Endocrinology Vol. 141, No. 3 1127-1135
Copyright © 2000 by The Endocrine Society
Substrate-Induced Down-Regulation of Human Type 2 Deiodinase (hD2) Is Mediated through Proteasomal Degradation and Requires Interaction with the Enzymes Active Center1
Jaime Steinsapir,
Antonio C. Bianco,
Christoph Buettner,
John Harney and
P. Reed Larsen
Thyroid Division, Brigham and Womens Hospital, Harvard Medical
School, Boston Massachusetts 02115
Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., Brigham and Womens Hospital, Thyroid DivisionHIM 550, 77 Avenue Louis Pasteur; Boston, Massachusetts 02115. E-mail:
Larsen{at}rascal.med.harvard.edu
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Abstract
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Type 2 iodothyronine deiodinase (D2) catalyzes the first step in
thyroid hormone action, the deiodination of T4 to
T3 . Endogenous D2 activity is posttranslationally
regulated by substrate that accelerates its degradation through the
ubiquitin-proteasome pathway. To understand how D2 activity correlates
with D2 protein during its normal decay and rT3-induced
down-regulation, HEK-293 cells, transiently expressing human D2, were
labeled with Na75SeO3 and then treated with 100
µM cycloheximide (CX), 30 nM rT3,
and/or 10 µM MG132, a specific proteasome inhibitor, for
24 h. D2 protein and enzyme activity changed in parallel,
disappearing with a half-life of 2 h in the presence of CX, or
1 h when CX + rT3 were combined. Treatment with MG132
blocked these effects. We created selenocysteine (Sec) 133 to cysteine
(Cys) or alanine (Ala) D2 mutants, without changing Sec 266. The CysD2
activity and protein levels were also parallel, with a similar
half-life of approximately 2 h, whereas the
rT3-induced D2 down-regulation required approximately
1000-fold higher rT3 concentration (30 µM)
due to a proportionally higher Michaelis constant of CysD2. In
similar experiments, the AlaD2 mutant retained the short half-life but
was not catalytically active and not susceptible to
rT3-accelerated degradation. We conclude that
substrate-induced loss of D2 activity is due to proteasomal degradation
of the enzyme and requires interaction with the catalytic center of the
protein.
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Introduction
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THE TYPE 2 iodothyronine deiodinase (D2)
(Mr
31 kDa) is the most recently cloned member
of the deiodinase family, which consists of three integral membrane
selenoproteins, types 1, 2, and 3 iodothyronine deiodinase (D1, D2, and
D3). Each of these contains the rare amino acid selenocysteine (Sec) in
the highly conserved active center (1, 2, 3, 4, 5). The presence of Sec accounts
for many of the biochemical properties that characterize D2 catalyzed
deiodination, including high catalytic efficiency and substrate
affinity (6).
D2 expression is tissue specific and can be regulated by
transcriptional and posttranscriptional mechanisms. For example, the
expression of the 7.5-kb D2 messenger RNA (mRNA) found in human brain
and pituitary gland has recently been shown to be inversely
proportional to thyroid status (7, 8, 9). In the brown fat, D2 mRNA is
markedly increased by the adrenergic stimulation during exposure of
rats to cold (7) and in the pineal gland the nocturnal increase in D2
activity is preceded by the increase in its mRNA (10). At the
posttranslational level, it has been known for a number of years that
D2 activity is rapidly down-regulated by iodothyronines (11, 12, 13, 14, 15, 16, 17, 18). The
substrate-induced down-regulation of D2 activity is apparently mediated
by posttranslational mechanisms rather than the rate of enzyme
synthesis because it occurs in the presence of inhibitors of
transcription or translation (15, 18). The isolation of D2 mRNA made it
possible to compare the effect of rT3 on D2 mRNA
and activity in pituitary tumor cells. Kim et al. found that
exposure of GH4C1 cells to 50 or 100 nM
rT3 caused a time-dependent 8090% reduction in
the D2 activity but no change in D2 mRNA (19).
