Identification of a Novel Partner of Duox
2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês
10.1074/jbc.m407709200
ISSN1083-351X
AutoresDantong Wang, Xavier De Deken, Milutin Milenkovic, Yue Song, Isabelle Pirson, Jacques E. Dumont, Françoise Miot,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoH2O2 is a crucial substrate of thyroproxidase (TPO) to iodinate thyroglobulin and synthesize thyroid hormones in thyroid. ThOX proteins (thyroid oxidase) also called Duox are believed to be responsible for H2O2 generation. Duoxs expressed in transfected cells do not generate an active system, nor permit their membrane localization suggesting that other proteins are required to fulfill these functions. In this study, we demonstrate interactions of Duoxs with TPO and with p22phox without any effect on Duox activity. By yeast two-hybrid method using EF-hand fragment of dog Duox1 as the bait we have isolated EFP1 (EF-hand binding protein 1), one partner of Duoxs that belongs to the thioredoxin-related protein family. EFP1 shares moderate similarities with other members of thioredoxin-related proteins, but the characteristic active site and the folding structures are well conserved. EFP1 can be co-immunoprecipitated with Duoxs in transfected COS cells as well as in primary cultured human thyrocytes. It interacts also with TPO but not thyroglobulin. Immunofluorescence studies show that EFP1 and Duox proteins are co-localized inside the transfected cells, suggesting that EFP1 is not sufficient to induce either the expression of Duox at the plasma membrane or to permit H2O2 production. EFP1 and Duox mRNA share similar distribution in nine different tissues. These results suggest that EFP1 could be one of the partners in the assembly of the multiprotein complex constituting the thyroid H2O2 generating system but is certainly not sufficient to permit H2O2 generation. H2O2 is a crucial substrate of thyroproxidase (TPO) to iodinate thyroglobulin and synthesize thyroid hormones in thyroid. ThOX proteins (thyroid oxidase) also called Duox are believed to be responsible for H2O2 generation. Duoxs expressed in transfected cells do not generate an active system, nor permit their membrane localization suggesting that other proteins are required to fulfill these functions. In this study, we demonstrate interactions of Duoxs with TPO and with p22phox without any effect on Duox activity. By yeast two-hybrid method using EF-hand fragment of dog Duox1 as the bait we have isolated EFP1 (EF-hand binding protein 1), one partner of Duoxs that belongs to the thioredoxin-related protein family. EFP1 shares moderate similarities with other members of thioredoxin-related proteins, but the characteristic active site and the folding structures are well conserved. EFP1 can be co-immunoprecipitated with Duoxs in transfected COS cells as well as in primary cultured human thyrocytes. It interacts also with TPO but not thyroglobulin. Immunofluorescence studies show that EFP1 and Duox proteins are co-localized inside the transfected cells, suggesting that EFP1 is not sufficient to induce either the expression of Duox at the plasma membrane or to permit H2O2 production. EFP1 and Duox mRNA share similar distribution in nine different tissues. These results suggest that EFP1 could be one of the partners in the assembly of the multiprotein complex constituting the thyroid H2O2 generating system but is certainly not sufficient to permit H2O2 generation. The biosynthesis of thyroid hormones by thyroid cells requires oxidation of iodide, its binding to tyrosines of thyroglobulin (Tg) 1The abbreviations used are: Tg, thyroglobulin; TPO, thyroproxidase; ThOX, thyroid oxidase; Trx, thioredoxin; TRP, thioredoxin-related protein; TMX, thioredoxin-related transmembrane protein; Nrx, nucleoredoxin; PDI, protein disulfide isomerase; ER, endoplasmic reticulum; aa, amino acids; DBD, DNA-binding domain; EFP1, EF-hand fragment partner 1; DEFP1, dog Duox EF-hand partner 1; IP, immunoprecipitation; mAb, monoclonal