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Thyroid Section (G.D.V.S., C.C.-M., J.W.H., C.L., P.R.L., A.C.B.), Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; Laboratory of Endocrine Neurobiology (B.G., A.Z.), Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest H-1083 Hungary; Department of Structural Biology (I.C., J.-P.M.), Institut de Minéralogie et de Physique des Milieux Condensés, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7590, Universities Paris 6 and Paris 7, Paris, France; and Division of Endocrinology (M.A.M., S.A.H.), Children’s Hospital Boston, Harvard Medical School Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Thyroid Section, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Harvard Institutes of Medicine Building, Suite 643, Boston, Massachusetts 02115. E-mail: abianco{at}deiodinase.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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D3) abrogated dimerization and deiodinase activity except when coexpressed with full-length catalytically inactive deiodinase, thus assembled as D3:D3 dimer; thus the D3 globular domain also exhibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The deiodinases are the products of three different genes, and each enzyme has distinct substrate affinities and physiological roles (6). Whereas all three deiodinases are known to be integral membrane proteins with a single transmembrane domain within the first 30–40 amino-terminal residues (7, 8, 9), there are clear differences with respect to their molecular and catalytic properties (3, 10, 11, 12). Unique among the deiodinases, D3 has no introns and has recently been found to be paternally imprinted (13). D3 (molecular mass, 32 kDa) recycles between the plasma membrane and the early endosomes, with a less clear orientation. Whereas immunofluorescence and biotinylation studies indicate a catalytic globular domain located in the extracellular space (14), functional data indicate that D3-mediated catalysis takes place inside the cell (15). At the same time, it is clear that D1 also resides in the plasma membrane (catalytic globular domain in the cytosol) (7, 8) and D2 is an endoplasmic reticulum-resident protein (catalytic globular domain in the cytosol) (8).
Early attempts to purify the deiodinases identified activity in higher molecular weight forms than predicted from their respective deduced amino acid sequences (16, 17). Subsequent studies using three different approaches confirmed that D1, D2, and D3 form homodimers (18). The evidence includes identification of monomeric bands for each deiodinase by Western blot analysis along with additional higher molecular weight bands of appropriate size for a putative dimeric enzyme, coimmunoprecipitation of 75Se- and FLAG-tagged deiodinases using anti-FLAG antibodies, and immunodepletion of D1 and D2 activities from lysates of cells coexpressing inactive FLAG-tagged deiodinase and the respective unflagged wild-type enzymes (D1 or D2). D1 and D2 dimerization was confirmed by other groups in subsequent studies (19, 20, 21).
Recently, we used fluorescence resonance energy transfer (FRET) and bioluminescence fluorescence energy transfer (BRET) to study D2 dimerization in live human embryonic kidney (HEK)-293 cells (22). Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2’s globular interacting surfaces that are critical for dimerization and catalytic activity (22).
Discovering this mechanism of D2 inactivation led us to consider the possibility that homodimerization of D3, the deiodinase capable of thyroid hormone inactivation, might also modulate its enzymatic activity. In the present investigation, we show that D3 and D1, the other deiodinases capable of thyroid hormone inactivation, also exist as homodimers maintained by transmembrane and globular interacting surfaces, and that there is a small degree of heterodimerization between D3 and the other two deiodinases. Dimerization is thus a shared regulatory mechanism for the activating and inactivating deiodinases.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, A and B), a value well above the limit of detection for this technique, estimated at 3–4% (23). For the purpose of standardizing the results, in each experiment the FRET signal obtained with the fused CFP-YFP was assigned a value of 100%, and signal values below 20% were considered background. Although the focus of the present studies was on D3, as much as possible we also included D1 and D2 in the experiments to compare and contrast the deiodinase properties. The signal obtained in cells expressing only CFP and YFP, or either chromophore-fused D3 or -D1 alone, common negative controls used in these kinds of studies, remained well below 20% (Fig. 1C). At the same time, coexpression of CFP- and YFP-fused deiodinases resulted in a strong whole-cell FRET signal, reaching approximately 75% of the positive control (Fig. 1C). Whereas in these experiments both deiodinases were found in the plasma membrane (14), some expression was also noted intracellularly (Fig. 2, A and B). However, FRET remained strong regardless of whether the cell area analyzed contained only plasma membrane or the whole cell (data not shown), the latter being performed throughout the study.
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). In addition, replotting of the data indicate that FRET signal for both deiodinases was independent of acceptor (YFP) expression levels (data not shown). Both findings indicate that FRET resulted from deiodinase dimerization and not from overexpression of randomly distributed molecules, densely packed in the proximity of one another as a result of overexpression (23). Thus, these amounts of plasmid DNA were used in all subsequent FRET studies if not indicated otherwise.