The mechanism for substrate-induced inactivation of D2 has been studied
in both rat pituitary tumor cells and primary cultures of rat glial
cells. In GH3 pituitary cells, D2 activity has a half-life of 50 min
that is reduced to 26 min by rT3 (18). Other D2
substrates (T4 and iopanoic acid) have similar
effects. Acceleration of degradation was enhanced by diamide which
depletes the cell of reduced thiols and D2 was regenerated more rapidly
in cells exposed to DTT (18), suggesting that D2 inactivation is
accelerated by oxidation of the active site by substrate. In
hypothyroid rat glial cells, the D2 activity is 2- to 5-fold increased
over the levels found in cells grown with normal serum. The addition of
cycloheximide (CX) or rT3 rapidly decreases D2
activity confirming the short half-life and the substrate-induced down
regulation of D2. In these cells it was also found that D2 degradation
is not affected by lysosomotrophic agents such as chloroquine or
NH4Cl but was partially blocked by ATP-depletion
(20).
The rapid turnover rate of D2 and the observation that ATP-depletion
partially blocks loss of D2 activity prompted us to investigate the
role of the ubiquitin (Ub)-proteasome pathway in D2 degradation. The
proteasome is a large complex of proteases (26S) present in all
eukaryotes to which ubiquitination targets proteins for degradation
(21). Indeed, we have found that in rat pituitary tumor cells the short
half-life of endogenous D2 is the result of its degradation by the
Ub-proteasome system. Enzyme activity in the presence of CX was
sustained for several hours by MG132 or lactacystin, specific
inhibitors of the proteasomes. In addition, the substrate
(rT3)-induced reduction of D2 half-life was also
blocked in the presence of these proteasome inhibitors (22).
The first goal of the present studies is to substantiate that the short
half-life of D2 activity and its down-regulation by substrate are
intrinsic properties of the enzyme per se, and this can be
observed with transiently expressed protein in cells not expressing
endogenous D2. If so, then by labeling the protein with
75Se we can determine whether substrate-induced
loss of D2 activity is consequent to enzyme degradation in the
Ub-proteasome system or to D2 inactivation or some other effect. The
second task is to examine the role of substrate interaction with the
enzyme during the process of substrate-induced D2 down-regulation by
studying transiently expressed mutant D2 proteins in which the
Sec-encoding TGA codon at position 133 in the active center of the
enzyme has been changed to one encoding cysteine (Cys) or alanine
(Ala). The results of such studies will provide the first molecular
insights as to the mechanism by which substrate reduces D2
activity.
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Materials and Methods
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Reagents
MG132 was obtained from the Peptide Institute, Inc.
(Osaka, Japan) and dissolved in DMSO. Cycloheximide (CX) and
rT3 were from Calbiochem (La Jolla,
CA). CX was dissolved in DMSO and rT3 in 70%
ethanol. Pansorbin was from Calbiochem. Outer ring-labeled
[125I]-T4 specific
activity: 4400 Ci/mmol) was from DuPont (Boston, MA).
Na75SeO3 was kindly
provided by the University of Missouri Research Reactor, courtesy of
Drs. Marla Berry and Dolph L. Hatfield. All other reagents were of
analytical grade.
Preparation of D2 expressing plasmids and their transient
expression
A D10 eukaryotic expression vector containing KD2-SelP [K
indicates the presence of a Kozak consensus sequence 5' to the
initiator ATG of the human D2 (hD2) coding region and SelP a SECIS
element from the SelP gene (23)] was used for transient transfection
of human embryonic kidney epithelial cells (HEK-293). Alternatively,
mutant D2 complementary DNAs (cDNAs) were prepared in which a Cys or an
Ala were substituted for Sec 133. These cDNAs were placed in the same
vector.
Mutagenesis
Overlap-extension PCR was used to convert the Sec-encoding TGA
codon at position 133 in the hD2 sequence to either TGC (Cys) or a GCA
(Ala) codon. A 459-bp AccI fragment containing these
mutations was then exchanged for the wild-type fragment in KD2-SelP in
the D10 vector (5). These mutant D2 enzymes are referred to as Cys or
AlaD2. The Sec residue at position 266 was not altered, thus permitting
labeling of Cys or AlaD2 with
75Se-selenocysteine. A version of CysD2 not
containing a Sec 266 residue was generated by removing the Xba fragment
containing the SelP SECIS element. In this construct, the TGA at 266
becomes a stop codon. The sequences of the mutated cDNAs were verified
by manual and automated sequencing. Kinetic studies showed the Ala 133
was inactive and the Sec 133 Cys mutation had an approximately 500-fold
increase in the apparent Michaelis constant
(Km) (6).
