.P. Tucholska M. Pawson T. Gingras A.C. Incorporating DNA shearing in standard affinity purification allows simultaneous identification of both soluble and chromatin-bound interaction partners.J. Proteomics. 2014; 100: 55-59Crossref PubMed Scopus (25) Google Scholar). In this work, we applied proximity-dependent biotin identification (BioID) to reveal the agonist-specific protein-protein interactome of the full-length GR and that of the full-length AR. We fused the GR and the AR with a mutated form of the E. coli biotin ligase (BirA*) that freely attaches biotin to primary amines within 10 nm of the ligase (25.Choi-Rhee E. Schulman H. Cronan J.E. Promiscuous protein biotinylation by escherichia coli biotin protein ligase.Protein Sci. 2004; 13: 3043-3050Crossref PubMed Scopus (158) Google Scholar, 26.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar, 27.Kim D.I. Birendra K.C. Zhu W. Motamedchaboki K. Doye V. Roux K.J. Probing nuclear pore complex architecture with proximity-dependent biotinylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E2453-E2461Crossref PubMed Scopus (301) Google Scholar). Biotin labeling in intact human cells exposed to hormonal agonist, antagonist or vehicle was followed by affinity purification and detection of the biotinylated proteins by MS. Approximately one third of the identified high-confidence agonist-specific GR-interacting proteins (10 of 33) have been previously identified as GR-interacting proteins (GR interactors) by other means. Interestingly, practically all of these interactions were dependent on intact DNA-binding function of the receptor, suggesting that they take place on chromatin. Moreover, the interactome of agonist-bound AR was in qualitative terms highly like that of the GR, and the interactomes of antagonist RU486-bound GR and antiandrogen enzalutamide-bound AR were largely devoid of the proteins interacting with the agonist-bound receptors. Overall, these first unbiased high confidence steroid receptor interactomes provide novel insights into the molecular mechanisms of GR and AR action, which can be applied in the pharmaceutical targeting of glucocorticoid and androgen signaling. For generation of the expression vector for tetracycline-inducible expression of N-terminally BirA*-tagged GR, AR and enhanced green fluorescence protein (EGFP), cDNA of the human GR isoform alpha (GR), human AR (AR), or EGFP were transferred with Gateway-cloning (Invitrogen, Carlsbad, CA) to the destination vector pcDNA5-FRT-TO-HA-BirA-GW. The GR DNA-binding mutant R447A was generated by mutagenesis PCR using QuickChange II XL Site-Directed Mutagenesis Kit (forward primer, 5′-GCTGTAAAGTTTTCTTCAAAGCAGCAGTGGAAGGACAGCAC-3′; reverse primer, 5′-CTGTCCTTCCACTGCTGCTTTGAAGAAAACTTTACAGCTTC-3′) and transferred to the same vector. Flp-In 293 T-REx™ cells (Invitrogen) containing a single genomic FRT site and stably expressing the Tet repressor were grown in Dulbecco's modified Eagle medium (DMEM, Gibco, Invitrogen, 41965–039) supplemented with 10% (v/v) FBS, 25 U/ml penicillin, 25 μg/ml streptomycin, 100 μg/ml zeocin (Invitrogen), and 15 μg/ml blasticidin (Invitrogen) (antibiotics were excluded prior to transfection). For cell line generation, Flp-In 293 T-REx™ cells were cotransfected with pcDNA5-FRT-TO-HA-BirA-GR, pcDNA5-FRT-TO-HA-BirA-GR-R447A, pcDNA5-FRT-TO-HA-BirA-AR, or pcDNA5-FRT-TO-HA-BirA-EGFP expression plasmids together with pOG44 vector (Invitrogen) for coexpression of the Flp-recombinase using the TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI). Two days after transfection, the cells were selected with 50 μg/ml hygromycin-B (Invitrogen) and 15 μg/ml blasticidin for 3 weeks. Cells were grown in DMEM supplemented with 2.5% (v/v) charcoal-treated FBS (steroid-depleted medium) before treatments. In all experiments, hormone/antagonist concentrations were as follows: 100 nm dexamethasone (dex, Sigma-Aldrich, St. Louis, MO), 1 μm RU486 (mifepristone, Sigma-Aldrich), 100 nm 5α-dihydrotestosterone (DHT, Steraloids Inc., Newport, RI), 10 μm enzalutamide (MDV3100, Medeia Therapeutics Ltd., Kuopio, Finland) or vehicle (etoh). Cell harvesting, lysis and immunoblotting was performed as described (28.Rytinki M.M. Kaikkonen S. Sutinen P. Palvimo