Endoplasmic Reticulum-Associated Degradation of the Human Type 2 Iodothyronine Deiodinase (D2) is Mediated via an Association between Mammalian UBC7 and the Carboxyl Region of D2

Brian W. Kim, Ann M. Zavacki, Cyntia Curcio-Morelli, Monica Dentice, John W. Harney, P. Reed Larsen and Antonio C. Bianco

Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital and Harvard Medical School

Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Women’s Hospital; HIM Building, Room 566, 77 Louis Pasteur Avenue, Boston, Massachusetts 02115. E-mail: abianco{at}partners.org.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The type 2 iodothyronine selenodeiodinase (D2) is an endoplasmic reticulum (ER)-resident selenoprotein that activates T4 to T3, playing a critical role in thyroid homeostasis. D2 has an approximately 45-min half-life due to selective ubiquitin-mediated ER-associated degradation (ERAD), a process of particular interest because it is accelerated by exposure to D2 substrates, T4 or rT3. The present in vitro binding studies indicate that glutathione-S-transferase (GST)-human D2 fusion proteins specifically associate with a mammalian homolog of the ubiquitin conjugase UBC7 (MmUBC7), with localization to amino acids 169–234 of D2. Coexpression of D2 with an inactive D2 mutant or a truncated version containing amino acids 169–234 stabilizes D2 half-life, supporting the importance of the carboxyl region of D2 for ERAD. Mammalian UBC6 (MmUBC6) does not directly associate with D2 but can associate with a complex containing UBC7 and D2. At the same time, functional studies in human embryonic kidney-293 cells indicate that D2 activity half-life and protein levels are stabilized only when inactive mutants of both UBC6 and UBC7 are overexpressed with D2, suggesting that redundancy may exist at the level of the E2 for both basal and substrate-accelerated D2 ERAD. In conclusion, D2 ERAD in human cells proceeds via an association between UBC7 and the carboxyl region of D2, a unique mechanism for the control of thyroid hormone activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THYROID HORMONE ACTION is initiated by the activation of T4 to T3, an outer ring monodeiodination reaction that is catalyzed by the type 1 or type 2 iodothyronine selenodeiodinase (D1 or D2). Irreversible inactivation of T4 and T3 occurs via inner ring monodeiodination catalyzed by the type 3 iodothyronine selenodeiodinase (D3). Whereas thyroid status depends on the combined actions of the three deiodinases, the substantial physiological plasticity exhibited by D2 suggests that it is the critical T3-producing deiodinase in the adaptive responses to changes in iodine supply, to cold exposure, and to changes in thyroid gland function (1).

The three selenodeiodinases are membrane-anchored proteins of 29–33 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 {alpha} and CD3-{delta} 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Vitro Interactions of MmUBC6 and MmUBC7 with D2
The possibility of complex formation involving D2 with MmUBC6 and/or MmUBC7 was tested by incubating in vitro synthesized [35S]methionine-labeled E2 proteins (Fig. 1AGo) with full-length or truncated GST-tagged D2 proteins that were expressed in Escherichia coli (Fig. 1BGo). GST pull-down and SDS-PAGE with autoradiography were used to identify D2-E2 association. [35S]MmUBC7 specifically associated with full-length GST-D2 [amino acids (aa) 1–273] (Fig. 2AGo). In contrast, there was no association between the E2s and GST-Arabidopsis thaliana ß-glucouronidase (GUS) or GST-D3. The association of [35S]MmUBC7 and D2 localized to the carboxyl region of the D2 molecule (Fig. 2AGo) and the use of additional truncated GST-D2 fusion proteins indicated that a region containing residues 169–234 of D2 is sufficient for MmUBC7 association (Fig. 2BGo). There was no significant association of [35S]MmUBC6 with GST-D2 (Fig. 2AGo). To assess whether MmUBC6 could participate in a complex with D2 and MmUBC7, the experiment was repeated using a mixture of 35S-labeled MmUBC6 and MmUBC7. Under these conditions, a small amount of MmUBC6 binding was visualized (Fig. 2CGo). In contrast, neither MmUBC7 nor MmUBC6 associated significantly with GST-GUS. Translation of [35S]MmUBC6 or [35S]MmUBC7 in the presence of canine microsomal membranes did not alter the intensity of these interactions (data not shown). In vitro binding was also assayed using an inactive mutant form of MmUBC6 in which Ser replaces Cys94 (MmUBC6M), and an inactive mutant form of MmUBC7 in which Ser replaces Cys89 (MmUBC7M). The in vitro binding results using these mutant constructs were identical to those obtained for the wild-type constructs, with [35S]MmUBC7M associating specifically with the 169–234 region of D2 (Fig. 2AGo).