Studies of D2 transfected cells
The hD2 or mutated proteins were transiently expressed by
introducing expression vectors containing the wild-type or mutant D2
cDNA into HEK-293 cells. To obtain uniform expression of D2 in all
plates in an experiment, we used the following batch type approach to
the transfection for studies on D2 activity. HEK-293 cells grown in
T-75 flasks were suspended in 5 ml of PBS (pH 7.3). Transfections were
then performed in each batch. Plasmid DNA was precipitated in ethanol
and then redissolved in 0.25 M CaCl2
in HEPES buffer and added to each cell batch. Twenty micrograms of D10
vector containing wild-type D2 or mutant D2 were transfected together
with 8 µg of a D15 vector in DMEM with 10% FBS. Cells and plasmid
DNA were allowed to stand for 2030 min at room temperature.
Transfected cell batches from several T-75s were then pooled and cells
seeded in 60-mm dishes. In an alternative approach, HEK-293 cells were
initially plated in 60-mm dishes and grown until confluence in DMEM
supplemented with 10% FBS. Plasmid DNA was then transfected as CaP
precipitates in pairs of plates and incubated for approximately 10
h. Cells from 1620 plates were then resuspended in PBS, pooled, and
seeded again in 60-mm plates to maximize the homogeneity of
transfection expression between plates.
Studies on D2 activity
Each experiment was performed with triplicate dishes for each
condition. This was done in serum-free DMEM supplemented with 0.1% BSA
to reduce nonspecific binding of rT3. The final
concentrations of DMSO and ethanol used to add CX,
rT3, and MG132 were 0.2% and 0.1%,
respectively, and were present in all plates as vehicle. At the
appropriate time, cells were harvested and D2 activity measured. D2
activity was measured as described previously (22). Briefly, cells were
harvested, washed, sonicated briefly in 0.1 M potassium
phosphate-1 mM EDTA, pH 6.9 (PE buffer) containing 10
mM DTT and 0.25 M sucrose. Cell homogenates
were then assayed for deiodination of freshly purified 2 nM
[125I]-T4. Incubations
were carried out for 2 h at 37 C using 300 µg of protein per
sample. Protein determinations in duplicate were by Bradford using BSA
as standard. D2 activity is reported as fmol of
T4 deiodinated/mg·min.
Production of anti-D2 antisera
We examined the amino acid sequence, surface probability,
antigenic index,
, ß, and turn regions of D2 and selected four
peptide sequences that were synthesized and combined with KLH by
Research Genetics, Inc., Huntsville, AL. The KLH-peptides
were emulsified by mixing with an equal volume of Freunds adjuvant
and injected into 34 sc dorsal sites of 3- to 9-month-old New Zealand
white rabbits (Research Genetics, Inc.), for a
total volume of 1 ml (0.1 mg of peptide) per immunization. Bleedings
were performed before immunization and 4, 8, 10, and approximately 14
weeks afterwards (see Fig. 5B
). Boost injections were given after 2 and
6 weeks. The antipeptide antibody titer was determined by ELISA with
free peptide on the solid phase (1 µg/well). Only the antisera with
the highest titers from each rabbit were used.

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Figure 5. D2 antisera used for IP of D2 protein from
75Se-labeled HEK-293 cells transiently expressing hD2. A,
Hypothetical representation of D2 protein and its relationship with the
ER membrane. The Sec amino acids are flagged. The peptide sequences
were used to immunize rabbits and generate antisera used for D2 IP. The
numbers under each peptide sequence identify each antiserum. B, HEK-293
cells were transfected with hD2 or CysD2 Xba, a D2 mutant construct,
which contains no SECIS element and in which Cys was substituted for
Sec 133. All plates were labeled with 75Se. The
autoradiograph of the 12% SDS-PAGE indicate the specific (hD2) and
nonspecific (CysD2) precipitation of 75Se-proteins by the
eight antisera. All antisera were used at a 1:100 dilution.
