| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Thyroid Division (L.A.J., J.W.H., A.C.B.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Institute of Experimental Medicine (B.G.), Department of Neurobiology, Budapest H-1078, Hungary
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Women’s Hospital, Harvard Institutes of Medicine Building, Room 550, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: abianco{at}partners.org.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
30 min). Clasto-lactacystin ß-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T4. Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated. Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants. Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein. In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Because of its physiological plasticity D2, not D1, is considered the critical homeostatic deiodinase during adaptation to iodine deficiency, cold exposure, or changes in thyroid status. In constrast to D1, D2 has a relatively low Km (Michaelis-Menten constant; T4) and a short half-life, enabling it to respond within minutes to neural and endocrine stimuli (see Ref. 1 for review). D2 is a 32-kDa type 1, endoplasmic reticulum (ER)-resident membrane protein that has a short lumenal NH2 terminus and a single-transmembrane domain within its first 40 amino acids. The bulk of the enzyme is in the cytosol, including its Sec-containing catalytic active center (2). In immunocytochemical studies using confocal microscopy, its distribution is typically perinuclear and it colocalizes with ER-resident binding protein when transiently expressed in HEK-293 and NB-2A cells, and in a human mesothelioma cell line (MSTO-211) where it is endogenously expressed (2, 3).
D2 is an unstable protein with a half-life of approximately 45 min due to its susceptibility to ubiquitination and proteasomal degradation (4, 5, 6). Both D2 activity and protein levels are rapidly stabilized in GH4C1 rat pituitary tumor cells and MSTO-211 cells by treatment with proteasome inhibitors (3, 4). Proteasomal uptake of D2 requires accessibility to its COOH terminus because its fusion to the FLAG epitope (but not to the NH2 terminus) increases severalfold the pool of ubiquitinated D2 and prolongs its half-life by 3- to 4-fold (6). A unique feature of D2 is a further enhancement of its intrinsic metabolic instability after exposure to its substrates, T4 or rT3. This substrate-induced loss of D2 activity and protein was first demonstrated in vivo (7) and subsequently confirmed in a number of cell systems (3, 4, 8). Interaction with the Se-containing enzyme’s active center accelerates, by unknown mechanisms, D2 ubiquitination and subsequent proteolysis (5). There have been reports of proteasome-mediated, catabolite-induced enzyme inactivation (9, 10), but D2 displays this unique mechanism of substrate-induced selective proteolysis that regulates its own levels and hence thyroid hormone activation. In contrast, the proteasome system does not appear to play a significant role in the posttranslational regulation of the other two members of the selenodeiodinase family (6).
In eukaryotes, normal ER-resident proteins such as D2 are degraded by the ER-associated degradation (ERAD), which also functions as cellular protein quality control (11, 12). Selective proteolysis occurs by the 26S proteasome complex after retrograde transport to the cytosol and ubiquitination. ERAD involves a variety of components, among which enzymes of the ubiquitin system are pivotal. Selection of specific proteins for proteolysis is usually achieved at the level of ubiquitin conjugation to the target, a process that is coordinated by the combined actions of a series of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). This system is very well characterized in the yeast Saccharomyces cerevisiae where approximately a dozen E2s or E2-related proteins are known (13). A conserved catalytic domain of approximately 150 amino acids characterizes E2s. Individual E2s are involved in different cellular processes and, therefore, in the ubiquitination of different classes of substrate proteins. E3s, on the other hand, more abundant and with no overt sequence homology, are thought to be largely responsible for the high degree of specificity of protein ubiquitination (14). Few examples of E3s participating in ERAD are available but it is likely that more exist (15, 16, 17, 18, 19, 20).
In yeast, a number of ER-resident proteins that are ERAD substrates undergo ubiquitination, including Sec61p (21) and carboxypeptidase Y (22), as well as hydroxy-3-methylglutaryl-coenzyme A-reductase (23). Three E2s, namely Ubc1p, Ubc6p, and Ubc7p, have been implicated in ERAD (15). Deletion of UBC6 and UBC7 genes stabilizes a mutant Sec61p, Sss1p, carboxypeptidase Y, Pdr5, and uracil (URA) permease (21, 22, 24, 25). Ubc6p is a C-terminal anchored membrane protein whose catalytic site faces the cytosol (26). Unlike Ubc6p, Ubc7p lacks a membrane anchor but associates with an ER-bound protein, Cue1p (27). Ubc1p is part of an E2 group that includes Ubc4p and Ubc5p, essential for cell growth, viability, and in-bulk turnover of short-lived and abnormal proteins (16, 19, 28, 29).
