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Thyroid Division, Brigham and Women’s Hospital, Harvard Medical School, Boston Massachusetts 02115
Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., Brigham and Women’s Hospital, Thyroid Division—HIM 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 80–90% 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 20–30 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 16–20 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 Freund’s adjuvant
and injected into 3–4 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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Statistical analysis
Results of D2 assays were expressed as mean ±
SD of the plates studied for each condition (n = 6–12
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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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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). 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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). 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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). 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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).
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 10–15%, 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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, 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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). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 enzyme’s 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.
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Footnotes |
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Received September 21, 1999.
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