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75Se incorporation studies and D2 IP
Transfected HEK-293 cells were labeled in vivo with
46 µCi of
Na2[75Se]O3/dish
on day 2 after transfection in the presence of DMEM supplemented with
10% FBS. On day 3, the cells were lysed for 23 h at 4 C using a
lysis buffer 1% Triton X-100, 1% bovine hemoglobin, 1
mM iodoacetamide, 0.2 U aprotinin/ml, 1
mM PMSF in TSA buffer (0.01
M Tris-HCl, pH 8.0, 0.14 M
NaCl, 0.025% NaN3)] 0.51 ml/dish. After
centrifugation of the lysate at 1,000 rpm for 5 min each 0.51 ml
supernatant was incubated for 1224 h at 4 C with preimmune rabbit
sera to a final dilution of 1:100. One hundred microliters of a 10%
Pansorbin suspension were then added per tube and incubated under slow
agitation for 20 min at 4 C. After centrifugation at 1,000 x
g for 7 min, the supernatants were incubated for 2448 h at
4 C with one of several D2 rabbit antisera (see above) at a final
dilution of 1:100. Immunoprecipitates were obtained following the
addition of 100 µl of a 10% Pansorbin suspension and centrifugation
at 1,000 rpm for 7 min. The pellets were then washed four times with a
dilution buffer (0.1% Triton X-100, 0.1% bovine hemoglobin in TSA),
then once in TSA buffer and once with 0.05 M
Tris-HCl, pH 6.8. Pellets were then heated at 95 C for 7 min in sample
loading buffer, spun at top speed in a microfuge for 5 min, and 30 µl
of the supernatants analyzed.
Statistical analysis
Results of D2 assays were expressed as mean ±
SD of the plates studied for each condition (n = 612
replicates) in three separate experiments. Because there were
variations in basal D2 activities among various groups of cells in
different experiments (from 5.0 ± 0.5 to 14.2 ± 1.5 fmol
T4 deiodinated/mg·min for wild-type D2 and from
0.3 ± 0.02 to 0.8 ± 0.12 fmol T4
deiodinated/mg·min for the CysD2 mutant), we normalized results for
each experiment to the mean of the control values for that experiment.
A one-way ANOVA with the Newman-Keuls test for multiple comparisons was
used to assess the statistical significance of a given treatment.
P < 0.05 was considered significant.
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Results
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Transiently expressed D2 in HEK-293 cells is inactivated in
proteasomes
The first experiments characterized the pathway of degradation of
transiently expressed D2. When CX was added to cells transfected
24 h earlier with D2 cDNA, D2 activity was decreased to
approximately 50% 2 h later, indicating a half-life of
approximately 2 h. Over the next 2 h, the activity fell
another 30%, indicating that the decrease in D2 activity was not log
linear over 4 h in the presence of CX (Fig. 1
). Transfected HEK-293 cells were next
incubated with 50 nM rT3 for 15 or
240 min and processed for D2 activity. After 4 h incubation with
50 or 100 nM rT3, D2 activity was
approximately 45% of control. There was no effect of a 15-min
incubation, indicating that the loss of activity is time dependent and
not explained by dilution of the T4 substrate by
the rT3 added to induce the effect (Fig. 2
). The minimum rT3
concentration required to obtain an effect was 20 nM.
T4, 50 nM, also caused a significant
decrease in D2 activity, but a formal dose-response comparison between
rT3 and T4 was not
performed.

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Figure 1. Disappearance of D2 activity in HEK-293 cells
transiently expressing wild-type D2 in the presence of 100
µM CX. Activity is expressed relative to the mean of the
vehicle (23 µl of DMSO in 2 ml of medium) ± SD in
three separate experiments, each performed in triplicate. The D2
activity in the control group was 8.8 ± 4.5 fmol of
T4 deiodinated/mg·min. *P < 0.05
vs. vehicle.
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Figure 2. Effects of time and rT3 concentration
on transiently expressed D2 activity in HEK-293 cells. Activity is
expressed relative to the mean of the vehicle (23 µl of 40
mM NaOH in 2 ml of medium) ± SD of two
separate experiments, each performed in duplicate. The D2 activity in
the control group was 8.8 ± 4.5 fmol of T4
deiodinated/mg·min. *P < 0.05 vs.
vehicle and 15-min time point.