FRET signal was readily detected if the cells expressing D3 (or D1) fused to the appropriate chromophores (CFP or YFP) in the same orientation, either amino (N) or carboxyl (C) (Fig. 3A). However, if the cells expressed deiodinases fused to chromophores in the opposite termini, energy transfer decreased dramatically. For D3, coexpression of D3N and D3C consistently resulted in much low, but higher than background, energy transfer, at about 20% of the positive control (Fig. 3A). For D1, coexpression of CFP-D1 fused to the N terminus (D1N) and YFP-D1 fused to the C terminus (D1C) resulted in background FRET levels (Fig. 3A).
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). Notably, the FRET signal for the D2:D3 heterodimer was stronger when the chromophores were fused to the C terminus of both deiodinases (Fig. 3B).
Next, BRET was used to study cells transiently expressing deiodinases fused to Renilla luciferase (RLuc) or YFP. Homodimerization was evident for D3 and D1, as the BRET ratio reached values between 0.2–0.3 (Fig. 4, A and B). Transfection with increasing amounts of YFP-deiodinase (D3 or D1) plasmid DNA progressively increased energy transfer, up to a maximum when 2.0 µg of plasmid DNA was used (Fig. 4, A and B). As with the FRET studies, measurable D3:D1 and D3:D2 heterodimerization was detected, but the D1:D1 and D2:D2 dimers were not affected by overexpression of other deiodinases, indicating a preference for homodimerization (Fig. 4, C and D).
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). Notably, these single transmembrane segments possess potentially charged residues (D, E, K, R, and H), a feature that is not found in other nondimeric membrane-anchored members of the thioredoxin-fold superfamily [GPX family (9)]. In the deiodinases, these residues interact most likely through electrostatic bridges to neutralize their polarity within the highly hydrophobic membranous environment. In D3, the amino acids of a conserved couple (D37/K43) are separated by six amino acids, which in an 
-helix conformation would have a nearly opposite direction with respect to the helix core. In D1, a similar role is played by indirect contacts between E18 and K27. Notably, these transmembrane segments lack proline residues, and only a few G residues are present, suggesting that deiodinases possess regular, unbent (or poorly bent) transmembrane helices. Intermolecular contacts between the histidine side chains of H44 in D3 with main chain atoms may also occur, compatible with a - dimeric architecture. Moreover, for D3, R42 is, as K43, directed toward D'37, supporting the predicted K/D bridge. In D1, E18 and K'27 are further apart (
10 Å), favoring the existence of an ionic pocket filled with a water molecule, which may be readily stabilized by the proximal H22 and not constrained by the relatively small V23, which occupies the D position in D3.
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) supports the hypothesis of direct contacts between inversely charged residues, indicating that the deiodinase models of transmembrane segments are particularly stable and energetically favored entities, fully compatible with homo- or heterodimerization, supporting the active participation of these regions in the dimerization process.
Globular Surfaces Mediate D3 Homodimerization
To test experimentally the role of the D3 transmembrane domain in deiodinase dimerization, we truncated D3 at the predicted amino acid (residue 55) that emerges from the membrane generating D3; truncating residue 50 in D1 generates D1. The two proteins were transiently expressed in HEK-293 cells and found predominantly in the cytosolic fraction (Fig. 6, A and B). When expressed alone, D3 or D1 did not homodimerize, as assessed by FRET (Fig. 6C) and BRET (data not shown) and displayed no catalytic activity (Fig. 6D). These findings are compatible with the idea that the transmembrane domains are critical for the deiodinase dimerization. However, we sought to determine whether dimerization between truncated deiodinases and their respective full-length counterparts could occur. This was tested by transiently coexpressing each individual truncated deiodinase molecule fused to YFP at the C terminus with a full-length inactive deiodinase, i.e. D3-YFP with D3-CFP or D1-YFP with D1-CFP. Inactivation of the full-length D3 (and D1) was achieved by replacing the Sec residue at the respective active centers with Ala. Notably, such combination of expressed proteins resulted not only in significant dimerization, i.e. D3:D3 and D1:D1 (Fig. 6C), but also in D3 and D1 catalytic activities, respectively (Fig. 6D). Although maximum velocities were substantially low, the affinities [Michaelis-Menten constant (Km)] for their preferred substrates, respectively T3 and rT3, were indistinguishable from those of the corresponding full-length enzymes (Fig. 7, A and B).
