Identification of a Novel Partner of Duox EFP1, A THIOREDOXIN-RELATED PROTEIN*

H 2 O 2 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 H 2 O 2 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 p22 phox 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 thiore-doxin-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 H 2 O 2 production. EFP1 and Duox mRNA share similar distribution

The biosynthesis of thyroid hormones by thyroid cells requires oxidation of iodide, its binding to tyrosines of thyroglobulin (Tg) 1 and the oxidative coupling of iodotyrosines into io-dotyronines. These reactions are catalyzed by thyroperoxidase (TPO) in the follicular space close to the apical pole of the cells. H 2 O 2 is a crucial substrate and limiting factor of TPO for the iodination reactions (1). The thyroid H 2 O 2 generating system has been widely characterized in in vitro biochemical studies (2)(3)(4)(5)(6)(7). The enzymatic activity has been purified as a Ca 2ϩ -dependent flavoprotein presenting an oxidase activity using NADPH as coenzyme (8 -13). Recently the corresponding cDNA has been cloned starting from this purified enzymatic activity (14). 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 H 2 O 2 generating systems, two cDNAs encoding two proteins of 1551 and 1548 amino acids, respectively, called ThOX1 and ThOX2 were cloned (15). 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 (16,17). Duox cDNA-transfected cell lines express only the immature form of the protein and no H 2 O 2 production can be detected (16) 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 gp91 phox (also referred to as NOX2), the NADPH oxidase moiety of the superoxide anions generator, needs p22 phox to be processed to the membrane (18 -20). Moreover NOX2 is active only when several cytosolic components (p47 phox , p67 phox , Rac, and p40 phox ) are recruited to form a complex at the membrane (21)(22)(23)(24)(25). Recently homologs of p47 phox and p67 phox , NOXO1 and NOXA1 (also called p41 nox and p51 nox ), have been found by several groups (24, 26 -28). These two proteins are able to activate NOX1, the NADPH oxidase responsible for O 2 . production mainly in the colon. The unsuccessful reconstitution of the H 2 O 2 -generating capacity in PLB-XCGD (gp91 phox -knock-out PLB) cells with Duox1 and Duox2 suggests that other thyroid-specific factors are required to permit H 2 O 2 production in thyroid (16). The interaction of Duox with thyroid proteins was investi-gated 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 H 2 O 2 . 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 (29,30). The maintenance of thioredoxin in its active reduced form is carried out by thioredoxin reductase in the presence of NADPH forming the thioredoxin system (29). 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 (31). Thioredoxin is also an antioxidant participating in the reduction of H 2 O 2 , scavenging free radicals and protecting against oxidative stress (32)(33)(34). It can modulate the activity of some transcription factors, such as AP-1 (35) and NF-B (36). A number of mammalian proteins containing thioredoxin domains have been identified, including human Trx, Trx2 (37,38), TRP32 (39) (thioredoxin-related protein), TRP14 (40), TMX (thioredoxin-related transmembrane protein) and TMX2 (41,42), Nrx (nucleoredoxin) (43,44), 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 (42). 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 (46). 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 (37,39,40,43,44). 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.

EXPERIMENTAL PROCEDURES
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 (47). The yeast host strain pJ69 -4A was used for the screening and the reconstruction steps as described previously (48). 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 (␣Leu␣Trp) were transformed with plasmids from the bait and the library. Such transformants were first selected on medium lacking His (␣Leu␣Trp␣His) and then on the medium lacking His and Ade (␣Leu␣Trp␣His␣Ade). DH10B bacteria were transformed by electroporation with the cDNAs coming from yeasts growing on ␣Leu␣Trp ␣His␣Ade 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).