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We next evaluated the effects of MG132 on the CX and
rT3-induced decrease in D2 activity. Cells
transiently expressing wild-type D2 were incubated with CX or
rT3 in the presence or absence of MG132, a
specific inhibitor of the Ub-proteasome pathway (Fig. 3
). Incubation with 100 µM
CX for 4 h reduced D2 activity by approximately 50%, an effect
that was completely blocked when MG132 was also present in the
incubation medium. Furthermore, the approximately 60% reduction in D2
activity during exposure to 100 nM
rT3 was also blocked by MG132. Altogether, the
data indicate that the D2 transiently expressed in HEK-293 cells has
similar characteristics to the endogenous D2 in GH4C1 cells.

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Figure 3. Effects of rT3, CX and/or MG132 on D2
activity in HEK-293 cells transiently expressing hD2. MG132 (10
µM) was introduced 10 min before CX (100
µM). rT3 (100 nM) was introduced
simultaneously with CX. Treatments lasted 2 h. Results are
expressed as percentage of the activity in vehicle-treated cells
± SD of two to four different experiments, each performed
in triplicate. D2 activity in vehicle was 8.5 ± 1.9 fmol of
T4 deiodinated/mg·min. *P < 0.05
vs. vehicle.
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Identification and quantification of D2 by IP
The decrease in D2 activity induced by substrate and by blockade
of protein synthesis could be due to degradation of D2 per
se or to loss of some other protein required for enzyme function.
To clarify this issue, we needed a method to identify D2 independently
of its catalytic activity. For these experiments, we took advantage of
the presence of the two Sec residues at position 133 and 266 in D2, and
the availability of 75Se as
Na75SeO3. We also prepared
rabbit antisera directed against four different D2 epitopes. One day
after transfection of D2 expressing plasmid, 75Se
was added to each plate, and after another day the cells were harvested
using lysis buffer and subsequently processed for IP. Radioautography
of the lysates showed at least 6 75Se-labeled
bands, all of which were present in control nontransfected cells except
a poorly visualized band at approximately 31 kDa (Fig. 4
). This band, the predicted size of hD2,
was substantially decreased by IP with a D2 antiserum and was enriched
in the precipitate of transfected cell lysates using eight different
antisera against four different D2 epitopes, two from the
NH2-terminal portion of the protein and two from
the COOH- terminal region (Figs. 4
and 5
). The CysD2
XBa construct will not
encode a seleno-D2 protein because the SECIS element has been deleted
and therefore serves as a negative control (Fig. 5
). The only nonD2
related 75Se-labeled protein in the
immunoprecipitated material from both control and D2-transfected cells
was one of about 15 kDa, which is nonspecific (Fig. 4
). These results
establish the identity of the 31-kDa protein as hD2.

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Figure 4. IP of D2 protein from 75Se-labeled
HEK-293 cells transiently expressing hD2. HEK-293 cells were
transfected with hD2 and labeled with 75Se. IP was carried
out as described in Materials and Methods using anti-NH2
antiserum no. 85254 at a dilution of 1:100. Five microliters of total
lysate (1 ml; lanes 12) or IP supernatant (1 ml; lanes 34) were
loaded on each lane. Sixty-five percent of the immunoprecipitates were
loaded on lanes 56.
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We then used this tool to determine if the changes in D2 activity
induced by CX or rT3 were accompanied by
equivalent changes in the amount of D2 protein. In these experiments,
pairs of cell plates were transfected with wild-type D2, labeled with
75Se, and 24 h later treated with 100
µM CX, 50 nM rT3,
and/or 10 µM MG132 for 4 h, exactly as for the
activity experiments. The cells were immediately lysed and processed
for IP using the anti-NH2-terminal or
anti-COOH-terminal antisera (Fig. 6
). The
first two pairs of lanes show that rT3 or Cx
causes an approximately 50% reduction in 75Se-D2
protein, in agreement with their effects on D2 activity. Treatment with
MG132 blocked both the CX- and rT3-induced loss
of D2 protein, again in parallel with effects on D2 activity (see Fig. 3
: pairs 1 vs. 3 and 2 vs. 4). The pairs in lanes
5 show that MG132 also increases the basal D2 as would be expected if
it blocked D2 degradation. This phenomenon is seen with both antisera.
These data establish that the loss of D2 activity following CX or
rT3 is due to D2 degradation in proteasomes.

