Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Brigham and Womens Hospital and Harvard Medical School
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Womens Hospital; HIM Building, Room 566, 77 Louis Pasteur Avenue, Boston, Massachusetts 02115. E-mail: abianco{at}partners.org.
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ABSTRACT |
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INTRODUCTION |
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The three selenodeiodinases are membrane-anchored proteins of 2933 kDa that share substantial sequence homology and catalytic properties (1). D1 and D3 have long half-lives and are located in the plasma membrane, with D3 undergoing endosomal recycling (2). D2, on the other hand, is an endoplasmic reticulum (ER)-resident protein (3) with an approximately 45-min half-life (2, 4, 5). The shorter half-life of D2 results from the fact that D2 is a substrate for ER-associated degradation (ERAD) (6, 7), i.e. D2 is selectively targeted for ubiquitination and subsequent proteasomal degradation (6, 7, 8). Once ubiquitinated, D2 becomes inactive, existing in a pool that may potentially be deubiquitinated (with reactivation of the enzyme) or degraded in the proteasomes. That ERAD plays a critical role in thyroid homeostasis is further emphasized by the observation that the ubiquitination of D2 is accelerated by substrate: experiments in which D2 has been transiently expressed in either human embryonic kidney (HEK-293) or Chinese hamster ovary cells have shown that increasing substrate (T4 or rT3) concentration accelerates D2 ubiquitination (8), with a corresponding decrease in D2 half-life (4, 9).
According to the classical model, ubiquitination occurs via a multistep process involving the actions of a series of enzymes including ubiquitin-activating enzymes (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3) (10). For most proteins, a combination of E2 and E3 enzymes determines the specificity of ubiquitination (10, 11). Studies utilizing heterologous expression of D2 in yeast suggest that UBC6 and UBC7, two ERAD-associated E2s (11, 12, 13, 14, 15), are required in D2 ubiquitination (16). However, because elimination of misfolded or unfolded proteins is one aspect of ERAD, studies utilizing heterologous expression of human proteins in yeast may be limited in their ability to discriminate between normal D2 ubiquitination and any potential nonspecific processing of misfolded D2. Furthermore, as opposed to our findings with D2 in yeast, there is evidence that other human ERAD substrates such as T cell antigen receptor subunits and CD3-
may be processed preferentially by UBC7 when expressed in the setting of a human cell (10).
The recent identification of murine homologs of UBC6 and UBC7, MmUBC6 and MmUBC7 (10), has made it feasible to investigate the relevance of UBC6 and UBC7 for D2 ERAD in mammalian cells. In the current investigation, our goals were to elucidate the mechanism of D2 ERAD in human cells by studying binding and functional interactions of D2 with these mammalian E2s. We found that MmUBC7 is present in the D2 ubiquitinating complex as evidenced by a strong specific association between MmUBC7 and the carboxyl region of D2 in vitro. Using overexpression of inactive mutant forms of MmUBC6 and MmUBC7 in a functional assay, we found that D2 half-life can only be stabilized when both E2s are neutralized.
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RESULTS |
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HEK-293 cells were cotransfected with plasmid DNA encoding D2 and the wild-type or inactive mutant E2s. In this transient expression system (7), D2 processing by the ubiquitin-proteasome complex was shown to have the same properties as when D2 is endogenously expressed in a human mesothelioma cell line (MSTO211H) (18) or in a rat pituitary tumor cell line (GH4C1) (6). Transient expression of the E2 proteins was documented by Western analysis (Fig. 4A). Coexpression of D2 with the wild-type E2s either individually or in combination did not reduce D2 activity (Fig. 4B
). Similarly, there was no increase in D2 activity during coexpression with either of the mutant E2s alone. However, coexpression with the combination of MmUBC6M and MmUBC7M caused a 2.5- to 3-fold increase in D2 activity compared with cells expressing D2 alone (Fig. 4
, BD, and Fig. 5A
). As an indication that this is a competitive process, the magnitude of the effect decreased when the ratio of transfected D2 plasmid DNA to mutant E2 DNA was increased from 1:5 to 2:1 (Fig. 4C
). When CysD2, a mutant D2 that is expressed at approximately 100-fold higher levels than wild-type D2 (3) was expressed, the effect of mutant E2 coexpression was lost (Fig. 4C
).