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Fig. 1. GST-D2 Constructs and in Vitro Translated [35S]E2s

A, SDS-PAGE of in vitro translation in reticulocyte lysates of MmUBC7, MmUBC7M, MmUBC6, and MmUBC6M in the presence of [35S]Met and [35S]Cys. B, Schematic of GST-D2 full-length fusion protein with amino acid residues indicated by numbers. Residues 1–42 correspond to the single putative transmembrane domain (TM). Cys replaces Sec133 to allow for expression in bacteria.

 


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Fig. 2. Association of 35S-Labeled in Vitro Translated E2s with GST-D2 Fusion Proteins

A, 12% SDS-PAGE of GST pull-down pellets. For each pull down, truncated D2 (amino acid sequences indicated by numbers) fused to GST is shown above each lane. Negative controls include GST-D3 and GST-GUS. The E2s were 35S labeled during in vitro translation (IVT); np, not performed. B, Same as panel A, with different GST-D2 truncated proteins used. C, Same as panel A, with a mixture of MmUBC6 and MmUBC7 used as the target.

 
When full-length GST-D2 and the reticulocyte lysates-translated [35S]MmUBC7 were incubated for binding studies, a ladder pattern of high molecular weight bands was visualized (Fig. 3AGo). These bands, which were not present when GST-GUS was used in place of D2, developed in a time-dependent manner (Fig. 3BGo) when [35S]MmUBC7 was incubated with D2. Visualization of these bands is dependent on the presence of the [35S]MmUBC7 signal. Given that MmUBC7 is only approximately 19 kDa, these bands likely represent protein complexes containing [35S]MmUBC7 along with the substrate D2 and possibly other components of the ubiquitinating complex. The varying sizes of the bands may reflect the degree of ubiquitination of D2: a similar pattern of high molecular weight bands containing ubiquitinated D2 has been documented to occur when D2 is translated in reticulocyte lysates (17) or when D2 is transiently expressed in HEK-293 cells (8).



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Fig. 3. Visualization of MmUBC7-Containing High-Molecular Weight Bands

A, 12% SDS-PAGE of GST pull-down pellets of 35S-labeled MmUBC7-containing reticulocyte lysates that had been incubated overnight with GST-D2 or GST-GUS. UNP, Unprogrammed lysate. B, Time course experiment; same as in panel A, but incubation time was 1–8 h and pellet was resolved by 10% SDS-PAGE for resolution of the high-molecular weight bands.

 
Overexpression of Both MmUBC6M and MmUBC7M Is Required to Stabilize D2 in Vivo
In prior studies we have determined that ubiquitinated D2 is inactive (8). If UBC6 or UBC7 are critical in ERAD of D2, then overexpression of these E2s could accelerate D2 ubiquitination and degradation. Conversely, competition for UBC6- or UBC7-D2 interactions by overexpression of cognate mutant enzymes might prolong substrate half-life, as has been previously demonstrated for T cell receptor subunits (10). We sought to confirm the functional significance of the interaction between MmUBC7 and MmUBC6 with D2 in vivo by analyzing these possibilities.