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Figure 6. Effects of rT3, CX and/or MG132
on 75Se-D2 in 75Se-labeled HEK-293 cells
transiently expressing hD2. Pairs of cell plates were transfected with
wild-type hD2 and labeled with 75Se. Indicated treatments
lasted for 2 h and were performed as in legend to Fig. 3 . IP was
carried out as described in Materials and Methods using
anti-NH2 antiserum no. 85253 or anti-COOH antiserum no. 45618. For each
pair of cell plates, the intensities of the D2 bands were quantified by
densitometry and the ratios (treatment/control) are as follows:
Anti-NH2 terminalPair 1, 0.35; Pair 2, 0.48; Pair 3, 1.1; Pair 4,
1.1; Pair 5, 2.6. Anti-COOH terminalPair 1, 0.48; Pair 2, 0.66; Pair
3, 1.3; Pair 4, 1.1; Pair 5: 1.7.
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Correlation between D2 enzyme activity and D2 protein in a batch
transfection system
We next took advantage of a batch transfection system to compare
the changes in D2 protein with D2 activity in a pool of cells
transiently expressing D2. Addition of CX for 2 h reduces D2
activity by approximately 40% (Fig. 7A
).
The fall in D2 activity was paralleled by a similar decrease in
75Se-D2 protein as measured by IP confirming it
is due to D2 degradation (Fig. 7B
). When MG132 was added with CX, the
loss of both activity and D2 protein were blocked proportionally. This
result also implies that D2 protein accumulating in the presence of
MG132 is enzymatically intact. However, because most of the
75Se-D2 is 31 kDa (but not heavier), it suggests
that the Ub-D2 pool is small probably because D2 is constantly and
rapidly deubiquitinated by the Ub-isopeptidases present in most cells.
When rT3 was added in the presence of CX, D2
activity decreased even more (by approximately 50%), though the
75Se-D2 level was only slightly affected. MG132
blocked this effect, the D2 activity decreasing by only 1015%, with
a similar modest change in D2 protein. Altogether, the data indicate
the D2 protein per se is degraded in proteasomes, and its
degradation is accelerated by exposure to
rT3.

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Figure 7. Effects of rT3, CX and/or MG132 on D2
activity (A) and 75Se-D2 (B) in HEK-293 cells transiently
expressing hD2. Cells were batch transfected with wild-type hD2 and
treated as in legend to Fig. 3A .Results are expressed as percentage of
vehicle-treated cells in three different experiments. D2 activity in
the vehicle group was 12.1 ± 6.6 fmol of T4
deiodinated per mg·min. *P < 0.05
vs. vehicle; **P < 0.05
vs. CX. B, Some plates were labeled with
75Se and processed for IP. The lower bar
graph indicates the intensity of each D2 band by densitometry.
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An hD2 containing an Sec 133 to Cys mutation is also degraded by
proteasomes and responds to substrate
The Sec133Cys substitution (CysD2) was created by PCR mutagenesis
and placed in the D10 eukaryotic expression vector. Importantly, the
Sec at position 266 was not changed, and the SECIS element remained
intact. This allows 75Se labeling of the
transiently expressed CysD2. Kinetic studies showed the apparent
Km for rT3 and
T4 of this mutant D2 was approximately 500-fold
higher than for the wild-type as discussed elsewhere [(6); data not
shown]. The same type of study as shown in Fig. 7
, comparing activity
with 75Se-D2 was performed using either 30
x 10-9 M rT3
or 30 x 10-6 M
rT3, the latter in light of much higher
Km of the CysD2 mutant. Incubations with CX or
rT3 were for 4 h, and the results are shown
in Fig. 8
. Basal levels of activity for
the CysD2 mutant were 0.61 ± 0.25 fmol/mg·min (Fig. 3
),
approximately 10 times lower than basal levels for wild-type D2. The
half-life of the CysD2 activity in the presence of CX was again about
2 h. There was no effect of the lower dose of
rT3 (data not shown), but incubation with 30
x 10-6 M rT3
decreased D2 activity by approximately 40%. Figure 8A
also shows that,
as with the wild-type D2, incubation with 10 µM MG132
increased basal CysD2 activity. The peptide aldehyde also blocked the
loss of D2 activity in the presence of CX and the acceleration of D2
disappearance in the presence of rT3. The changes
in D2 activity under these circumstances were mirrored by the IP
results (Fig. 8B
), indicating that changes in enzyme activity during CX
or rT3 treatment are the result of changes in the
quantity of CysD2 protein.