antibody; HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; MTN, multiple tissue Northern; CHO, Chinese hamster ovary; DEF, dog Duox fragment containing EF-hand domains; HEF, human Duox fragment containing EF-hand domains.1The abbreviations used are: Tg, thyroglobulin; TPO, thyroproxidase; ThOX, thyroid oxidase; Trx, thioredoxin; TRP, thioredoxin-related protein; TMX, thioredoxin-related transmembrane protein; Nrx, nucleoredoxin; PDI, protein disulfide isomerase; ER, endoplasmic reticulum; aa, amino acids; DBD, DNA-binding domain; EFP1, EF-hand fragment partner 1; DEFP1, dog Duox EF-hand partner 1; IP, immunoprecipitation; mAb, monoclonal antibody; HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; MTN, multiple tissue Northern; CHO, Chinese hamster ovary; DEF, dog Duox fragment containing EF-hand domains; HEF, human Duox fragment containing EF-hand domains. and the oxidative coupling of iodotyrosines into iodotyronines. These reactions are catalyzed by thyroperoxidase (TPO) in the follicular space close to the apical pole of the cells. H2O2 is a crucial substrate and limiting factor of TPO for the iodination reactions (1Taurog A. Braverman L.E. Utiger R.D. Werner and Ingbars's The Thyroid. Lippincott, Philadelphia1996: 61-85Google Scholar). The thyroid H2O2 generating system has been widely characterized in in vitro biochemical studies (2Bjorkman U. Ekholm R. Endocrinology. 1984; 115: 392-398Crossref PubMed Scopus (98) Google Scholar, 3Bjorkman U. Ekholm R. Mol. Cell. Endocrinol. 1995; 111: 99-107Crossref PubMed Scopus (54) Google Scholar, 4Corvilain B. Van S e J. Laurent E. Dumont J.E. Endocrinology. 1991; 128: 779-785Crossref PubMed Scopus (148) Google Scholar, 5Corvilain B. Collyn L. Van Sande J. Dumont J.E. Am. J. Physiol. 2000; 278: E692-E699Crossref PubMed Google Scholar, 6Raspe E. Dumont J.E. Endocrinology. 1995; 136: 965-973Crossref PubMed Scopus (54) Google Scholar, 7Raspe E. Laurent E. Corvilain B. Verjans B. Erneux C. Dumont J.E. J. Cell. Physiol. 1991; 146: 242-250Crossref PubMed Scopus (59) Google Scholar). The enzymatic activity has been purified as a Ca2+-dependent flavoprotein presenting an oxidase activity using NADPH as coenzyme (8Dupuy C. Deme D. Kaniewski J. Pommier J. Virion A. FEBS Lett. 1988; 233: 74-78Crossref PubMed Scopus (31) Google Scholar, 9Dupuy C. Kaniewski J. Deme D. Pommier J. Virion A. Eur. J. Biochem. 1989; 185: 597-603Crossref PubMed Scopus (49) Google Scholar, 10Dupuy C. Virion A. Ohayon R. Kaniewski J. Deme D. Pommier J. J. Biol. Chem. 1991; 266: 3739-3743Abstract Full Text PDF PubMed Google Scholar, 11Gorin Y. Ohayon R. Carvalho D.P. Deme D. Leseney A.M. Haye B. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar, 12Gorin Y. Leseney A.M. Ohayon R. Dupuy C. Pommier J. Virion A. Deme D. Biochem. J. 1997; 321: 383-388Crossref PubMed Scopus (23) Google Scholar, 13Leseney A.M. Deme D. Legue O. Ohayon R. Chanson P. Sales J.P. Pires D.C. Dupuy C. Virion A. Biochimie (Paris). 1999; 81: 373-380Crossref PubMed Scopus (55) Google Scholar). Recently the corresponding cDNA has been cloned starting from this purified enzymatic activity (14Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). It encoded a protein of 138 kDa, which was the truncated form of Duox2. At the same time, based on the functional homology existing between the leukocyte and the thyroid H2O2 generating systems, two cDNAs encoding two proteins of 1551 and 1548 amino acids, respectively, called ThOX1 and ThOX2 were cloned (15De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). These ThOX proteins belong to the family of NADPH oxidases and were later called Duox1/Duox2 because their expression is not restricted to the thyroid tissue and because they present peroxidase like domain at their N-terminal ends. In the thyroid Duox1/Duox2 show two N-glycosylated states: the fully glycosylated mature form (190 kDa) expressed at the plasma membrane and the high mannose glycosylated immature form (180 kDa) expressed exclusively