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). Fortyeight 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 TM ) 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, 5Ј-GTGTCTGAGAAGCTCGTGGGA-3Ј (forward) and 5Ј-GG-GAGGGTCTGTGTCCATGG-3Ј (reverse); Duox2, 5Ј-ATGGGACTTCT-GCGTGCGCTG-3Ј (forward) and 5Ј-AATACCATTTCCACCTCCACC-3Ј (reverse); EFP1, 5Ј-CCACTTCTCTACATCCCATCTC-3Ј (forward) and 5Ј-GGCATCAACCTCACATTTCTTC-3Ј (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 g 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, 5Ј-TTTCCC-CGAGACTCGCAGAAC-3Ј; T1-R: 5Ј-AAGCTGGGCAGCCACTCATAC-3Ј) corresponding to the common region of Duox1 and Duox2 (53). 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 g 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 ϫ 10 6 transformants growing on ␣Leu␣Trp medium were screened with the bait. Forty-two and 14 positive clones were isolated after plating on ␣Leu␣Trp␣His and ␣Leu␣Trp␣His␣Ade selective media, respectively. The cDNAs of potential partners of the positive clones were purified and re-introduced in yeasts in pPC97 vectors expressing Gal4 DBDcontaining 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 (Gen-Bank TM 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 TM 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 (54); both of them comprise ␤-␣-␤ structure followed by ␤-␤-␣ structure, and the active cysteines were located in the loop separating a ␤-strand and an ␣-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 (ALESTSS-PRALVSFTGEWHLETKIYVL), 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 normal- ized 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 confirm the RT-PCR results, a human MTN blot was hybridized first with an EFP1 specific probe and then with a probe from a PCR amplification product that corresponds to a common region of Duox1 and Duox2. EFP1 mRNA was detected in all the tissues of the panel. In agreement with the results of RT-PCR, thyroid and prostate presented the highest signal intensity. Duox probe revealed a signal at 5-6 kb in thyroid, prostate, and colon. The EFP1 probe detected EFP1 messenger at 3 kb.
Cellular Localization of EFP1-The cellular localization of EFP1 protein was studied by immunofluorescence. CHO cells were co-transfected with both EFP1 in pcDNA3.1/HIS and human Duoxs cDNA in pcDNA3.1. After permeabilization anti-HIS/anti-mouse-FITC and anti-Duox/anti-rabbit-Texas Red were used to detect these two proteins. The localization was analyzed by confocal microscopy. As already known, Duox, which is partly expressed at the plasma membrane but mainly associated with the ER in thyrocytes is exclusively expressed inside the cell when transfected in CHO cells (16). When Duox and EFP1 were transfected together in CHO cells, signals of both EFP1 and Duox proteins were co-localized inside the cell associated with the ER when they expressed in the same cell (Fig. 5). Thus the co-transfection of Duoxs together with EFP1 did not modify the localization of Duoxs from the ER to the plasma membrane.
Interaction between EFP1 and Human Duox Proteins-To verify the interaction between EFP1 and human Duox1/Duox2, co-immunoprecipitation experiments were carried out. The full-length cDNA of the human EFP1 was subcloned into pcDNA3.1/HIS vector. HIS-tagged EFP1 was co-expressed in COS cells with either human Duox1 or Duox2 alone or both. The expression of proteins of interest was checked in total lysates with anti-HIS and anti-Duox antibodies. The polyclonal antibody anti-Duox recognizes both Duox1 and Duox2, but the affinity is higher for Duox1 than Duox2. This explains why the signal corresponding to Duox2 is systematically weaker. Immunoprecipitation was performed using anti-Duox antibody or preimmune serum and immunodetected first with anti-HIS and after with anti-Duox antibodies. EFP1 and Duox1/Duox2 were correctly expressed in COS cells (Fig. 6A) and EFP1 co-immunoprecipitated with both human Duox1 and Duox2 (Fig. 6B). As the bait was chosen in the intracellular part of Duox containing EF-hand motives, we looked too see if the interaction between EFP1 and Duox was Ca 2ϩ -dependent. Duox1-EFP1 co-transfected COS cells were lysed in IP lysis buffer in the presence of EGTA (10 mM) or Ca 2ϩ (10 M) or both and incubated overnight at 4°C (55). As shown in Fig. 6C the interaction was not affected by any of the treatments.