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If the increase in D2 activity due to combined E2 mutant coexpression was due to impaired ERAD of D2, then the half-life of D2 should be prolonged. We therefore measured the effects of E2 mutant coexpression on both D2 activity half-life and protein half-life. To determine the effect on D2 activity half-life, HEK-293 cells transiently expressing D2 with or without the E2 mutants were processed for D2 activity under basal conditions and after 2 h exposure to cycloheximide (CX) or rT3 treatment. In control cells expressing D2 alone, D2 activity fell by approximately 50% after 2 h of CX treatment. The effect of the mutant E2s was measured using two different ratios of transfected D2 plasmid DNA to E2 mutant DNA. In cells transfected with a 1:5 ratio, D2 activity fell by only approximately 20% after CX (Fig. 5A), indicating a prolongation of its activity half-life, whereas a 2:1 ratio resulted in a decrease in D2 activity of approximately 40% (1.0 ± 0.03 vs. 0.59 ± 0.05 fold). Coexpression of the E2 mutants also blunted the substrate-accelerated loss of D2 activity after treatment with 30 nM rT3 (Fig. 5A
).
To determine whether the changes in D2 activity half-life reflected changes in D2 protein concentration, we measured the effect of combined mutant E2 coexpression on D2 protein half-life using pulse-chase 75Se labeling of FLAG-tagged D2 transiently expressed in HEK-293 cells (Fig. 5B). Given the short half-life of the D2 mRNA (19) and the general inefficiency of selenoprotein synthesis (20), this required cells to be transfected with at least 10 µg D2 plasmid DNA (18). In these experiments, the amount of 75Se-labeled D2 decreased by approximately 50% after a 2-h chase period with media containing 1 µM Na2SeO3 (Fig. 5B
), consistent with the approximately 2-h half-life of D2 in this system (7). When 10 µg D2 plasmid DNA were cotransfected with 5 µg each of MmUBC6M and MmUBC7M, the basal amount of D2 protein was increased by approximately 20%, and the half-life was significantly prolonged, i.e. 75Se-D2 protein fell only by approximately 30% over 2 h (Fig. 5B
). The magnitude of these changes in protein half-life was similar to those observed for D2 activity half-life when the same ratio of transfected DNA was used (Fig. 4C
).
D2 Activity Is Stabilized during Coexpression with a Truncated Carboxyl-Terminal D2
If UBC7-mediated ubiquitination is critical for D2 inactivation, then it should be possible to inhibit this process by titrating the limiting components of the ERAD pathway (UBC7 and E3) with the fragment of D2 containing the residues (aa 169234) that physically associate with the ERAD complex. Initially, a full-length inactive mutant D2 in which Sec 133 was replaced with Ala (AlaD2) was evaluated to determine whether wild-type D2 activity would be affected in this system (Table 1A). The Ala for Sec133 substitution does not affect the kinetics of D2 degradation, suggesting that the basal ubiquitination mechanism is not affected by this change (7). When AlaD2 was coexpressed with wild-type D2, however, the resulting D2 activity at 48 h was increased 1.7- to 2.2-fold as compared with controls (Table 1A
).
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To confirm that the coexpression with AlaD2 led to stabilization of D2 as opposed to affecting D2 by another mechanism, D2 activity half-life was measured after CX exposure in the presence or absence of AlaD2. The decrease in D2 activity after 2 h when expressed with the empty vector was approximately 45% (Table 1B). The specificity of this stabilization was confirmed by comparing the effect of AlaD2 on wild-type D2 activity with that of AlaD1 and AlaD3 (the analogous inactive D1 and D3 constructs with Ala in place of Sec in the active center). Neither AlaD1 nor AlaD3 coexpression affected wild-type D2 activity (with AlaD1, activity fell
54 ± 12%; with AlaD3, activity fell
47 ± 8%; both P < 0.05 vs. vehicle).