J.J. Analysis of androgen receptor SUMOylation.Methods Mol. Biol. 2011; 776: 183-197Crossref PubMed Scopus (16) Google Scholar). Anti-GR (sc-1003), anti-GAPDH (sc-25778), anti-Lamin B1 (sc-6216) and anti-CoREST (sc-376567) were from Santa Cruz Biotechnology (Dallas, TX), anti-IRF2BP2 (A303–190A), anti-BCOR (A301–673A), anti-RAI1 (A302–317A), and anti-NUP98 (A301–786A) were from Bethyl Laboratories (Montgomery, TX), anti-LSD1 (ab17721) was from Abcam (Cambridge, United Kingdom), and anti-AR as described (29.Karvonen U. Kallio P.J. Janne O.A. Palvimo J.J. Interaction of androgen receptors with androgen response element in intact cells. roles of amino- and carboxyl-terminal regions and the ligand.J. Biol. Chem. 1997; 272: 15973-15979Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). After primary antibody incubation and washes, membranes were incubated in blocking buffer containing horseradish peroxidase (HRP)-conjugated secondary antibody (Life Technologies, Carlsbad, CA) and detected with chemiluminescence reagent (Pierce, Thermo Fisher Scientific, Waltham, MA). For immunoblotting of the biotinylated proteins, membranes were blocked in 2.5% (w/v) BSA in PBS supplemented with 0.4% (v/v) Triton X-100 and incubated with streptavidin-HRP (Molecular Probes, Life Technologies). Confocal microscopy was used to analyze the cellular localization of the biotinylated proteins. Flp-In T-Rex 293 cells expressing BirA*-fused GR, GR-R447A or AR were seeded to coverslips and grown for 24 h. Medium was replaced with steroid-depleted medium and cells were induced with 0.03 μg/ml tetracycline for 24 h before fixing. Cells were treated with 50 μm biotin for 6 h before fixing. In the case of BirA*-GR and BirA*-GR-R447A cell lines, cells were additionally treated with dex, RU486, or vehicle for 6 h before fixing. For the BirA*-AR cell line, cells were treated with DHT or vehicle. Cells were washed with PBS, fixed with 4% (w/v) formaldehyde-PBS for 20 min, and permeabilized with permeabilization buffer (PBS supplemented with 0.1% [v/v] Triton X-100 and 0.5% [w/v] BSA) for 20 min. Coverslips were incubated in primary antibody in permeabilization buffer for 1 h, washed three times for 5 min with PBS, incubated in secondary antibody in permeabilization buffer for 1 h, and washed three times for 5 min with PBS. BirA*-GR, BirA*-GR-R447A and BirA*-AR were detected with anti-HA (MMS-101P, Nordic BioSite AB, Taby, Sweden), biotin with fluorescence-coupled streptavidin DyLight 488 (SA-5488, Vector Laboratories Inc., Burlingame, CA), and lamin with anti-Lamin B1 (sc-6216, Santa Cruz Biotechnology). Rhodamine Red-X (715-295-150, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and Cy5 (705-175-003, Jackson ImmunoResearch Laboratories Inc.) were used as secondary antibodies. After immunolabeling, coverslips were mounted with ProLong Diamond antifade reagent (Thermo Fisher Scientific) and imaged on Zeiss LSM 800 confocal microscope. For the functional validation of the novel coregulators, A549 cells were seeded to 6-well plates in steroid-depleted medium and transfected with 20 nm ON-TARGETplus SMARTpool siRNAs (Dharmacon) against BCOR (l-004584–01-0005), RAI1 (l-012295–00-0005), RCOR1 (l-014076–00-0005), KDM1A (l-009223–00-0005), IRF2BP2 (l-007177–02-0005) for 72 h using Lipofectamine RNAiMAX (Invitrogen) reagent, and cells exposed to dex or vehicle for 6 h before collecting. ON-TARGETplus Non Targeting Pool (GE Dharmacon, Lafayette, LA) was used as the control siRNA. GR-expressing HEK293 cells were transfected as above, but seeded to their regular growth medium 1 day prior to change to steroid-depleted medium. RNA extraction, cDNA synthesis and fold change calculations were done as previously described for the GR (30.Paakinaho V. Kaikkonen S. Makkonen H. Benes V. Palvimo J.J. SUMOylation regulates the chromatin occupancy and anti-proliferative gene programs of glucocorticoid receptor.Nucleic Acids Res. 2014; 42: 1575-1592Crossref PubMed Scopus (60) Google Scholar) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA levels for normalization. RT-qPCR primer sequences are available upon request. GR, AR or EGFP -expressing Flp-In 293 T-REx™ cells were grown for 72 h, washed with PBS and the medium was