Co-immunoprecipitation of EFP1 with Duox was also performed in human thyrocytes. Due to the lack of anti-EFP1 antibody, a polyclonal anti-human thioredoxin (anti-hTRX) antibody was used to detect EFP1 after immunoprecipitation with anti-Duox as described above. The polyclonal antibodies against thioredoxin reveal a main protein at a molecular mass of 125 kDa in COS transfected with EFP1. After immunoprecipitation with Duox antibody this protein of 125,000 appears to be associated with Duox. TPO was also co-immunoprecipitated with Duox, and this interaction was confirmed by coimmunoprecipitation of Duox with anti-TPO antibody (Fig.  6D). Surprisingly EFP1 was co-immunoprecipitated with anti-TPO antibody ( fig 6D) but not with anti-Tg antibody (data not shown). p22 phox could be also co-immunoprecipitated with anti-Duox and anti-TPO antibodies (Fig. 6D).
Despite the interactions between Duox and EFP1, TPO, and p22 phox , no production of H 2 O 2 could be detected after cotransfection of the cDNAs coding these proteins (data not shown).

DISCUSSION
ThOX1/Duox1 and ThOX2/Duox2 are believed to be responsible for the H 2 O 2 generation in thyrocytes. Our previous studies (15,16) showed that the majority of Duox proteins are accumulated inside the cell in the endoplasmic reticulum, and only a small fraction is expressed at the apical cell surface. In the thyrocyte Duox proteins present two N-glycosylation states. The highly glycosylated 190-kDa form corresponding to the matured active protein is expressed at the plasma membrane and the high mannose 180-kDa form corresponding to the immature inactive protein is still inside the ER. As only the latter form is expressed in the transfected cells at the level of ER, the lack of H 2 O 2 production observed in these transfected cells could be therefore related to the absence of expression of the active form at the plasma membrane. Very recently (56) it has been shown that human and pig Duox2 possess regions responsible for the retention of the protein in the ER: one in the first transmembrane part of human Duox2 and one in the first intracellular loop of both Duox2. When the protein corresponding to the extracellular part, first transmembrane domain, and first intracellular loop of pig Duox2 is expressed in HEK cells, it is blocked in the ER; this is contrary to the protein corresponding to only the extracellular part and first transmembrane domain, which is correctly processed to the plasma membrane. In addition if the four cysteine residues located in the extracellular part of Duox2 (aa 351, 370, 568, and 582), which are well conserved in human and pig and predicted to form disulfide bonds, are mutated, the protein is completely blocked in the ER. These data suggest that the folding of the protein or its interaction with ER-localized proteins may play a critical role in the export of the protein from ER to Golgi apparatus. So additional factors seem to be required to correctly process Duox and permit NADPH oxidase activity. During this study, several groups have found in human and mouse that NOXA1 and NOXO1, homologs of p67 phox and p47 phox , respectively, activate the NADPH oxidase I, NOX1 (24, 26 -28). As p67 phox and p47 phox failed to activate Duox in PLB-XCGD cells transfected with Duoxs, and as NOXA1 and NOXO1 did not seem to contribute to H 2 O 2 production in cells co-transfected with Duoxs (data not shown), other factors or mechanisms must be considered to explain the activation of Duox.