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. 4AGo). Coexpression of D2 with the wild-type E2s either individually or in combination did not reduce D2 activity (Fig. 4BGo). 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. 4Go, B–D, and Fig. 5AGo). 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. 4CGo). 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. 4CGo).



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Fig. 4. Coexpression of Active and Inactive MmUBC6 and/or MmUBC7 with D2 in HEK-293 Cells

A, HEK-293 cells transiently expressing wild-type or mutant E2s were processed for Western analysis using anti-HA (MmUBC6 and MmUBC6M) or anti-MYC (MmUBC7 and MmUBC7M) antibodies. A is MmUBC7, B is D10 vector, C is MmUBC7M, D is MmUBC6, E is D10 vector, and F is MmUBC6M. Plasmid DNA (5 µg) was transfected per 60-mm plate. B, D2 activity in HEK-293 cells transiently expressing D2 and either vector (D10) or E2(s) as indicated. Results are expressed as D2 activity normalized for GH expression, relative to activity with D10. D2 plasmid DNA (1 µg) was transfected into all cells along with 5 µg of each E2/E2 mutant or D10 plasmid DNA as indicated. Values are mean ± SD of three to six plates. *, P < 0.05 by ANOVA vs. D10. C, Effect of changing the ratio of transfected D2 to mutant E2 DNA on D2 activity. The experiment was performed as in panel B except that the ratio of D2 plasmid DNA to E2 mutant DNA was progressively increased from 1:5 (1 µg D2) to 2:1 (10 µg D2). In the two far right bars the 2:1 DNA ratio was applied but 10 µg CysD2 plasmid DNA was used. All values were normalized to results with D10. *, P < 0.05 by Student’s t test vs. the corresponding control (D10). D, Deiodinase activities in HEK-293 cells transfected with either D1, D2, or D3 plasmid DNA with or without the combined (MmUBC6M + MmUBC7M) mutant E2 DNAs. All cells were transfected with 1 µg deiodinase plasmid DNA. The amount of E2 mutant DNA transfected is indicated; if not indicated, the amount is the same as in panel B. Values are mean ± SD of three to six plates. *, P < 0.05 by ANOVA vs. D10; **, P < 0.05 by ANOVA vs. 1 µg of mutant E2 DNA.

 


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Fig. 5. Effect of MmUBC6M and MmUBC7M Combined Overexpression on D2 activity and Protein Half-Life and Response To Substrate

A, Experiments were performed as in Fig. 4BGo, except that cells were treated with vehicle, 30 nM rT3, or 100 µM CX for 2 h before harvesting. Values are mean ± SD of three to six plates. *, P < 0.05 by ANOVA vs. vehicle. B, 10% SDS-PAGE of immunoprecipitated [75Se]D2 from HEK-293 cells transiently expressing wild-type FLAG-tagged D2 with or without MmUBC6M and MmUBC7M (E2 mutants) as indicated. Cells were labeled with 75Se overnight and chased for 2 h with media containing 1 µM unlabeled Se. Anti-FLAG antibody was used for immunoprecipitation. The bands were quantified and the results are expressed in densitometric units (DU) as average ± SD relative to D2 density at time 0. All results are significantly different from each other by ANOVA (P < 0.05). The two lanes on the far right were digitally moved for logical presentation of the data.

 
To confirm the specificity of these results on D2, the experiments were repeated using the nonubiquitinated deiodinases D1 and D3 (Fig. 4DGo). Coexpression of the E2 mutants had no significant effect on either D1 or D3 activity.

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. 5AGo), 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. 5AGo).

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. 5BGo). 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. 5BGo), 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. 5BGo). 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. 4CGo).

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 169–234) 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 1AGo). 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 1AGo).