Having confirmed that the cellular ERAD machinery can be saturated using an inactive D2 mutant, we tested this possibility using the inactive truncated carboxyl region D2 (CC27, aa 129273 of D2). When CC27 was coexpressed with wild-type D2, D2 activity increased to a similar magnitude as with AlaD2 (Table 1C). This increase in activity also occurred on the basis of an increase in D2 activity half-life (data not shown). Thus, coexpression of the truncated carboxyl region of D2 was sufficient to stabilize wild-type D2 activity in HEK-293, indicating the importance of this region in D2 ERAD.
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DISCUSSION |
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To understand the individual role of each of these E2s in D2 ubiquitination, we pursued a previously described functional strategy in which the ERAD mechanism is saturated via overexpression of inactive UBC6 and/or UBC7 mutants (10). In this setting D2 activity was stabilized to a degree that can be explained by changes in D2 protein half-life (Figs. 4 and 5
). That both UBC6 and UBC7 must be neutralized to achieve D2 stabilization (Fig. 4B
) suggests that redundancy exists at the level of the E2, i.e. either UBC6 or UBC7 can catalyze D2 ubiquitination. Thus, coexpression of the individual E2 mutants had no effect on D2 ERAD. However, the fact that MmUBC6 does not bind with the same affinity to the D2 ERAD complex as does MmUBC7 (Fig. 1B
) suggests that there may be a preference for UBC7-mediated D2 ERAD. In fact, it has been suggested that UBC7, rather than UBC6, is dominant in ERAD (10, 25, 28), and controversy exists as to the role of UBC6 in the ubiquitination of some ERAD substrates including the T cell receptor subunits
and CD3-
(29).
On the other hand, the small amount of MmUBC6 visualized when GST-D2 and MmUBC7 were present in the binding assay (Fig. 1D) supports the hypothesis that both E2s could theoretically coexist in the D2-ERAD complex. This would be consistent with a model in which both E2s are present in the ubiquitinating complex, as may occur in yeast D2 ERAD (16). Such a model, in which multiple E2 enzymes can affect rates of ERAD, has been proposed for other substrates, but limited mechanistic data are available (30). However, the finding that individual neutralization of either E2 does not stabilize D2 is not consistent with this hypothesis (Fig. 3B
). In addition, our data do not allow determination of whether MmUBC6 is binding to a putative E3 or, as has been suggested, to MmUBC7 (12).
The novelty of the present functional data becomes apparent when it is considered in light of our previous studies in yeast strains lacking either UBC6 or UBC7 activity (16). Contrary to the situation in yeast, the neutralization of individual E2 activities in HEK-293 cells did not stabilize D2 (Fig. 3B). Whereas ERAD mechanisms are highly conserved from yeast to mammals in a general sense, recent data indicate that increased complexity exists in the mammalian pathways. For example, two mammalian subfamilies of UBC6-related proteins were recently identified, raising the possibility that the function of the single yeast UBC6 is split between the two mammalian subfamilies (29). Although at the present time we do not fully understand these differences, the D2-ERAD model may lend itself for future comparison between yeast and mammalian ERAD.
It is also interesting to note that overexpression of wild-type MmUBC6 or MmUBC7 did not decrease D2 activity. A similar pattern was seen using this system and T cell receptor subunits (10). One interpretation of this finding is that the cellular concentration of these proteins per se is not the critical determinant for initiation of D2 ERAD, i.e. UBC6 and UBC7 activity do not direct D2-ERAD. Rather, the signals for D2 ubiquitination could be intrinsic to D2, such as a change in conformation or redox status or a change in phosphorylation that could increase binding affinity with the putative D2-specific E3, as is seen with HECT-E3s (21).