In this study we try to analyze the interaction of Duox with proteins present in the thyroid. p22 phox is present in the thyroid but is not sufficient to confer any activity to Duox when cotransfected in CHO cells (16). It has been shown for a long time that p22 phox forms a heterodimer with NOX2, and more recently it is found that p22 phox interacts directly with NOX1 and NOX4 (57). By co-immunoprecipitation experiments in thyrocytes we were able to demonstrate an interaction between Duox and p22 phox , Duox and TPO, and p22 phox and TPO. These membrane proteins may participate in the multimeric complex and are therefore perhaps necessary but not sufficient to produce H 2 O 2 . That is why the yeast two-hybrid method was used to search for other proteins interacting with Duox. In this study we found a novel protein, EFP1, interacting with both human and dog Duoxs. EFP1 was described by Andersson and Yu (58,59) in the context of shotgun library construction as a sequence presenting thioredoxin domains with unknown function (hypothetical protein LOC51061 of Homo sapiens, GenBank TM accession number NP_056998). After expression and reconstruction in yeast, we found that EFP1 interacted specifically with Duox first intracellular loop containing the EF-hand domains. The region of EFP1 interacting with Duox would be located between aa 744 and 874 of EFP1, since that is the overlapping region of two clones interacting with the bait (DEF). A mutagenesis study is required to define the precise interacting site.
We have shown that EFP1 interacted with Duox by immunoprecipitation experiments in transfected COS cells in a Ca 2ϩ -independent manner. The association between these two proteins was also detected in primary cultured human thyrocytes confirming the existence of a physiological interaction. EFP1 interacts also with TPO, as shown by co-immunoprecipitation experiments, but not with Tg. TPO and Duoxs are closely related proteins in thyrocytes in both functions; they participate in the biosynthesis of thyroid hormones and localization, and they co-localize at the apical pole of the thyrocyte (15). Presumably the H 2 O 2 -generating system in the thyroid requires, as in the leukocyte, the assembly of a supramolecular complex. Such a complex is different from the leukocyte complex as expression of Duox in XCGD-PLB cells, which contain all the elements of the superoxide anion generating system except NOX2, does not permit activity or membrane expression. The present data suggest that by its interaction with Duoxs and TPO, EFP1 may play an important role in the formation of the multimeric complex and could participate in the thyroid H 2 O 2 generating system. The lack of specific antibody against EFP1 till now did not allow us to study the regulation and localization of this protein in native thyrocytes. EFP1 contains two thioredoxin domains. Thioredoxins are small enzymes involved in redox reactions via a reversible reduction of the disulfide bond in the active center: WCGPC. The WCXXC active site motif is well conserved through the members of the thioredoxin superfamily, including thioredoxin, glutaredoxin, and PDI. PDIs mediate in the ER the oxidation of free sulfhydryl residues and the isomerization of existing disulfide bonds leading to new disulfide bonds. The thioredoxinrelated proteins seem thus to be involved in various redox regulations, but the precise biological function of most of them FIG. 6. Interaction of EFP1 and Duoxs in COS transfected cells and human thyroid cells in primary culture. COS cells were cotransfected with human Duox1/Duox2 and HIS-tagged EFP1 cDNA. COS cells were harvested after 48-h transfection, and human thyrocytes in primary culture were harvested after 4 days of culture, in Laemmli lysis buffer for total lysates and nondenaturing lysis buffer for IP experiments, respectively. A, protein expression in COS co-transfected with EFP1 and Duox1, Duox2, or both Duox1 and Duox2. 10 g of each total lysate proteins (20 g for COS cells transfected with Duox2) were subjected to 4 -6% SDS-PAGE. Human thy., total lysates from human thyrocytes as control. B, IP in COS cells transfected with Duox1/Duox2 and/or EFP1. IP experiments were performed using 1 mg of protein precipitated with anti-Duox antibody or preimmune serum. C, IP in COS cells co-transfected with Duox and EFP1. Cells were lysed in IP lysis buffer in the presence of EGTA (10 mM), Ca 2ϩ (10 M), or both and incubated overnight at 4°C. The membranes of A-C were first immunoblotted with anti-Duox/anti-Rabbit Ig-HRP and then with anti-HIS/anti-mouse Ig-HRP after stripping treatment. D, IP experiment performed using 1 mg of protein extract from primary cultured human thyrocytes with anti-Duox, preimmune serum, and anti-TPO mAb15, respectively. Antibodies used for immunodetection were indicated on the right side of the panel. Human Thy. and COS-Duox-TPO-EFP1, total protein extracts from human thyrocytes and COS cells co-transfected with Duox, TPO, and EFP1, respectively, as controls.