inside the cell in the endoplasmic reticulum (16De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (155) Google Scholar, 17Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (65) Google Scholar). Duox cDNA-transfected cell lines express only the immature form of the protein and no H2O2 production can be detected (16De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (155) Google Scholar) suggesting the involvement of other proteins in the maturation process of Duoxs to permit the capacity of the oxidase system. It is well known that in the leukocyte gp91phox (also referred to as NOX2), the NADPH oxidase moiety of the superoxide anions generator, needs p22phox to be processed to the membrane (18Yu L. DeLeo F.R. Biberstine-Kinkade K.J. Renee J. Nauseef W.M. Dinauer M.C. J. Biol. Chem. 1999; 274: 4364-4369Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 19Yu L. Quinn M.T. Cross A.R. Dinauer M.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7993-7998Crossref PubMed Scopus (175) Google Scholar, 20Dahan I. Issaeva I. Gorzalczany Y. Sigal N. Hirshberg M. Pick E. J. Biol. Chem. 2002; 277: 8421-8432Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Moreover NOX2 is active only when several cytosolic components (p47phox, p67phox, Rac, and p40phox) are recruited to form a complex at the membrane (21DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (455) Google Scholar, 22Dang P.M. Cross A.R. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3001-3005Crossref PubMed Scopus (96) Google Scholar, 23Dang P.M. Cross A.R. Quinn M.T. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4262-4265Crossref PubMed Scopus (77) Google Scholar, 24Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 25Sarfstein R. Gorzalczany Y. Mizrahi A. Berdichevsky Y. Molshanski-Mor S. Weinbaum C. Hirshberg M. Dagher M.C. Pick E. J. Biol. Chem. 2004; 279: 16007-16016Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Recently homologs of p47phox and p67phox, NOXO1 and NOXA1 (also called p41nox and p51nox), have been found by several groups (24Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 26Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 27Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 28Geiszt M. Lekstrom K. Witta J. Leto T.L. J. Biol. Chem. 2003; 278: 20006-20012Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). These two proteins are able to activate NOX1, the NADPH oxidase responsible for O2˙− production mainly in the colon. The unsuccessful reconstitution of the H2O2-generating capacity in PLB-XCGD (gp91phox-knock-out PLB) cells with Duox1 and Duox2 suggests that other thyroid-specific factors are required to permit H2O2 production in thyroid (16De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (155) Google Scholar). The interaction of Duox with thyroid proteins was investigated by the yeast two-hybrid method to isolate potential partners that are able to play a physiological role in the maturation of protein leading to the generation of H2O2. The method allowed us to isolate a protein containing thioredoxin domains. Thioredoxins (Trx) are small proteins that were first identified in Escherichia coli. They are characterized by an evolutionary conserved active cysteine site, CGPC, which undergoes reversible oxidation of the two cysteine residues from dithiol to disulfide form (29Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 30Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). The maintenance of thioredoxin in its active reduced form is carried out by thioredoxin reductase in the presence of NADPH forming the thioredoxin system (29Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). In eukaryotic cells thioredoxin has been implicated in a wide variety of biochemical functions. It functions as a hydrogen donor and facilitates the folding of disulfide containing proteins (31Holmgren A. Methods Enzymol. 