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Figure 8. Effects of rT3, CX and/or MG132 on D2
activity in HEK-293 cells transiently expressing CysD2. A, MG132 (10
µM) was introduced 10 min before cycloheximide (100
µM). rT3 (30 µM) was added
simultaneously with cycloheximide into the media. Results are expressed
as percentage of controls (Vehicle) ± SD of three
different experiments, each performed in triplicate. D2 activity in the
vehicle group was 0.61 ± 0.25 fmol of T4 deiodinated
per milligrams of protein per minute. *P < 0.05
vs. vehicle; **P < 0.05
vs. rT3. (B) Some plates were labeled with
75Se and processed for IP. The lower bar
graph indicates the intensity of each D2 band by densitometry.
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An Ala 133 mutant of hD2 is also degraded by proteasomes but does
not respond to substrate
Interestingly, the AlaD2 mutation, which can be easily
visualized by Western blots (data not shown), does not have catalytic
activity. Therefore, our analysis was limited to the quantification of
the D2 protein by IP. In these experiments, a paired transfection
rather than a batch approach was used. Results of a typical experiment
show that rT3 had no effect on D2 protein level
but that the rate of disappearance in the presence of CX of AlaD2
protein was identical to wild-type and CysD2 (Fig. 9
). This same result was found in two
other experiments. Thus, the change in D2 due to the substitution of
Ala for Sec blocks both enzymatic activity and substrate-induced
degradation of the protein suggesting that there is a causal
relationship between these two events.

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Figure 9. Effects of rT3, CX and/or MG132 on
75Se-D2 from 75Se-labeled HEK-293 cells
transiently expressing AlaD2. Pairs of plates containing
75Se-labeled HEK-293 cells transiently expressing AlaD2
were treated as in the legend to Fig. 8 . After 2-h treatment, cells
were harvested and processed for IP with anti-d2 antiserum. The
bar graph indicates the density of the 75Se-alaD2 band
relative to that in the cells of the respective control plate for each
pair.
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Discussion
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D2 activity is regulated at both the transcriptional and the
posttranslational level. Early evidence for the posttranslational
regulation derived from studies first in hypothyroid animals and later
in cell culture systems showing that the D2 activity has a short
half-life and that high affinity substrates for D2, namely
rT3 and T4, were more
potent down-regulators of D2 activity than was T3
(11, 12, 13, 14, 15, 16, 17, 18).
Cloning of the D2 mRNA has allowed demonstration that
T3 will suppress D2 mRNA levels, probably by
suppression of transcription (7, 8, 9). In some tissues, such as cerebral
cortex, the posttranslational effects are more important than are
transcriptional changes (7). We recently showed that the decrease in
endogenous D2 activity by rT3 or CX in GH4C1
cells is blocked by MG132 establishing the physiological significance
of proteasomes in the posttranslational regulation of D2 activity (22).
While this observation is an important step to understanding the
mechanism responsible for the short half-life and for the substrate
effect on D2, the GH4C1 cell does not lend itself to experiments
designed to probe those structural features of D2 that dictate the
proteasomal dependence of its degradation and the mechanism(s) by which
rT3 accelerates this. Furthermore, with respect
to the primary effect of MG132 to block D2 degradation, we did not have
formal proof that it was the degradation of D2 per se that
was accelerated by rT3 and blocked by MG132. The
present results show that while the t1/2 of transiently expressed D2
activity is about 2 h, twice as long as that in GH4C1 cells (Fig. 1
), with respect to the qualitative effects of
rT3 (Fig. 2
) and MG132 (Figs. 1
and 3
),
transiently expressed D2 behaves identically to the endogenous D2 in
GH4C1 cells. Furthermore, because it is possible to label the protein
with 75Se, we were able to demonstrate a tight
correlation between changes in wild-type D2 activity and in the
immunoprecipitated 75Se-D2 (Fig. 6
).