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Table 1. D2 Activity in HEK-293 Cells Cotransfected with Wild-Type D2 (wtD2) and Inactive Forms of D2

 
As previously mentioned, selenoprotein synthesis is inefficient, such that cognate mutant proteins with substitution of Sec with Ala or Cys have approximately 100-fold greater expression (vs. wild type) at equal ratios of transfected plasmid (20). Thus, the ratio of AlaD2 to wild-type D2 protein is likely to be on the order of approximately 2000:1 when 10 µg AlaD2 plasmid DNA and 0.5 µg wild-type D2 plasmid DNA are cotransfected and the cells harvested at 48 h. A 4-fold reduction of this ratio (5 µg AlaD2 to 1 µg wild-type D2 plasmid DNA) did not reduce the effect on D2 activity (Table 1AGo).

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 1BGo). 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 129–273 of D2). When CC27 was coexpressed with wild-type D2, D2 activity increased to a similar magnitude as with AlaD2 (Table 1CGo). 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present studies provide the first details of the ERAD mechanism for D2 in mammalian cells. The major novel finding is that there is a high-affinity, specific physical association between MmUBC7 and a carboxy region (aa 169–234) of the D2 enzyme (Fig. 1Go), a region that is exposed to the cytosol according to prior topological analysis (3). The functional stabilization of wild-type D2 in HEK-293 by coexpression of an inactive carboxyl region-truncated D2 (CC27) (Table 1CGo) also suggests that a critical interaction occurs in this region. While direct binding of E2s has been proposed for certain substrates (11, 21, 22, 23), current models of ubiquitination suggest that E2s play only a secondary role in substrate recognition, with E3 enzymes being the major specificity determinant (11). It is therefore likely that the D2-MmUBC7 association is mediated by an as-yet-unknown E3 or other adaptor protein(s) present in the reticulocyte lysate. Although several E3s important in ERAD have been identified (24, 25, 26, 27), these are largely membrane proteins that would not be expected to be present in microsome-free reticulocyte lysate.

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. 4Go and 5Go). That both UBC6 and UBC7 must be neutralized to achieve D2 stabilization (Fig. 4BGo) 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. 1BGo) 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 {alpha} and CD3-{delta} (29).

On the other hand, the small amount of MmUBC6 visualized when GST-D2 and MmUBC7 were present in the binding assay (Fig. 1DGo) 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. 3BGo). 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. 3BGo). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
CX and type III protease inhibitors were obtained from Calbiochem (La Jolla, CA) and dissolved in dimethylsulfoxide. Triton X-100 and anti-FLAG M2 antibody were obtained from Sigma Chemical Co. (St. Louis, MO). BM chemiluminescence Western blotting kit (mouse/rabbit) was from Roche Clinical Laboratories (Indianapolis, IN). Polyvinylidene fluoride (Immobilon) membrane was from Millipore Corp. (Bedford, MA). TnT Quick Coupled Transcription/Translation System and Canine Pancreatic Microsomal Membranes were from Promega Corp. (Madison, WI). E. coli BL21 DE3 pLysS cells were obtained from Stratagene (La Jolla, CA). Glutathione sepharose 4B resin was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Protein G Plus/Protein A Agarose suspension was obtained from Oncogene Research Products (Boston, MA). L-[35S]methionine (Easytag) was obtained from Perkin Elmer (Boston, MA). Na2(75Se)O3 was kindly provided by the University of Missouri Research Reactor, courtesy of Drs. Marla Berry and Dolph L. Hatfield. Gateway cloning and expression systems were obtained from Invitrogen (Carlsbad, CA). Autofluor was obtained from National Diagnostics (Atlanta, GA). All other reagents were of analytical grade.

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 42–104 (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 194–273 (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 234–273 (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 169–234 (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 manufacturer’s 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 129–273), 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 1–5 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 4–6 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.


    ACKNOWLEDGMENTS
 
We thank Drs. Swati Tiwari and Allan Weissman for the plasmids encoding wild-type and mutant MmUBC6 and MmUBC7.


    FOOTNOTES
 
This work was supported by NIH Grant DK58538. B.W.K. was a recipient of a grant from the Endocrine Fellows Foundation.

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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