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), there is currently no obvious three-dimensional (3D) template that may directly suggest an overall architecture for this rather long segment, which is likely to possess an intrinsic flexibility favoring mutual fitting of the two moieties of the deiodinase dimer. In this regard, the recent model of -L-iduronidase (25) does not shed light on this problem because the iduronidase ADPLVGWSLPQP segment, which matches the deiodinase sequence, constitutes only the first half of the active site insertion (9). Moreover, the 3D context of this segment is likely to be different between deiodinases and iduronidase and, given its predicted flexibility, this segment may indeed fold upon contact with other local structures.
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B, the disposition of which matches those of the strand β7 and 7 helix of the clan GH-A-fold of glycoside hydrolases (9). Immediately after the core β2 (deiodinase) or β7 (iduronidase) strands, a conserved E residue is found in both enzyme families (E156, E163, and E174 in D1, D2, and D3, respectively), the mutation of which inactivates the enzymes. In the D2 model, E163 is about 10 Å from the essential Sec133 within the catalytic site. In the thioredoxin templates, there is also often an acidic E or D residue in this position. Next, in the deiodinases, there are six amino acids, small (A), neutral (H), or belonging to the strong loop-forming amino acid class PGDNS (9). This high concentration of P, S, D, or G strongly suggests a loop linking β2 to a short hydrophobic cluster (WAF, WAI, and WVTT for D1, D2, and D3, respectively), which is typically associated with β-strands (26). Downstream, a second loop is likely to occur (KNN, PGDSSLS, DSP sequences, respectively), leading to another short hydrophobic cluster (MDI, FEV, YII, respectively), which is, like the first cluster, typical of a β-strand. Next, a loop (RNHQN, KKHQN, PQHRS sequences, respectively) is likely to precede the core B-helix. We refer to the two above putative short β-strands as βd1 and βd2, respectively, as they may participate in a small β-sheet within the dimeric interface of deiodinases (22).
Next, the role of specific amino acid residues within the proposed globular dimerization surfaces (20, 21), was tested by creating D1, D2, and D3 mutants fused to YFP at the respective C termini in which the corresponding positions 152–154 (IYI) in D1, 159–161 (VYI) in D2, and 170–172 (IYI) in D3 were mutated to Ala (AAA). Indeed, FRET studies indicated that the energy transfer between these mutant dimer counterparts was only about one third of that observed in the wild-type proteins, confirming a role for these globular sequences in dimerization (Fig. 8B). However, despite this residual FRET signal, we found all these mutants to be catalytically inactive, indicating that these residues are also critical to maintain catalytic activity.
Loss of Energy Transfer between D3:D3 Results in Loss of Deiodinase Activity
In D2, homodimerization is critical for catalytic activity (22). To test whether this is the case also for D3, we sought to interfere with the D3:D3 dimers by exposing sonicates of cells transiently expressing the appropriately chromophore-fused D3 (or D1) proteins to increasing concentrations of urea, a known caotropic agent, while monitoring BRET and enzymatic activity. Exposure to urea resulted in progressive loss of energy transfer emanating from D3:D3 and D1:D1 dimers (Fig. 9, A and B), which was followed by proportional loss of deiodinase activity (Fig. 9, C and D). Notably, exposure to urea did not interfere with D3-RLuc or D1-RLuc activities or with direct D3-YFP or D1-YFP fluorescence (data not shown). This suggests that a proper fit between the two counterparts in D3:D3 (and D1:D1) is critical for catalytic activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), the significance of which begs further investigation.
Although the FRET and BRET studies were performed in cells transiently expressing the different chromophore-fused deiodinases, the intrinsic properties of these enzymes, such as subcellular localization and affinity for their preferred substrates, are indistinguishable from endogenously expressed D3 or D1. Protein modeling identified potential dimerization surfaces in the transmembrane and globular domains, the latter capable of supporting D3:D3 or D1:D1 dimers. Lastly, exposure to a high concentration of urea impaired dimerization and deiodinase activity, indicating that homodimerization is critical for catalysis.
Given our previous findings with D2 (22), the present study indicates that all deiodinases have two dimerization surfaces: one in their transmembrane domain and the other in their globular catalytic domain modeled to the iduronidase-like active site insertion situated near the selenocysteine-containing active site. Interestingly, it is clear that for both inner- and outer-ring deiodinases the globular dimerization is sufficient for catalytic activity. As the truncated deiodinases are inactive and do not homodimerize (i.e. D3-D3 or D1-D1) (Fig. 6), the interaction between these two truncated molecules containing only the globular domain does not mediate stable dimerization. However, D3 and D1 still dimerize with their full-length counterparts, which results in catalytic activity (Fig. 6). Whereas it is unclear why there is no interaction between two D3 or D1 globular domains in the cytosol, the finding of dimerization between the globular domains of D3-D3 and D1-D1 indicates that membrane insertion of at least one of the dimer counterparts is required to accommodate globular dimerization.