In conclusion, the present data support a mechanism for the physiological regulation of D2 and thyroid hormone activation in mammals via specific ubiquitination mediated by either UBC6 or UBC7. Based on the fact that UBC7 associates with the carboxyl region of D2, it is likely that UBC7 is the primary E2 in the D2 ubiquitination complex. The functional data confirm that UBC7 is involved in D2 ubiquitination but suggest that it can be replaced by UBC6 in both constitutive and substrate-accelerated ERAD. The physiological significance of this E2 redundancy remains the subject of active investigation.
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MATERIALS AND METHODS |
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Constructs
The constructs GST-D1, GST-D2, GST-D3, GST-GUS, and BG121 were previously described (31). GST-D1 is an NH2-terminal GST-tagged rat D1 with Cys replacing selenocysteine (Sec)126 (GST-D1). GST-D2 is an NH2-terminal GST-tagged human D2 with Cys replacing Sec133 (GST-D2). GST-D3 is an NH2-terminal GST-tagged human D3 with Cys replacing Sec145 (GST-D3). GST-GUS is an NH2-terminal GST-tagged Arabidopsis Thaliana ß-glucouronidase (GST-GUS). BG121 is a COOH-terminal FLAG-tagged human D2.
GST fusions of D2 fragments in an E. coli expression vector were constructed as follows. BG94 is a carboxy-terminal FLAG-tagged human D2 with Cys replacing Sec133 and Sec266 that has been previously described (8). BG94 was selected as a PCR template given the inability of bacteria to express selenoproteins (32). PCR products containing D2 fragments flanked by attB1 sites were created by amplifying BG94 via the Expand High Fidelity PCR System (Roche Clinical Laboratories) and the following primers: GST-D2 TM (5'-attB1 sense, GGGG ACAAGTTT GTAC AAAA AAGC AGGC TTGG GCTG GGCG ATAC CGGG GGAC; 3'-attB1 antisense, GGGG ACC ACT TTG TAC AAG AAA GCT GGG TT TCA CTC TCC GCG AGT GGA CTT G), GST-D2 NH2 42104 (5'-attB1 sense, GGGG ACA AGT TTG TAC AAA AAA GCA GGC TT CGC TCC AAG TCC ACT CGC GGA G; 3'-attB1 antisense, GGGG ACC ACT TTG TAC AAG AAA GCT GGG TT TCA CTC CTG GGT ACC ATT GCC), GST-D2 COOH 194273 (5'-attB1 sense, GGGG ACAA GTTT GTAC AAAA AAGC AGGC TTAG CAGC CCAG CAGC TTCT GGAG; 3'-attB1 antisense, GGGG ACCA CTTT GTAC AAGA AAGCTGGGTTTA ACCAGCTA ATCT AGTT TT), GST-D2 COOH 234273 (5'-attB1 sense, GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTG AGA CAG AAA ATT GCT TAT CTG; 3'-attB1 antisense, GGGG ACCA CTTT GTAC AAGA AAGCTGGGTTTA ACCAGCTA ATCT AGTT TT), GST-D2 COOH 169234 (5'-attB1 sense GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT GGG CTG GGC GAT ACC GGG GGA C; 3'-attB1 antisense GGGG ACC ACT TTG TAC AAG AAA GCT GGG TT TCA CTG CAC AAT GCA CAC ACG). The resulting products were used to create pDONR201 constructs using the BP reaction of the Gateway PCR system (Promega Corp.) following the manufacturers instructions. This vector was then used to generate E. coli expression vectors (pDEST15 constructs) containing an NH2-terminal GST fusion via the LR reaction. Constructs were confirmed by sequencing.