replaced with steroid-depleted medium. After growing in serum-depleted medium for 24 h, cells were induced with tetracycline (0.03 μg/ml, Sigma-Aldrich) for the next 18 h, after which biotin (50 μm, Sigma-Aldrich) with either vehicle, dex, RU486, DHT or enzalutamide was added for 6 h. The cells were washed with ice-cold PBS supplemented with 0.1 mm MgCl2 and 0.1 mm CaCl2, harvested in PBS supplemented with 1 mm EDTA, snap frozen in liquid nitrogen and stored at −70 °C until purification. For affinity purification, ∼1 × 108 cells were lysed in 3 ml of lysis buffer (50 mm Hepes-NaOH [pH 8.0], 5 mm EDTA [pH 8.0], 150 mm NaCl, 50 mm NaF, 0.5% Nonidet P-40, 1 mm PMSF, 1.5 mm Na3VO4, 0.1% SDS, 83 U/ml benzonase, and 0.1× protease inhibitor mixture from Sigma-Aldrich) and sonicated with a probe sonicator (Branson Digital Sonifier) in ice-water bath using eighteen 30 s bursts with 60 s pauses with 30% amplitude. The lysates were then centrifuged at 16,000 × g for 15 min at 4 °C, transferred to new tubes and centrifuged for an additional 10 min at 16,000 × g at 4 °C. The cleared lysates were loaded on spin columns (Bio-Rad Laboratories, Hercules, CA) containing 400 μl Strep-Tactin beads (2-1201-010, IBA Lifesciences, Goettingen, Germany) prewashed in lysis buffer and the beads were washed three times with 1 ml of wash buffer 1 (lysis buffer without SDS and benzonase) and four times with 1 ml of wash buffer 2 (50 mm Hepes-NaOH [pH 8.0], 5 mm EDTA [pH 8.0], 150 mm NaCl, 50 mm NaF). Proteins were eluted twice with 300 μl of 0.5 mm biotin in wash buffer 2, and frozen at −20 °C until further processing. Eluates were neutralized with 100 mm NH4HCO3. Cysteines were reduced with 5 mm Tris(2-carboxyethyl)phosphine and alkylated with 10 mm iodoacetamide. The proteins were then trypsinized to peptides by adding 1 μg trypsin (Promega, Madison, WI). After overnight incubation at 37 °C, samples were quenched with 10% trifluoroacetic acid, purified with C18 Micro SpinColumns (The Nest Group Inc., Southborough, MA) according to the manufacturer's instructions and re-dissolved in 30 μl buffer A (0.1% trifluoroacetic acid and 1% acetonitrile in LC-MS grade water). The MS analysis was performed on Orbitrap Elite hybrid mass spectrometer coupled to EASY-nLC II -system using the Xcalibur version 2.7.0 SP1 (Thermo Fisher Scientific). 4 μl of the tryptic peptide mixture was loaded into a C18-packed pre-column (EASY-Column™ 2 cm x 100 μm, 5 μm, 120 Å, Thermo Fisher Scientific) in 10 μl volume of buffer A and then to C18-packed analytical column (EASY-Column™ 10 cm x 75 μm, 3 μm, 120 Å, Thermo Fisher Scientific). Sixty-minute linear gradient at the constant flow rate of 300 nl/minute from 5 to 35% of buffer B (98% acetonitrile and 0.1% formic acid in MS grade water) was used to separate the peptides. Analysis was performed in data-dependent acquisition: one high resolution (60,000) FTMS full scan (m/z 300–1700) was followed by top20 CID- MS2 scans in ion trap (energy 35). Maximum FTMS fill time was 200 ms (Full AGC target 1,000,000) and the maximum fill time for the ion trap was 200 ms (MSn AGC target of 50,000). Precursor ions with more than 500 ion counts were allowed for MSn. To enable the high resolution in FTMS scan preview mode was used. Proteins were identified using Proteome Discoverer™ software with SEQUEST search engine (version 1.4, Thermo Scientific). Thermo .raw files were searched against the human component of the UniProt-database (release 2014_11; 20130 entries) complemented with trypsin, BSA, GFP and tag sequences. Trypsin was used as the enzyme specificity. Search parameters specified a precursor ion tolerance of 15 ppm and fragment ion tolerance of 0.8 Da, with up to two missed cleavages allowed for trypsin. Carbamidomethylation (+57.021464 Da) of cysteine residues was used as static modification whereas oxidation (+15.994491 Da) of methionine and biotinylation (+226.078 Da) of lysine residues or N terminus were used as dynamic modification. Peptide false discovery rate (FDR) was calculated using Percolator node of software and set to <0.01. Spectral counting was used to produce semiquantitative data. Identification metrics for each sample are listed in supplemental Table S1. For analyzing the