Notably, the fit between the globular domains in D3-D3 and D1-D1 support catalytic activity with an affinity for their respective preferred substrates that is indistinguishable from native enzymes (Fig. 7), strongly suggesting that dimerization is critical for catalytic activity. In fact, exposure of D3 (or D1) to increasing concentrations of urea, a caotropic agent, resulted in progressive loss of dimerization and proportional decrease of catalytic activity (Fig. 9). Even considering the lack of specificity of the conformational changes induced by urea, these data support a relationship between deiodinase dimerization and catalysis.
Because in D3-D3 and D1-D1, the respective D3 and D1 counterparts are inactive, it is likely that both Sec-containing active centers in each dimer function independently. In fact, we found no evidence of a cross talk between the active centers in both dimer counterparts of any deiodinase, suggesting that dimerization is critical for the conformation of the active center rather than cross talk between them. Given that transiently disrupting D2 dimerization via conjugation to ubiquitin is a switch that controls enzyme activity (22), it remains to be determined whether similar posttranslational mechanisms can interfere with dimerization and regulate the D3 enzyme activity.
Simpson et al. (21) claim to have identified and characterized essential residues critical for dimerization in the globular domain of the deiodinases. By overexpressing sequential alanine-substituted mutants of this domain fused to a green fluorescent protein, they showed that the sequence 152IYI154 was required for D1 assembly and that a catalytically active monomer was generated by a single I152A substitution. For D2, a similar strategy identified five residues (153FLIVY157) at the beginning and three residues (164SDG166) at the end of this region, which were required for dimerization. Although we find no evidence that either deiodinase monomer is active, we mutated such key residues [152–154 (IYI) in D1, 159–161 (VYI) in D2, and those in the corresponding position in D3 (170–172; IYI)] to alanines (AAA) and monitored the ability to homodimerize by FRET exhibited by each deiodinase. Our findings indicate that such mutants retain only about one third of their capacity to dimerize (Fig. 8), but most importantly, they were found to be catalytically inactive. These data would confirm our hypothesis that dimerization is critical for catalytic activity. However, given that these mutants are inactive, it is also conceivable that the amino acid substitutions resulted in deiodinase misfolding, which would minimize the physiological significance of these findings.
In conclusion, the present findings indicate that the thyroid hormone-inactivating deiodinases (D3 and D1) are assembled as homodimers stabilized by interacting surfaces at the transmembrane and globular domains. Whereas only D3 exhibits a limited tendency for heterodimerization with D1 and D2, homodimerization of all three deiodinases seems critical for catalytic activity. Regulation of dimerization could underlie a posttranslational mechanism to control D3- and/or D1-mediated catalysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA Constructs
All DNA fragments were generated with Vent PCR on templates containing the coding region of rat D1 (rD1) or human D3 (hD3) with a Cys-mutated active center. To fuse the deiodinase fragments to the carboxy portion of YFP or CFP, the generated D1 fragment was inserted between EcoRI and SalI, whereas D3 was cloned between EcoRI and BamHI of pEYFP-C1 or pECFP-C1 (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively. To fuse rD1 and hD3 to the amino portion of YFP or CFP, the PCR fragments were inserted between EcoRI and BamHI of pEYFP-N1 or pECFP-N1. C or N indicates the terminus of deiodinase used to fuse CFP or YFP.
Vent PCR-based strategy was used to fuse the transmembrane-less () versions of D1 and D3 to the N portion of YFP. Briefly, the first 50 and 55 residues were deleted from rD1 and hD3, respectively. All fragments were inserted between EcoRI and BamHI of pEYFP-N1. The human D2 (hD2) CFP/YFP fusions have been previously described (22). Overlap-extension PCR was performed to replace the following residues with A: amino acids 152–154 IYI in rD1, amino acids 159–161 VYI in hD2, and amino acids 170–172 IYI in hD3. All of these deiodinase fragments contained Cys instead of selenocysteine and were inserted between EcoRI and BamHI of pEYFP-N1.
Cell Culture and Transfections
HEK-293 epithelial cells were plated in 60-mm plates and grown until midconfluence in DMEM (without phenol red) supplemented with 10% fetal bovine serum. HEK-293 cells were transfected using Lipofectamine 2000, as per the manufacturer’s instructions. Human GH (thymidine kinase promoter-driven GH) was used as a control for monitoring the transfection efficiency, as described previously (30). The cotransfection ratios of the experimental constructs, truncated N termini, and alanine and cysteine mutants varied depending on the experiment. Usually, 2 µg of each plasmid was transfected, unless otherwise mentioned in the figure legend. Cells were gently washed twice in PBS, 48 h after transfection, and live-cell FRET imaging was performed as described in subsequent sections.