AlaD1, AlaD2, and AlaD3 have been previously described (31, 33). Briefly, AlaD1 (also called BG132) is an NH2-terminal FLAG-tagged human D1 with Ala replacing Sec126. AlaD2 (also called CC16) is an NH2-terminal FLAG-tagged human D2 with Ala replacing Sec133. AlaD3 (also called CC9) is an NH2-terminal FLAG-tagged human D3 with Ala replacing Sec145. Wild-type human D2 (hD2SelP) has been previously described (34). CC27 is a truncated human D2 (aa 129273), with Cys replacing Sec133 and Sec266. Briefly, the oligos Bp155 and Bp85 (Bp155 sense, ggaa ttca ttAT GGGC TCAG CCAC TTGt CCTC CTTT CA, Bp85 antisense ttcc cggc cgct atgg ccga cgtc gac ttaa ccag ctaa tcta gttt tctt tcat ct) were used for vent PCR with BG94 template. The resulting vent fragment was cut with EcoRi/XbaI and put into EcoRi/XbaI of BG94. CC27 was confirmed by sequencing.
Mammalian E2 constructs were kindly provided by Dr. Swati Tiwri and Dr. Allan Weissman (10). MmUBC6 is an NH2-terminal hemagglutinin-tagged murine UBC6 in pCI vector. MmUBC7 is an NH2-terminal Myc tagged murine UBC7 in pcDNA3 vector. The construct C94SMmUBC6, designated as MmUBC6M in this communication, is MmUBC6 with Ser replacing Cys 94 in pCI. The construct C89SMmUBC7, designated as MmUBC7M in this communication, is MmUBC7 with Ser replacing Cys 89 in pcDNA3.
E. coli Overexpression of GST-Deiodinase Fusion Proteins
GST-tagged constructs were overexpressed as described previously (31). Briefly, transformed cultures of E. coli BL21 DE3 pLysS were grown until log phase, and then induced with 0.2 mM isopropyl-ß-D-thiogalactopyranoside for 3 h at 37 C. Cells were pelleted and stored at -20 C. Pellets were thawed on ice and resuspended in cold extraction buffer composed of 25 mM Tris (pH 7.5), 1 mM EDTA, 20 mM NaCl, 20% glycerol, plus type III protease inhibitors (Calbiochem) and sonicated. This lysate was spun at 16,000 x g for 15 min at 4 C, and the supernatant was stored at -70 C. To quantitate the amount of protein expressed, 50 µl of the crude bacterial extract were incubated for 1 h at room temperature with 30 µl of glutathione sepharose resin that had been prewashed twice with 1 ml of 1 x PBS. After incubation, the resin mixture was washed with 1 ml of 25 mM Tris buffer (pH 7.0), containing 0.5% Triton X-100, 300 mM NaCl, 1 mM CaCl2 (wash buffer 1) followed by 1 ml of 25 mM Tris buffer (pH 7.0), containing 140 mM NaCl, 1 mM CaCl2 (wash buffer 2). The pellets were then resuspended in SDS-PAGE buffer, resolved using 12% SDS-PAGE, and the proteins were visualized via Coomassie blue staining. The extracts were then normalized for GST-fusion protein concentration via addition of extraction buffer as necessary.
GST Pull-Down Assays
E2 constructs (MmUBC6, MmUBC6M, MmUBC7, MmUBC7M) were translated in vitro using a reticulocyte lysate kit (Promega Corp.). One microgram of plasmid DNA was used per 50-µl reaction along with 2 µl of [35S]methionine. Sixty microliters of the GST-tagged protein bacterial cell extracts were incubated with 30 µl of glutathione sepharose resin for 1 h at room temperature with gentle agitation. These mixtures were then centrifuged at 10,000 x g for 1 min and the supernatant discarded. Ten microliters of the in vitro translation mixture or mixtures were then added to the pellet, with incubation overnight at 4 C with gentle agitation. After centrifugation at 10,000 x g for 1 min, pellets were washed with 1 ml of wash buffer 1 and 1 ml of wash buffer 2. Loading buffer was then added to the resulting pellet, boiled for 5 min, and protein resolved via 12% SDS-PAGE.