is not yet understood. The first thioredoxin domain of EFP1 (aa 124 -211) contains only one cysteine. The second thioredoxin domain (aa 665-772) contains the conserved active site motif WCGFC (aa 691-695). Although similarity in amino acid sequences is low, the domain structures are almost identical among the thioredoxin-related proteins. The structure analysis of these proteins and of EFP1 shows a ␤-␣-␤ structure followed by ␤-␤-␣ structure with the active cysteines always located in the loop separating a ␤-strand and a ␣-helix. These properties are well conserved in the thioredoxin family (60). Therefore EFP1 can be considered as a member of the thioredoxin-related protein family.
Several hypotheses can be proposed for the role of EFP1 in the thyroid H 2 O 2 generating system. EFP1 could as PDIs induce a conformational change allowing the export of the protein. PDIs are multidomain proteins containing two or more active thioredoxin sites. Five domains have been identified, a, b, bЈ, aЈ, and c. The a and aЈ domains are clearly containing thioredoxin active sites WCGHC, and they are believed to be involved in thiol-disulfide exchange reactions (61). But the fact that isolated a and aЈ domains are less efficient catalysts than the intact PDI suggests that other domains also play important roles in the catalytic process. b and c domains do not have the active Trx sites, but they are responsible for the interaction with other non-native proteins or peptides (62,63). PDIs may act on both reduction and oxidation. The lack of a cysteine in the first thioredoxin domain of EFP1 and of b, bЈ, and c domains makes EFP1 different from PDI. Nevertheless based on the published experimental data on thioredoxin-related protein and structure analysis, EFP1 could function as redox regulator involved in Duox folding. The interaction of EFP1 occurs with the first intracellular loop of Duox, which does not exist in the short NADPH oxidases-like NOX1, NOX2, NOX3, and NOX4. This mechanism of regulation could therefore be specific for H 2 O 2 generating system in thyroid or other cells expressing Duox. The interaction of EFP1 with both Duox and TPO would suggest that EFP1 could be a chaperone which plays a role in protein processing and would permit the assembling of the multiprotein complex at the membrane of the thyrocyte. However, EFP1 does not modify the localization of Duox in ER nor permit the production of H 2 O 2 in co-transfection (Duox, TPO, and EFP1) experiments in COS, CHO cells, and even in polarized Fisher rat thyroid cells. These results indicate that EFP1 may be involved in protein maturation but is certainly not sufficient to permit H 2 O 2 generation.
To iodinate Tg, the thyrocyte synthesizes a huge amount of H 2 O 2 at its apical pole. Since H 2 O 2 can diffuse freely into the cell, several anti-oxidant systems prevent cells from reactive oxygen species damage, e.g. catalase in peroxisome and GSH/ GSSG in cytoplasm (64,65). EFP1 could supplement these as an on site instant protector. According to the function of thioredoxin, we assume that EFP1 might be reduced by NADPH by an enzyme like thioredoxin reductase. In this way EFP1 could be the co-factor of a peroxidase inactivating H 2 O 2 , like glutathione peroxidase, and would detoxify the H 2 O 2 produced by Duoxs and leaking in the cell. Attempts to measure the NADPH oxidase activity of EFP1 have, however, been unsuccessful (data not shown).
The attempts to determine the real function of EFP1 are complicated without a functional Duox H 2 O 2 generating system. Nevertheless its function is now investigated by performing RNA interference experiments in thyrocytes with high transfection efficiency methods. Whatever the role of EFP1 in the thyroid H 2 O 2 generating system, it is peculiar to the long forms of NADPH oxidases like Duoxs, or perhaps NOX5 (45), since only these enzymes possess the intracellular portion of the protein containing EF-hand domains in the NOX family, to which EFP1 binds. However, it could be a new chaperone-like protein that plays a role in the protein processing as well.