1984; 107: 295-300Crossref PubMed Scopus (118) Google Scholar). Thioredoxin is also an antioxidant participating in the reduction of H2O2, scavenging free radicals and protecting against oxidative stress (32Zhang P. Liu B. Kang S.W. Seo M.S. Rhee S.G. Obeid L.M. J. Biol. Chem. 1997; 272: 30615-30618Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 33Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (201) Google Scholar, 34Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). It can modulate the activity of some transcription factors, such as AP-1 (35Karimpour S. Lou J. Lin L.L. Rene L.M. Lagunas L. Ma X. Karra S. Bradbury C.M. Markovina S. Goswami P.C. Spitz D.R. Hirota K. Kalvakolanu D.V. Yodoi J. Gius D. Oncogene. 2002; 21: 6317-6327Crossref PubMed Scopus (104) Google Scholar) and NF-κB (36Das K.C. J. Biol. Chem. 2001; 276: 4662-4670Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). A number of mammalian proteins containing thioredoxin domains have been identified, including human Trx, Trx2 (37Collet J.F. D'Souza J.C. Jakob U. Bardwell J.C. J. Biol. Chem. 2003; 278: 45325-45332Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 38Damdimopoulos A.E. Miranda-Vizuete A. Pelto-Huikko M. Gustafsson J.A. Spyrou G. J. Biol. Chem. 2002; 277: 33249-33257Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), TRP32 (39Lee K.K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (thioredoxin-related protein), TRP14 (40Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), TMX (thioredoxin-related transmembrane protein) and TMX2 (41Matsuo Y. Akiyama N. Nakamura H. Yodoi J. Noda M. Kizaka-Kondoh S. J. Biol. Chem. 2001; 276: 10032-10038Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 42Meng X. Zhang C. Chen J. Peng S. Cao Y. Ying K. Xie Y. Mao Y. Biochem. Genet. 2003; 41: 99-106Crossref PubMed Scopus (29) Google Scholar), Nrx (nucleoredoxin) (43Laughner B.J. Sehnke P.C. Ferl R.J. Plant Physiol. 1998; 118: 987-996Crossref PubMed Scopus (43) Google Scholar, 44Kurooka H. Kato K. Minoguchi S. Takahashi Y. Ikeda J. Habu S. Osawa N. Buchberg A.M. Moriwaki K. Shisa H. Honjo T. Genomics. 1997; 39: 331-339Crossref PubMed Scopus (97) Google Scholar), and PDI (protein disulfide isomerase). The active sites of human Trx, Trx2, and TRP32 are identical to that of E. coli Trx, while modified active site sequences are found in TRP14 (CPDC), TMX (CPAC), Nrx (CPPC), and PDI (CGHC). Human Trx and TRP32 are localized in cytoplasm, Trx2 (also called mTrx), and Nrx are localized in the mitochondria and the nucleus respectively. TMX2 possesses only one cysteine at the active site (SNDC) and a predicted endoplasmic reticulum (ER) membrane retention signal, KKXX-like motif, and is likely localized in ER (42Meng X. Zhang C. Chen J. Peng S. Cao Y. Ying K. Xie Y. Mao Y. Biochem. Genet. 2003; 41: 99-106Crossref PubMed Scopus (29) Google Scholar). PDI has been known for many years to assist proteins containing disulfide bonds to fold in ER to attain their native structure. Studies also suggest that some PDIs are located in non-ER fractions and presumably have different functions (46Turano C. Coppari S. Altieri F. Ferraro A. J. Cell. Physiol. 2002; 193: 154-163Crossref PubMed Scopus (396) Google Scholar). Most of the thioredoxin domain-containing proteins are widely expressed in human tissues and have been proven to have Trx-like reducing activities in vitro by insulin disulfide reduction assay, but their precise physiological function in the cell remains to be defined (37Collet J.F. D'Souza J.C. Jakob U. Bardwell J.C. J. Biol. Chem. 2003; 278: 45325-45332Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 39Lee K.K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 40Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 43Laughner B.J. Sehnke P.C. Ferl R.J. Plant Physiol. 1998; 118: 987-996Crossref PubMed Scopus (43) Google Scholar, 44Kurooka H. Kato K. Minoguchi S. Takahashi Y. Ikeda J. Habu S. Osawa N. Buchberg A.M. Moriwaki K. Shisa H. Honjo T. Genomics. 