Transfections and Assays
HEK-293 cells were transfected using the calcium phosphate method as previously described (35) with cotransfection of TKGH for control of transfection efficiency (36). One half to 10 µg of wild-type D1, D2, or D3 and 15 µg of E2 plasmid DNA were used as indicated. Unless specified, cells were harvested 48 h later. For activity half-life studies, the above protocol was modified by treatment either with vehicle (dimethylsulfoxide) or with 100 µM CX for 2 h before harvesting. For substrate-accelerated ubiquitination studies, the above protocol was modified by treatment with either vehicle (40 mM NaOH) or with 30 nM rT3 for 2 h before harvesting. For substrate competition studies, HEK-293 cells were cotransfected with wild-type D2 and with D10 (empty vector), AlaD2, or CC27, as indicated. Cells were harvested at 48 h and processed for D2 activity. For activity half-life studies, cells were treated with 100 µM CX or vehicle for 2 h before harvesting.
Deiodinase activities were assayed as described previously (6). D1 was assayed in the presence of 10 nM [125I]rT3, D2 in the presence of 2 nM [125I]T4, and D3 in the presence of 2 nM [125I]T3. The results are reported as % of converted substrate of fmol/min·mg protein. Western analysis was performed as previously described (8). Blots were probed using polyclonal anti-HA (CLONTECH, Palo Alto, CA) or monoclonal anti-MYC (CLONTECH) primary antibodies, and the signal was detected using the BM chemiluminescence Western blotting kit (Roche Clinical Laboratories) according to the instructions of the manufacturer.
D2 protein stability was assayed via immunoprecipitation of 75Se-labeled FLAG-tagged D2 protein as described previously (31) with the following modifications. Given the inefficiency of selenoprotein synthesis, 10 µg FLAG-tagged wild-type D2 plasmid DNA were used for transfection with/without 5 µg of each mutant E2 plasmid DNA as indicated. The transient expression protocol was modified by the addition of 100 nM unlabeled selenium to the media containing DMEM supplemented with 10% FBS, and 46 µCi Na2(75Se)O3/plate on d 2 after transfection. On d 3, half of the plates in each group were washed with media containing 1 µM unlabeled selenium, the cells were harvested and pelleted via centrifugation at 3000 x g for 5 min, and the dry pellets were frozen immediately at -80 C. The remaining plates were incubated for 2 additional hours in media containing 1 µM cold selenium before being pelleted and frozen. The frozen pellets were thawed and resuspended in 500 µl lysis buffer containing 0.1% Triton-X, 25 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, and type III protease inhibitor cocktail (Calbiochem). After vortexing, lysis was completed under slow agitation at 4 C for 2 h. The supernatant of 3000 x g for 5 min was incubated overnight with 5 µl of monoclonal anti-FLAG antibody. The supernatant of 1200 x g for 2 min was then incubated with 30 µl of Protein G Plus/Protein A agarose suspension (Oncogene Research Products, San Diego, CA) for 2 h, followed by centrifugation at 1200 x g for 2 min. The supernatant was removed, and the pellets were washed with the lysis buffer minus Triton X-100 and protease inhibitors, and then washed again with buffer containing 25 mM Tris-HCl, 140 mM NaCl, and 1 mM CaCl2. Pellets were resolved by 10% SDS-PAGE. Band quantification was performed using Typhoon 9410 gel and blot imager (Amersham International, Buckinghamshire, UK) and ImageQuant 5.2 software (Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical Analysis
All data presented are the mean ± SD. Comparisons were performed by one-way ANOVA followed by the Newman-Keuls test. We considered P < 0.05 for rejection of the null hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: aa, Amino acids; CX, cycloheximide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GST, glutathione-S-transferase; GUS, Arabidopsis thaliana ß-glucouronidase; HEK-293, human embryonic kidney-293.
Received for publication March 10, 2003. Accepted for publication August 11, 2003.
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REFERENCES |
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