effect of RU486 in GR target gene expression, GR-expressing HEK293 cells were seeded to 6-well plates and grown 24 h. Medium was replaced with steroid-depleted medium and the cells were grown for 48 h before collecting. Cells were treated with 100 nm dex, 1 μm RU486 or vehicle (ethanol) for 6 h before collecting. RT-qPCR analyses were otherwise performed as described for the siRNA experiments. RT-qPCR primer sequences are available upon request. Reporter gene assay for the GR DNA-binding mutant characterization was done similarly as described for the androgen receptor (31.Makkonen H. Jaaskelainen T. Rytinki M.M. Palvimo J.J. Analysis of androgen receptor activity by reporter gene assays.Methods Mol. Biol. 2011; 776: 71-80Crossref PubMed Scopus (11) Google Scholar, 32.Kaikkonen S. Paakinaho V. Sutinen P. Levonen A.L. Palvimo J.J. Prostaglandin 15d-PGJ(2) inhibits androgen receptor signaling in prostate cancer cells.Mol. Endocrinol. 2013; 27: 212-223Crossref PubMed Scopus (21) Google Scholar) with the following modifications. COS-1 cells on 12-well plates were cotransfected with pGRE4-tk-luc (Ikonen 1997; Tian 2002) reporter (100 ng/well) together with expression vectors encoding GR and GR-R447A (20 ng/well), and the cells were treated with 100 nm dexamethasone, 1 μm RU486 or vehicle (ethanol) before lysis in passive lysis buffer (Promega). pCMVβ (10 ng/well, Promega) was used for the transfection control. Luciferase and β-galactosidase activities were measured as described before (31.Makkonen H. Jaaskelainen T. Rytinki M.M. Palvimo J.J. Analysis of androgen receptor activity by reporter gene assays.Methods Mol. Biol. 2011; 776: 71-80Crossref PubMed Scopus (11) Google Scholar). Fluorescence recovery after photobleaching (FRAP) was used to study the mobility of the GR DNA-binding mutant. Flp-In T-Rex 293 cells were seeded to μ-slide 8-well chambers (Ibidi GmbH, Munich, Germany) and transfected with constructs expressing EGFP -tagged GR and GR-R447A. Cells were induced with 100 nm dex, and the nucleus was scanned using 488 nm excitation at 500-ms intervals with Zeiss LSM 700 microscope. After 10 scans, a high intensity bleach pulse at 488 nm was applied to a 1-μm wide rectangular area spanning the nucleus, and scanning of the nucleus was continued until equilibrium in fluorescence distribution was reached. The fluorescence recovery was analyzed from the bleached area. GR and AR-specific interactors from three biological replicates were discriminated from background contaminants by using 10 individual BirA*-EGFP control purifications as the control. Significance Analysis of INTeractome (SAINT) (33.Choi H. Larsen B. Lin Z.Y. Breitkreutz A. Mellacheruvu D. Fermin D. Qin Z.S. Tyers M. Gingras A.C. Nesvizhskii A.I. SAINT: Probabilistic scoring of affinity purification-mass spectrometry data.Nat. Methods. 2011; 8: 70-73Crossref PubMed Scopus (481) Google Scholar) V.2.5.0 with default settings was used to determine the statistical significance of the detected interactions. SAINT input and output files are in supplemental Table S2. Interactions with FDR < 0.05 were considered significant with the following exceptions: Acetyl-CoA carboxylase 2 (ACACB, endogenously biotinylated) (34.Chandler C.S. Ballard F.J. Distribution and degradation of biotin-containing carboxylases in human cell lines.Biochem. J. 1985; 232: 385-393Crossref PubMed Scopus (28) Google Scholar), keratins (KRT2, KRT5, KRT14) and trypsin (unspecific interactors), and tubulin beta-4A chain (TUBB4A, nonspecific mapping of peptides to different tubulin isoforms). To detect GR-interacting proteins with proximity-dependent biotin identification, we generated HEK293 flp-in T-REx™ cell lines that express BirA*-fused wild-type GR, a DBD-mutated GR (GR-R447A), or enhanced green fluorescent protein (EGFP, a control) in a tetracycline (tet)-inducible fashion (supplemental Fig. S1). The tet-induced expression of BirA*-GR that exceeded the level of endogenous GR in the HEK293 cells by ∼3-fold was used for further experiments. Immunoblotting confirmed biotinylation of endogenous proteins of various sizes when the BirA* -fused bait proteins were expressed in the presence of excess biotin (Fig. 1A). In the absence of ligand, b
Referência(s)