The goals of the present investigation were to express a functional D2 in S. cerevisiae and identify which E2s are involved in the normal and substrate-induced D2 ubiquitination and selective proteolysis. Here we report that a functional Sec133D2Cys in S. cerevisiae retains its metabolic instability displayed in mammalian cells and is further destabilized upon exposure to T4. Both natural decay and substrate- induced D2-selective proteolysis are specifically blocked by clasto-lactacystin ß-lactone and depend on UBC6 and UBC7 but not on the gene coding for the other E2 ERAD component, Ubc1p.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). Controls included uninduced transformed cells in which T4-to-T3 conversion was minimal at all times (not shown) and cells transformed with empty pYes2 vector in which deiodinase activity remained at background levels regardless of the presence of 2% galactose (Fig. 1A).
|
). One of the properties of D2 that differentiates it from D1 is its insensitivity to inhibition by PTU. To verify D2 identity, all assays of deiodinase activity in yeast cell extracts were performed in the presence of 1 mM PTU, which only reduced total deiodinase activity 5–10%. CysD2 identity was also confirmed by the kinetics of the deiodination to T4 (Fig. 1C). The calculated Km (T4) is approximately 2.5 µM, supporting that the T4 deiodinase activity found in the transformed yeast cells is authentic cysD2.
Evidence for Proteasomal Involvement in Basal and Substrate-Induced CysD2 Proteolysis in S. cerevisiae
To establish the relative CysD2 stability in S. cerevisiae, CysD2-expressing wild-type and cell wall- permeable Ise1 cells (30) were treated with 100 µg/ml cycloheximide (CX) for 30 min, harvested, lysed, and processed for CysD2 activity. Samples of similarly treated Ise1 cells were processed and analyzed by Western blot using anti-FLAG antibody to determine levels of FLAG-tagged CysD2 protein. Treatment with CX caused a rapid fall in CysD2 activity to 35–50% of control values (Table 1). We also tested whether the degradation of CysD2 in yeast is accelerated by substrate (30 µM T4). In both wild-type and in Ise1 cells D2 activity was rapidly lost after exposure to substrate (Table 1 and Fig. 2) but not to 30 µM T3 (not shown). Loss of D2 activity is likely the result of CysD2 proteolysis because CysD2 protein levels fell accordingly during basal and substrate exposure conditions (Fig. 2). In parallel experiments we used 30 µM Tetrac to induce CysD2 down-regulation, a T4 analog with reportedly greater permeability in yeast (31), but similar findings were obtained (not shown).
|
|
) due to a prolongation of its half-life (Fig. 5A). This is likely due to increased levels of D2 protein as indicated in the Western analysis (Fig. 2). Treatment with clasto-lactacystin ß-lactone also blocked the CX- and T4-induced loss of D2 activity (Table 1) and protein decay (Fig. 2).
|
). These bands are specific to D2 because they are absent from the lane loaded with lysates of cells transformed with empty pYes2. The identity of these high molecular mass bands was investigated after the cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and the pellets immunoblotted with anti-FLAG antibody or antiubiquitin antiserum after elution with an excess of FLAG peptide (Fig. 3). In the IP lane probed with anti-FLAG antibody, there is a perfect match with the bands detected in the total cell lysate, confirming the specificity of the anti-FLAG antibody. When IP pellets were probed with the antiubiquitin antiserum, the CysD2 band almost disappeared but the staining of the highest molecular mass bands, which matched those visualized with the anti-FLAG antibody, was even more intense, compatible with their identity as ubiquitin-CysD2 conjugates. It is notable that these ubiquitin-CysD2 conjugates are smaller than those of 100- to 300-kDa forms in HEK-293 cells (Fig. 3).