1997; 39: 331-339Crossref PubMed Scopus (97) Google Scholar). In this study we report a novel thioredoxin-related protein EFP1 (EF-hand binding protein 1), which interacts with the intracellular part of Duox1 and Duox2 containing two EF-hand domains. It may play a critical role in the protein processing. Two-hybrid Screenings and Constructs—The cDNA fragment (DEF) corresponding to the first intracellular loop of dog Duox1 protein containing the two EF-hand domains (aa 616–1043, Fig. 1) was cloned by PCR downstream from the Gal4 DBD (DNA-binding domain) in the yeast two-hybrid vector pPC97. The construction was checked by sequencing confirming the correct reading frame of the fusion protein. A dog thyroid cDNA library constructed in pPC86 vector fused to Gal4 transcription-activating domain has been used for yeast two-hybrid screening (47Arsenijevic T. Degraef C. Dumont J.E. Roger P.P. Pirson I. Biochem. J. 2004; 378: 673-679Crossref PubMed Google Scholar). The yeast host strain pJ69–4A was used for the screening and the reconstruction steps as described previously (48Vandenbroere I. Paternotte N. Dumont J.E. Erneux C. Pirson I. Biochem. Biophys. Res. Commun. 2003; 300: 494-500Crossref PubMed Scopus (56) Google Scholar). For the screening, the pJ69–4A yeast containing Gal4DBD-DEF was transformed with the dog thyroid cDNA library mentioned above. The yeasts growing on medium without Leu and Trp (αLeuαTrp) were transformed with plasmids from the bait and the library. Such transformants were first selected on medium lacking His (αLeuαTrpαHis) and then on the medium lacking His and Ade (αLeuαTrpαHisαAde). DH10B bacteria were transformed by electroporation with the cDNAs coming from yeasts growing on αLeuαTrp αHisαAde medium. The potential partner, DEFP1 (dog Duox EF-hand partner 1), was isolated. Reconstruction was performed to verify the specificity of the interaction. The same procedure was applied for another construction HEF corresponding to the human EF-hand-containing fragment of Duox (aa 616–1043). Identification and Subcloning of Human cDNA-encoding DEFP1 Homologous Protein—Searches of non-redundant sequence and expressed sequence tag data bases with DEFP1 sequence yielded an unknown human protein (EFP1) sharing 74% identity with DEFP1. Simple modular architecture research tool (SMART) was used to perform hydropathy plot and motif analysis. The secondary structure was analyzed using the PredictProtein server. The cDNA in pME18SFL3 encoding 958 aa was purchased from Takara Biomedical Europe and subcloned into pcDNA3.1/HIS between EcoRI and NotI sites (Invitrogen). Demonstration of Interaction by Co-immunoprecipitation Experiments—2 × 105 COS cells were transfected with the HIS-tagged EFP1 or/and human Duox cDNAs using FugENE 6 (Roche Applied Science). Cells were harvested 48 h after transfection in Laemmli buffer (2% SDS, 100 mm dithiothreitol) to obtain total lysates, and with nondenaturing lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 0.02 mm sodium azide, and Complete protease inhibitor mixture (Roche Applied Science)) for immunoprecipitation (IP) experiments. Thyrocytes in primary culture were obtained from follicles isolated by collagenase digestion and differential centrifugation from fresh human thyroid as described previously (49Roger P.P. Dumont J.E. FEBS Lett. 1982; 144: 209-212Crossref PubMed Scopus (97) Google Scholar, 50Roger P.P. Dumont J.E. Biochem. Biophys. Res. Commun. 1987; 149: 707-711Crossref PubMed Scopus (29) Google Scholar). The thyrocytes were harvested after 4 days of culture in lysis buffer described above. After preclearing, the extracts were immunoprecipitated with 5 μl of anti-Duox or anti-TPO mAb15 antibodies that had been prelinked to 30 μl of 50% protein A-Sepharose (16De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (155) Google Scholar). The immunoprecipitates were collected by centrifugation and analyzed by 4–6% SDS-PAGE and Western blotting. The membrane was immunodetected with anti-Duox (1/8000)/anti-rabbit Ig-HRP (1/10,000) and anti-TPO mAb47 (1/1000)/anti-mouse Ig-HRP (1/10,000), respectively. 