FRET Data and Image Acquisition
We used confocal-based FRET detection by acceptor photobleaching (22). This technique is based on the increase in the donor fluorescence (CFP) immediately after acceptor (YFP) photobleaching (31). Numerical data and image acquisition were obtained using Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc., Thornwood, NY), as described elsewhere (22). In brief, live HEK-293 cells in PBS were examined. Typically, a 2-µm optical slice was used to visualize a cell expressing the constructs of interest tagged with CFP and YFP. Dual excitation of CFP and YFP was achieved by using an argon laser with a 458-nm/514-nm dual dichroic filter (31). Optimized images were collected at 12-bit resolution over 512 x 512 pixels with a pixel dwell time of 1.6 sec (23). A cell was selected as the region of interest, which was then irradiated with the 514-nm laser line (100% intensity); the number of iterations varied, although the goal was to photobleach YFP as quickly as possible. Only cells that exhibited about 85% photobleaching were considered in the FRET studies (32). To appreciate the occurrence of FRET, caution was taken not to saturate the region of interest and to optimize the image carefully and reasonably. A minimum of at least five cells to a maximum of 14 cells per experiment was studied. To analyze a single parameter, typically 10–91 cells were studied in multiple experiments.
Postbleach images were acquired immediately after YFP photobleaching, a process known as "donor dequenching" (23). FRET efficiency was calculated by using the following equation: 100 x (CFP postbleach fluorescence intensity – CFP prebleach fluorescence intensity)/CFP postbleach fluorescence intensity. FRET efficiencies were calculated for all the constructs discussed in Results. Numerical data are normalized and presented as a percent of the positive control, a CFP-YFP fused tandem, because of experiment-to-experiment variability in the FRET efficiencies. D1N-CFP and D1C-YFP (fluorophores tagged in the opposite orientation of the protein are unlikely to yield FRET), and thus such a combination is used as negative control for each respective deiodinase.
BRET Assay
BRET was preformed, as described elsewhere (22). Briefly, HEK-293 cells were transfected with combinations of D1-humanized Renilla (RLuc) and D1-YFP or D3-RLuc and D3-YFP constructs, 1.5 µg each per plate. Cells were washed twice with PBS 48 h post transfection, detached in PBS/2 mM EDTA, centrifuged, and resuspended in PBS/0.1% glucose. Cells (100,000) were dispensed into each well of a 96-well plate with clear bottoms, which had dark surroundings to minimize interference caused by autofluorescence. Studies in cell sonicates were done by resuspending the cell pellet in 250 µl of PBS/0.1% glucose and by sonicating cells for 6 min. Protein was determined according to Bio-Rad protein assay, and equal amounts of protein were added to each well. Renilla was activated by DeepBlueC coelenterazine and read by a Fluostar Optima Fluorimeter (BMG Lab Technologies, Offenburg, Germany) equipped with filters with band pass for emission: 475–30 nm for RLuc luciferase and 535–30 nm for YFP at a set gain. BRET ratio is defined as the [(YFP Emission at 535–30 nm)/RLuc (475–30 nm) of the sample] – RLuc (475–300 nm) in a sample where RLuc construct is expressed alone.
Enzyme Assays of D1 and D3
Deiodinase activities were assayed as described earlier, in the presence of 20 mM dithiothreitol (18). D1 was assayed in the presence of 1 µM [125I]rT3 and D3 in the presence of 2 nM [125I]T3.
Sequence Analysis and Structure Modeling
Further sequence analysis has been conducted using hydrophobic cluster analysis as previously reported for the construction of the thioredoxin fold model of deiodinases (33, 34). 3D models were built and handled using the Swiss PDB viewer tool (35).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online March 20, 2008
1 G.D.V.S. and B.G. contributed equally to this paper and should be considered co-first authors. 
Abbreviations: BRET, Bioluminescence fluorescence energy transfer; CFP, cyan fluorescent protein; D1, D2, and D3, type 1, type 2, and type 3 deiodinase, respectively; 3D, three dimensional; FRET, fluorescence resonance energy transfer; HEK, human embryonic kidney; RLuc, Renilla luciferase; YFP, yellow fluorescent protein.
Received for publication October 24, 2007. Accepted for publication March 11, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-l-iduronidase: insights into human disease. Mol Genet Metab 85:28–37[CrossRef][Medline]NURSA Molecule Pages Link:
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