|
). Exposure to 2% galactose induced D2 activity by approximately 6.8-fold in wild-type cells but the induction in Ubc6p- and Ubc7p-deficient mutants was much higher, approximately 11- and 17-fold, respectively (Table 2). No increased induction was observed in the Ubc1p-deficient yeast mutants. The Western analysis of these cell lysates using anti-FLAG antibody revealed that the expression of D2 protein is much higher in the UBC6 and UBC7 mutant strains, particularly in the latter (Fig. 4), in agreement with the higher D2 activity observed in this strain. Interestingly, the intensity of the Ub-D2 bands is decreased in lysates of UBC6 and UBC7 mutants, supporting impaired D2 ubiquitination (Fig. 4). On the other hand, D2 levels are normal, and the pattern of high molecular mass ubiquitin-D2 conjugates seen in wild-type cells is present in the lysate of UBC1 cells, suggesting that the deletion of the third Ubcp involved in ERAD, Ubc1p, did not stabilize CysD2 (Fig. 4).
|
|
). A similar result was observed in the mutant strains. In the first 10 min after dextrose addition, there was a transient 20–40% increase in CysD2 activity possibly due to dextrose being a superior carbon source than galactose (Fig. 5, B–D). After that, however, CysD2 activity disappeared in the wild-type cells with a half-life of approximately 23 min. The levels of CysD2 protein, as detected by Western analysis using anti-FLAG antibody, were also rapidly decreased after Dio2 transcription was blocked (Fig. 5E). Interestingly, in both UBC6 and UBC7 strains there was a prolongation of the D2 activity half-life to more than 60 min, whereas no change in activity half-life was detected when CysD2 was expressed in the UBC1 strain (Fig. 5, B–D). This correlates with the levels of CysD2 protein, stabilized in the UBC6 and UBC7 mutants after inhibition of Dio2 transcription when compared with wild-type and UBC1 cells. These results confirm that a prolongation in the CysD2 half-life caused by an abrogation of its ubiquitination and degradation is the cause of elevated CysD2 activity and protein levels in Ubc6p- and Ubc7p-deficient strains. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and 2). The CysD2 properties in yeast were similar to that in mammalian cells in a number of respects, indicating that this model system is physiologically relevant. First, CysD2 activity is localized in the microsomal fraction of yeast cell lysates, indicating membrane localization, and it displays kinetic properties indistinguishable from transiently expressed CysD2 in mammalian cells (Fig. 1). Second, CysD2 protein levels correlate with enzymatic activity (Figs. 2 and 4). Third, the short half-life of CysD2 activity and protein (30 min), as assessed by either CX-induced arrest in protein synthesis or by dextrose-induced transcriptional suppression, are remarkably similar to the values found in mammalian cells (3, 5) (Figs. 2 and 5). Under both conditions the rapid turnover rate is stabilized by treatment with proteasome inhibitors (Figs. 2 and 5). Fourth, yeast CysD2 is susceptible to the unique and highly specific substrate-induced acceleration of proteolysis mediated by the proteasome pathway (Fig. 2 and Table 1). These four points indicate that D2 ubiquitination and proteolysis in yeast is due to its intrinsic instability and substrate interaction, and not due to generalized nonspecific activation of the unfolded-protein response (UPR) due to the expression of a heterologous protein.
There is considerable evidence that D2 is degraded by proteasomes in mammalian cells (3, 4, 5, 6). The present study provides additional confirmation that proteasomes mediate selective D2 proteolysis. Galactose-induced CysD2 activity (Table 1) and protein levels (Fig. 2) are increased in Ise cells treated with lactacystin b-lactone, a yeast wall-permeable proteasome inhibitor. This increase in CysD2 activity is due to stabilization of D2 half-life, which was doubled in the presence of proteasome inhibitor (Fig. 5E). Accordingly, treatment with this proteasome inhibitor also blocked the CX-induced loss of CysD2 activity (Table 1).
Ubiquitination is required for the proteasomal degradation of D2 (6). Ubiquitin-D2 conjugates were first identified in HEK-293 cells transiently expressing CysD2-FLAG after immunoprecipitation with anti-FLAG antibody and Western analysis with antiubiquitin antiserum (6). In the present investigation, a similar strategy was used to establish the identity of the abundant FLAG-containing higher molecular mass bands detected in the Western analysis of the yeast cell lysates (Fig. 3). It is interesting that, contrary to the ubiquitin-CysD2 bands seen in HEK-293 cells, these ubiquitin-CysD2-containing yeast bands are abundant and display a ladder pattern (Fig. 3). The reasons for this discrepancy are not clear but could indicate that the proteasomal uptake of the ubiquitin-CysD2 conjugates is less efficient in yeast so that a larger more evident pool of these conjugates can build up.