12% SDS-PAGE was used to detected p22phox with anti-p22 mAb448 (1/200)/anti-mouse Ig-HRP (1/10,000). After stripping treatment (2% SDS, 100 mm dithiothreitol, 30 min at 60 °C), the membrane was immunodetected with monoclonal anti-HIS (1/10,000)/anti-mouse Ig-HRP (10,000) in transfected cells, with polyclonal anti-hTRX (2 μg/ml)/anti-goat biotinylated (1/10,000)/strepavidin (1/2000) in human thyrocyte co-immunoprecipitation. Anti-Duox antibodies were produced by our group (15De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar); anti-TPO mAb15 and mAb47 and anti-p22phox mAb448 were kindly given by Dr. J. Ruf (51Finke R. Seto P. Ruf J. Carayon P. Rapoport B. J. Clin. Endocrinol. Metab. 1991; 73: 919-921Crossref PubMed Scopus (60) Google Scholar) and Dr. D. Roos (52Verhoeven A.J. Bolscher B.G. Meerhof L.J. van Zwieten R. Keijer J. Weening R.S. Roos D. Blood. 1989; 73: 1686-1694Crossref PubMed Google Scholar), respectively; anti-HIS and anti-human TRX antibodies were purchased from BD Biosciences; anti-rabbit Ig-HRP, anti-mouse Ig-HRP, anti-goat biotinylated, and streptavidin were purchased from Amersham Biosciences. Immunofluorescence—CHO-K1 cells were seeded on a cover glass (Corning Glass) and transfected with the HIS-tagged EFP1 and/or human Duox cDNAs using FuGENE 6 (Roche Applied Science). Forty-eight hours after transfection, the cells were fixed with 4% paraformaldehyde on ice during 15 min. After permeabilization with 1% Triton X-100 and blocking with 5% horse serum in phosphate-buffered saline, the cells were incubated with the polyclonal antibodies against Duox (1/2000) or/and monoclonal antibody against HIS tag (1/200) in 0.3% dithiothreitol-bovine serum albumin. These primary antibodies were finally detected using anti-rabbit-Texas Red (1/50) or/and anti-mouse-FITC (1/50, Amersham Biosciences). The observations were made on an Axioplan2 imaging fluorescence inverted microscope (Zeiss) and a confocal microscope (MRC 1024, Bio-Rad) fitted on an inverted microscope (Axiovert 100; Zeiss). Expression Profile of EFP1 mRNA in Human Tissues—An equal amount of cDNA from multiple tissue cDNA (MTC™) panel II (BD Biosciences) and of cDNA resulting from reverse transcription of total RNA prepared from human thyrocytes in primary culture were used to perform PCR with the specific human Duoxs and EFP1 primers (for Duox1, 5′-GTGTCTGAGAAGCTCGTGGGA-3′ (forward) and 5′-GGGAGGGTCTGTGTCCATGG-3′ (reverse); Duox2, 5′-ATGGGACTTCTGCGTGCGCTG-3′ (forward) and 5′-AATACCATTTCCACCTCCACC-3′ (reverse); EFP1, 5′-CCACTTCTCTACATCCCATCTC-3′ (forward) and 5′-GGCATCAACCTCACATTTCTTC-3′ (reverse)). The amount of amplified cDNA was normalized using the PCR amplification of G3PDH (glyceraldehyde-3-phosphate dehydrogenase) with the primers provided in the kit. To confirm the RT-PCR results, a human multiple tissue Northern (MTN) blot (Clontech) and a blot with 2 μg of human thyroid mRNA were hybridized with an EFP1-specific probe obtained by PCR using EFP1F and EFP1R primers as described above. The blots were re-hybridyzed with a probe obtained by PCR (T1-F, 5′-TTTCCCCGAGACTCGCAGAAC-3′; T1-R: 5′-AAGCTGGGCAGCCACTCATAC-3′) corresponding to the common region of Duox1 and Duox2 (53Pachucki J. Wang D. Christophe D. Miot F. Mol. Cell. Endocrinol. 2004; 214: 53-62Crossref PubMed Scopus (64) Google Scholar). Total RNA from human thyroid tissue was isolated with TRIzol (Invitrogen) and purified with RNeasy mini kit (Qiagen). The mRNA was prepared using PolyATtract mRNA isolation systems (Promega) and 2 μg were quantified spectrophotometrically. Isolation of EFP1 as a Partner of Duox1 by Yeast Two-hybrid Screening—The cDNA corresponding to the first intracellular loop of dog Duox1 protein containing two EF-hand domains (DEF, aa 956–1248; Fig. 1) was cloned by PCR downstream from the Gal4 DBD in the yeast two hybrid vector pPC97 and was used to screen a dog thyroid cDNA library cloned downstream from the Gal4 activation domain in the yeast two hybrid vector pPC86. 