We can therefore apply this system to begin to probe the one or more
mechanisms that confer metabolic instability to D2 and by which
substrates such as rT3 accelerate the proteasomal
degradation of this protein. We have first focused on the turnover rate
of the D2. Our previous finding that the rapid fall of D2 activity
following CX treatment is prevented by MG132 indicates that the normal
turnover of D2 is mediated via the Ub-proteasome system. In the present
investigation, the close correlation between D2 activity and
75Se-D2 protein confirms that, in fact, the rapid
disappearance of enzyme activity is due to proteasomal degradation of
the D2 per se. This seems to be independent of the
substrate-induced degradation of D2 because, as discussed below, the
AlaD2 mutation of the enzymes active center eliminating either
selenium or sulfur did not affect the rate of D2 degradation. This is
an indication that D2 is an intrinsically unstable molecule that is
rapidly targeted by the Ub-system. This depends basically on two steps,
conjugation of the substrate with Ub and interaction of the
Ub-conjugate with the proteasome. Ubiquitination of proteins occurs
only to lysine, of which there are 15 residues in D2 (24). It also may
require the presence of degradation signals within the protein molecule
to mark it for ubiquitination. Some degradation signals that confer
metabolic instability have been reported, e.g. N-degron
and PEST sequences (25). However, the hD2 sequence does not contain any
recognized destabilizing amino acid sequences.
We investigated the active center of the enzyme, changing the Sec to
Cys, in effect exchanging S for Se which increases the
Km of the enzyme for rT3
and T4 about 500-fold (6) to see if this affects
the response of D2 to its substrate. As a consequence, the
concentration of rT3 required to accelerate the
degradation of D2 is increased in a parallel fashion (Fig. 7
). This
suggests that catalysis somehow promotes the degradation of D2. This
hypothesis is further supported by the fact that when Ala is
substituted for Cys, rT3 no longer accelerates D2
degradation even though its half-life is not changed by this
substitution (Fig. 8
). The absence of an effect of
rT3 could be due to the lack of an oxidizable
nucleophile (Se or S) in the active center or to a lack of
rT3 binding due to changes in the shape of the
binding pocket of D2 secondary to the Ala substitution. Whichever the
explanation, the result shows the potential of the transient expression
system to address these issues.
A question raised by the present results with
75Se-D2 is why, when MG132 blocks degradation of
ubiquitinated D2, there is no accumulation of a ladder of
75Se-D2-ubiquitin conjugates of increasing
molecular size. This apparent paradox has been described in earlier
studies of other ubiquitinated proteins and can be explained by the
presence of cellular isopeptidases that rapidly deubiquitinate those
Ub-protein conjugates not degraded in the proteasomes (26). In
cell-free systems, the use of ubiquitin aldehyde (27), which blocks the
isopeptidases, allows demonstration of such Ub-D2 conjugates (28). This
compound, however, does not cross the cell membrane and therefore could
not be used in the present studies. The data in Figs. 4
and 5
showing
that D2 can be readily immunoprecipitated by 8 different antisera
directed against 4 different D2 epitopes further suggest that there is
not a large pool of a poly-ubiquitin D2 conjugate in the cell lysate.
In agreement with this is the fact that the activity of D2 is preserved
and parallels the changes in 75Se-D2 protein.
Another implication of the parallel increase in
75Se-D2 and D2 activity is that the
deubiquitinated D2 retains its catalytic activity and was not
irreversibly inactivated by interaction with substrate.
In conclusion, these results confirm the effect of
rT3 to accelerate the degradation of D2 via the
proteasome in an entirely independent system from the GH4C1 cells. The
high transient expression of D2 allows it to be labeled with
75Se and immunoprecipitated. Thus, a precise
correlation can be demonstrated between the effects of substrate on D2
activity and D2 protein confirming that it is D2 protein per
se which is more rapidly degraded during substrate exposure. This
system should lend itself to other perturbations of the D2 protein to
allow definition of the critical structural elements required for this
strikingly rapid nonnuclear mediated effect of
rT3 (and T4) to regulate
the concentration of this enzyme.
 |
Footnotes
|
|---|
1 Supported by NIH-R01-DK36256. J.S. was supported by a Minority
Supplement to NIH-R01-DK-36256. A.C.B. was supported in part by a
scholarship grant from Conselho Nacional de Pesquisa. C.B. was
supported by a Reimar Luest grant from the Koerber Foundation. 
Received September 21, 1999.
 |
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