Once the D2 yeast expression system was sufficiently characterized and convincing evidence of its similarity to the mammalian system and physiological relevance were obtained, mutant yeast strains that lack specific E2 enzymes of the ERAD were used to define which of these are rate limiting in D2 ubiquitination. In a previous study we employed a similar genetic analysis to establish the essential role of E1 and ubiquitination in D2 proteolysis (6). Two observations point to the involvement of Ubc6p and Ubc7p in regulating D2 proteolysis in yeast: the abundance of the ubiquitin-CysD2 conjugates is much lower and the levels of CysD2 protein are much higher than in wild-type or UBC1 cells (Fig. 4). Both results are supported by higher D2 activity (Table 1) and protein levels (Fig. 4) documented in these cells. These findings are explained by a prolonged activity and protein half-life in both UBC6 and UBC7 mutants, documented in experiments in which Dio2 transcription was blocked with dextrose (Fig. 5). This indicates that both Ubc6p and Ubc7p are directly involved in the normal turnover and ubiquitination of CysD2. Ubc1p, on the other hand, is not.
Substrate-induced loss of D2 activity and protein also occurs in yeast (Tables 1 and 2 and Fig. 2), indicating that it is mediated by intrinsic properties of the D2 protein rather than by vertebrate cell-specific mechanisms. Even though we do not yet understand the details of the molecular mechanisms involved, it is clear that substrate interaction with the enzyme’s active center accelerates ubiquitination and hence proteasomal degradation (5, 6). It is remarkable that this unique mechanism is lost in UBC6 and UBC7 mutants (Table 1). This is a clear indication that both Ubc6p and Ubc7p are involved in not only the normal turnover of D2 but also the substrate-induced CysD2 ubiquitination. On the other hand, CysD2 expression in the UBC1 mutant was indistinguishable from wild-type cells in terms of basal and galactose-inducible CysD2 activity, susceptibility to substrate exposure (Table 2), and half-life (Fig. 5). Because of the specific link between Ubc1p and UPR (16), this also supports the specificity of CysD2 proteolysis in our system, which is unrelated to UPR.
The rapid loss of D2 activity and protein could be explained by ubiquitination alone, with only a minor role played by the proteasomes. Previous studies indicate that D2 is inactive after conjugation to ubiquitin because the level of unconjugated, but not conjugated, D2 protein correlates with D2 activity (6). Given the large pool of ubiquitin-D2 conjugates, the loss of D2 activity and protein levels reflects ubiquitination and not proteolysis per se. This observation is supported by the increased D2 activity and protein in the UBC6 and UBC7 cells (Table 1 and Fig. 4) that display impaired D2 ubiquitination.
Although we do not fully understand the mechanism by which substrate interaction increases ubiquitination, it is intriguing to speculate that other Ubc6p and Ubc7p substrates might also be subject to a similar regulatory mechanism. In the case of D2, this process is a potent homeostatic regulator of the biological activity of secreted thyroid hormone.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Yeast Strains, Plasmids, Transformation, and Culture Media
The strains of S. cerevisiae used in the present investigation are described in Table 3. All were generous gifts of Dr. Alfred Goldberg (Harvard Medical School, Boston, MA). UBC1 genotype was confirmed by PCR using both primers from the UBC1 locus (32).
|
All media and related products were obtained from BIO101 (Carlsbad, CA). Yeast cells were made competent and transformed using the S.c. EasyComp Transformation Kit (Invitrogen) according to the instructions of the manufacturer. Transformed cells were grown in agar plates of synthetic defined medium (SD) containing complete supplement mixture (CSM-URA) and 2% dextrose. Single colonies were grown exponentially [optical density at 600 nm (OD600), 0.8–1.0] at 30 C in liquid CSM-URA containing 2% dextrose.
Experimental Procedures
For D2 induction studies, cells were grown exponentially at 30 C in SD containing CSM-URA and 2% galactose (16 h to OD600 0.8). In the time course experiment, induction was stopped at specific time points. In the CX- and T4-induced decay experiments, approximately 15 OD600 log-phase wild-type and Ise1 cells were harvested and resuspended in 4 ml of prewarmed SD medium containing CSM-URA, 2% galactose, and 100 µg/ml CX or 30 µM T4. CX-treated cells were harvested after 30 min and T4-treated cells were harvested after 2 h. Some cells were treated for 2 h with 20 µM clasto-lactacystin ß-lactone. At the end of all treatments cells were either processed for D2 activity or Western analysis (see below). When exposed to T4, cells were washed once in media containing 20% horse serum to deplete the cell pellet of T4. In the dextrose-induced Dio2 gene shut-off experiments, approximately 15 OD600 log-phase cells were collected by centrifugation and then shifted to a prewarmed CSM-URA medium containing 2% dextrose to shut off Dio2 gene transcription. Aliquots were taken at the indicated times and either processed for D2 activity or Western analysis.