1.8 × 106 transformants growing on αLeuαTrp medium were screened with the bait. Forty-two and 14 positive clones were isolated after plating on αLeuαTrpαHis and αLeuαTrpαHisαAde selective media, respectively. The cDNAs of potential partners of the positive clones were purified and re-introduced in yeasts in pPC97 vectors expressing Gal4 DBD-containing HEF (human EF-hand-containing sequence) or DEF to confirm interaction and p45 (an unrelated protein) to check the specificity of the interaction with the bait. After the reconstruction the positive clones were sequenced. As shown in Fig. 1, clones n1.5 and n5.1 interacted only with DEF and HEF baits. No interaction occurred with pPC97 alone nor with Gal4 DBD fused with an irrelevant protein p45. Sequence analysis revealed that the two cDNAs, n1.5 and n5.1, corresponded to a dog protein showing high similarity with the so called hypothetical human protein LOC51061 (GenBank™ accession number NP_056998). This human peptide sequence is a fragment of a 958-amino acid protein with a theoretical PI of 6.14 and a molecular mass of 107.511 kDa according to PeptideMass program. This protein shares 82.9% similarity with a mouse protein of 948 aa (GenBank™ accession number NP_083858). We called it EFP1. The gene encoding the protein EFP1 possesses 13 exons and is located on chromosome 16p13.13. N1.5 and n5.1 cDNAs encoded peptides corresponding to aa 744–958 and 644–874 of human EFP1 and sharing 67 and 81% identity in amino acids, respectively, with this human protein. The alignment of these two overlapping dog cDNAs showed 74% identity in amino acids with human EFP1 (Fig. 2A). Further sequence analysis predicted EFP1 as a protein without signal peptide but containing two thioredoxin domains. The second thioredoxin domain was located between aa 665 and 772 and contained the conserved active site motif WCGFC (aa 691–695). The first thioredoxin domain (aa 124–211) contained only one cysteine in the active site motif (WCGQS). The structure analysis of EFP1 using the Predict-Protein program showed that the thioredoxin domains shared the same secondary structure of thioredoxin (54Martin J.L. Structure (Camb.). 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar); both of them comprise β-α-β structure followed by β-β-α structure, and the active cysteines were located in the loop separating a β-strand and an α-helix (Fig. 2B). Caenorhabditis elegans and Drosophila data base screening (tblastn of Blast program) with the EFP1 protein sequence showed that the amino acid sequence around the potential active site (aa 683–747) of EFP1 shared 40% identity and 59% similarity with the C. elegans protein disulfide isomerase-3 (PDI-3) and 37% identity and 60% similarity with Drosophila D-ERp60 (PDI isoform) (Fig. 3A). A multiple amino acid sequences alignment showed that EFP1 shared 30–46% similarities with several other mammalian thioredoxin-related proteins (Fig. 3B). It is noteworthy that during the preparation of this manuscript, the sequence of protein LOC51061 has been modified. The most recent sequence corresponding to a protein of 985 amino acids instead of in the previous 958 amino acids with 27 amino acids insertion between aa 264 and 265 (ALESTSSPRALVSFTGEWHLETKIYVL), which does not contain particular domains or structure after analysis with the PredictProtein program. Interestingly the mouse homolog does not contain this 27-aa stretch. EFP1 Tissue Distribution—The tissue distribution of EFP1 was investigated by RT-PCR amplification on a panel of cDNAs. A human multiple tissue cDNA panel and cDNA from reverse-transcribed total RNA of human thyrocytes in primary culture were used. The amount of amplified cDNA was normalized and compared with the expression of G3PDH. As shown in Fig. 4, EFP1 and Duox2 shared the same tissue expression distribution, i.e. basal level expression was observed in all of the nine tissues (except Duoxs in leukocytes), and high expression of EFP1, Duox1, and Duox2 was found in thyroid and prostate. To confi
Referência(s)