Cell Harvesting and Lysis
Cells were collected in an equal volume of 30 mM sodium azide on ice and harvested by centrifugation at 2500 rpm for 10 min. The cell pellets were immediately frozen in liquid nitrogen and stored at -80 C. For cell lysis, pellets were resuspended in 1:1 vol phosphate buffer, pH 7.4, containing 110 mM NaCl, 5 mM EDTA, 10 mM dithiothreitol (DTT), and a number of protease inhibitors (400 µg/ml aprotinin, 145 mM benzamide, 30 µM leupeptin, 100 µg/ml pepstatin A, 1 mM phenylmethylsulfonylfluoride). An equal volume of glass beads (0.5 mm, Sigma) was added to the cell suspension which was followed by agitation. Cell disruption was achieved after 10 cycles of 1 min of blending (vortex) and 1 min cooling in ice. Lysates were then centrifuged at 3,000 rpm for 10 min and the supernatant transferred to a new tube containing sucrose in lysis buffer to a final concentration of 0.25 M and stored at -80 C until further processing. In one experiment cell lysates were processed for subcellular fractionation. These were centrifuged at 3,000 x g for 10 min. The supernatant was then transferred to a new tube and centrifuged at 100,000 x g for 60 min at 4 C. The cytosol (supernatant) was stored and the microsomal pellet washed once with lysis buffer. Both cytosol and pellet were later processed for D2 activity.
D2 Assay
D2 activity was measured as described (3). Essentially, about 300 µg total cell lysate protein were incubated for 3 h in the presence of 0.5–1 nM [125I]5'-T4, 20 mM DTT, and 1 mM PTU. Specific T4-to-T3 conversion was calculated by subtracting nonspecific deiodination in tubes containing the same amount of protein cell lysate obtained from cells of the same strain transformed with empty pYes2 vector. The background activity of these samples was less than 2%. Deiodinase activity was expressed as femtomoles T4/min·mg protein.
IP and Western Blot Analysis
In these studies cell lysates were obtained similarly as described above except that lysis buffer contained 0.5% Triton X-100 and no DTT was added (34). Total cell lysates (100 µg) were resolved by 12% SDS-PAGE and electrotransferred to a polyvinylidine difluoride membrane (Immobilon, Millipore Corp., Bedford, MA) for the Western analysis. Alternatively, cell lysates (400 µl) were immunoprecipitated with 30 µl of M2-anti-FLAG agarose affinity gel (Sigma) for 4 h at 4 C. After three successive washes with lysis buffer FLAG-tagged proteins were eluted with 50 µl of 100 µg/ml FLAG peptide. The supernatant (40 µl) was then mixed with gel loading buffer and resolved by SDS-PAGE as above. Western analysis used the chemiluminescence kit of Roche Molecular Biochemicals (Indianapolis, IN), according to the instructions of the manufacturer. The blots were probed with an anti-FLAG M2 antibody (1:3333, Sigma) or with a polyclonal antiubiquitin antibody (1:1000, Chemicon, Temecula, CA). In the Western analysis, we used a lysate of HEK-293 cells transiently expressing a FLAG-tagged D2 as positive control as described previously (6). The negative control was a lysate of yeast cells transformed with empty pYes2 vector. Protein concentrations were measured by Bradford (35).
Statistical Analysis
Data are presented as mean ± SD throughout the studies. ANOVA was used for comparative analysis; 5% was the level of significance required to reject the null hypothesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
B.G. is a Magyary Zoltán postdoctoral fellow of the Hungarian Education Ministry and supported by an Felsooktatási Kutatási és Fejlesztési Pályázat grant.
* Present address: Division of Endocrinology, Children’s Hospital Boston, Boston, Massachusetts 02115.
Present address: Alameda Rio Claro, 189, Apartment 1, 01332-010 São Paulo SP, Brazil.
Abbreviations: CSM, Complete supplement medium; CX, cycloheximide; CysD2, FLAG-tagged Sec133-to-Cys mutation of the human D2; D1, D2, D3, type 1, type 2, and type 3 monodeiodinase; DTT, dithiothreitol; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; ER, endoplasmic reticulum; ERAD, ER-associated degradation; IP, immunoprecipitated; PTU, propylthiouracil; SD medium, synthetic defined medium; Sec, selenocystine; UPR, unfolded protein response; URA, uracil.
Received for publication April 8, 2002. Accepted for publication June 3, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
2 repressor degradation. Genes Dev 15:2660–2674This article has been cited by other articles:
 |
|
 |
|
  A. M. Zavacki, R. Arrojo e Drigo, B. C. G. Freitas, M. Chung, J. W. Harney, P. Egri, G. Wittmann, C. Fekete, B. Gereben, and A. C. Bianco The E3 Ubiquitin Ligase TEB4 Mediates Degradation of Type 2 Iodothyronine Deiodinase Mol. Cell. Biol., October 1, 2009; 29(19): 5339 - 5347. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Gereben, A. M. Zavacki, S. Ribich, B. W. Kim, S. A. Huang, W. S. Simonides, A. Zeold, and A. C. Bianco Cellular and Molecular Basis of Deiodinase-Regulated Thyroid Hormone Signaling Endocr. Rev., December 1, 2008; 29(7): 898 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lamirand, G. Mercier, M. Ramauge, M. Pierre, and F. Courtin Hypoxia Stabilizes Type 2 Deiodinase Activity in Rat Astrocytes Endocrinology, October 1, 2007; 148(10): 4745 - 4753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fekete, B. C. G. Freitas, A. Zeold, G. Wittmann, A. Kadar, Z. Liposits, M. A. Christoffolete, P. Singru, R. M. Lechan, A. C. Bianco, et al. Expression Patterns of WSB-1 and USP-33 Underlie Cell-Specific Posttranslational Control of Type 2 Deiodinase in the Rat Brain Endocrinology, October 1, 2007; 148(10): 4865 - 4874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lerner, M. Corcoran, D. Cepeda, M. L. Nielsen, R. Zubarev, F. Ponten, M. Uhlen, S. Hober, D. Grander, and O. Sangfelt The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD Mol. Biol. Cell, May 1, 2007; 18(5): 1670 - 1682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kanou, A. Hishinuma, K. Tsunekawa, K. Seki, Y. Mizuno, H. Fujisawa, T. Imai, Y. Miura, T. Nagasaka, C. Yamada, et al. Thyroglobulin Gene Mutations Producing Defective Intracellular Transport of Thyroglobulin Are Associated with Increased Thyroidal Type 2 Iodothyronine Deiodinase Activity J. Clin. Endocrinol. Metab., April 1, 2007; 92(4): 1451 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Zeold, L. Pormuller, M. Dentice, J. W. Harney, C. Curcio-Morelli, S. M. Tente, A. C. Bianco, and B. Gereben Metabolic Instability of Type 2 Deiodinase Is Transferable To Stable Proteins Independently of Subcellular Localization J. Biol. Chem., October 20, 2006; 281(42): 31538 - 31543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. A. Gouveia, M. A. Christoffolete, C. R. Zaitune, J. M. Dora, J. W. Harney, A. L. Maia, and A. C. Bianco Type 2 Iodothyronine Selenodeiodinase Is Expressed throughout the Mouse Skeleton and in the MC3T3-E1 Mouse Osteoblastic Cell Line during Differentiation Endocrinology, January 1, 2005; 146(1): 195 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Kim, A. M. Zavacki, C. Curcio-Morelli, M. Dentice, J. W. Harney, P. R. Larsen, and A. C. Bianco 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 Mol. Endocrinol., December 1, 2003; 17(12): 2603 - 2612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Webster, S. Tiwari, A. M. Weissman, and R. J. H. Wojcikiewicz Inositol 1,4,5-Trisphosphate Receptor Ubiquitination Is Mediated by Mammalian Ubc7, a Component of the Endoplasmic Reticulum-associated Degradation Pathway, and Is Inhibited by Chelation of Intracellular Zn2+ J. Biol. Chem., October 3, 2003; 278(40): 38238 - 38246. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
