Selective Proteolysis of Human Type 2 Deiodinase: A Novel Ubiquitin-Proteasomal Mediated Mechanism for Regulation of Hormone Activation
Balázs Gereben,
Carla Goncalves,
John W. Harney,
P. Reed Larsen and
Antonio C. Bianco
Thyroid Division Department of Medicine Brigham and
Womens Hospital Harvard Medical School Boston Massachusetts
02115
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ABSTRACT
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We investigated the mechanism by which
T4 regulates its activation to
T3 by the type 2 iodothyronine deiodinase (D2).
D2 is a short- lived (t1/2 50 min), 31-kDa
endoplasmic reticulum (ER) integral membrane selenoenzyme that
generates intracellular T3. Inhibition of the
ubiquitin (Ub) activating enzyme, E1, or MG132, a proteasome blocker,
inhibits both the basal and substrate-induced acceleration of D2
degradation. Using a catalytically active transiently expressed
FLAG-tagged-NH2-D2, we found rapid synthesis of
high molecular mass (100300 kDa) Ub-D2 conjugates that are
catalytically inactive. Ub-D2 increases when cells are exposed to D2
substrate or MG132 and disappears rapidly after E1 inactivation. Fusion
of FLAG epitope to the COOH terminus of D2 prolongs its half-life
approximately 2.5-fold and increases the levels of active and,
especially, Ub-D2. This indicates that COOH-terminal modification
interferes with proteasomal uptake of Ub-D2 that can then be
deubiquitinated. Interestingly, the type 1 deiodinase, a related
selenoenzyme that also converts T4 to
T3 but with a half-life of >12 h, is
inactivated but not ubiquitinated or degraded after exposure to
substrate. Thus, ubiquitination of the ER-resident enzyme D2
constitutes a specific posttranslational mechanism for
T4 regulation of its own activation in the
central nervous system and pituitary tissues in which D2-catalyzed
T4 to T3 conversion is
the major source of intracellular T3.
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INTRODUCTION
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Various intracellular regulatory pathways can be modified by
selective proteolysis of key rate-limiting enzymes. This process is
frequently mediated by the proteasome system in which different
metabolic signals stimulate uptake and proteolysis by the proteasomes.
Well known examples include cytosolic enzymes such as ornithine
decarboxylase (1) and endoplasmic reticulum (ER) transmembrane protein
hydroxy-methylglutaryl-coenzyme A reductase (2). In many cases
selective proteolysis is preceded by conjugation to ubiquitin (Ub),
a step that is activated by exposure of cells to specific
substrates. In Saccharomyces cerevisiae, for example,
glucose-containing medium induces the metabolic transition from
gluconeogenesis to glycolysis by ubiquitination and degradation of
fructose-1,6-biphosphatase, a key regulatory enzyme of gluconeogenesis
that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate
fructose-6-phosphate and inorganic phosphate (3). This degradation
process has been termed "catabolite inactivation" and operates for
other key enzymes in this pathway as well, including cytosolic malic
enzyme, phosphoenolpyruvate kinase, and isocitrate lyase (4). The
specific mechanism(s) by which catabolites induce the proteolytic
cascade is poorly understood.
In vertebrate cells, basal and substrate-induced selective enzyme
proteolysis is also involved in the control of transformation of
T4 to the active hormone T3
, the first step in thyroid hormone action. Two enzymes, the types 1
(D1) and 2 (D2) iodothyronine deiodinases, can catalyze this reaction.
Along with type 3 deiodinase (D3) that inactivates
T4 and T3, these integral
membrane selenoproteins constitute a homeostatic system that controls
the intracellular concentration of active thyroid hormone within human
tissues. Whereas D1, expressed primarily in liver and kidney, is
considered to be the major source of circulating
T3, D2 catalyzes the local production of
T3 in the central nervous system, pituitary
gland, and brown adipose tissue (5). As a result, thyroid hormone
receptor (TR) occupancy by T3 is higher in these
tissues than is the case in cells in which plasma is the only source of
intracellular T3. In addition, the effects of a
decrease in plasma T4, such as occurs in iodine
deficiency, are mitigated by a rapid compensatory increase in D2
activity (5). One key feature of D2 that allows such plasticity is its
short half-life (<1 h) (6).
Considerable evidence indicates that D2 regulation by its substrates is
posttranslational (7, 8, 9) and recent data implicate the proteasome
system (10, 11). Indeed, in pituitary tumor cells, proteasomal
inhibitors (MG132 or lactacystin) stabilize D2 activity for several
hours in the presence of cycloheximide (CX) or the D2 substrates
T4 or rT3 (10). Parallel
reductions of transiently expressed 75Se-labeled
D2 and D2 activity occur after exposure of cells to CX and/or
rT3, indicating that this reduction is due to
catabolism of the protein rather than to an alteration in its structure
(11). Single amino acid changes in the active center of D2, which
either raise the Michaelis-Menten constant (Km)
for substrate approximately 1,000-fold or block its catalytic activity,
impair or eliminate substrate-induced loss of D2 (11). These results
suggest that interaction of substrate with D2 selectively targets the
protein for degradation by the proteasome system. Despite this, an
ubiquitinated D2 intermediate has not been identified nor has the role
of catalysis in its production been clarified.
Proteins containing selenocysteine (Sec) are synthesized
slowly due to the complex mechanisms required to suppress the stop
codon function of the Sec codon, UGA (12). This and its short half-life
result in low cellular D2 concentrations. D2 protein contains few
immunogenic peptide sequences, resulting in difficulties in the
generation of high-affinity antibodies suitable for Western blots (11).
Accordingly, to enhance the possibility of detecting ubiquitinated D2,
we employed a mutated D2 enzyme in which Cys is substituted for Sec 133
in the active center. This protein is transiently expressed at levels
approximately 100-fold higher than the native D2 but is catalytically
active and subject to the same substrate regulatory pathways as is the
native enzyme (11). We also labeled D2 with FLAG, an epitope for which
there is a highly specific antibody. By transiently expressing
catalytically active FLAG-tagged D2, we have identified
ubiquitinated-D2 and show that its levels are increased by exposure of
cells to substrate. We also found that the Ub-activating enzyme, E1, is
required for Ub conjugation to D2 and for the substrate-induced
acceleration of that process. Surprisingly, while we have confirmed
previous results showing that substrate also reduces D1 activity, this
T4 activating enzyme is not ubiquitinated. This
indicates that ubiquitination of D2 is a specific mechanism that
confers rapid posttranslational regulation by T4
of its activation to T3. This is a unique example
of substrate-induced selective proteolysis that involves ubiquitination
of an ER resident protein. To our knowledge, it is the first
demonstration that such a regulatory pathway controls activation of a
hormone.
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RESULTS
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An Active E1 Ub-Activating Enzyme Is Required for D2
Proteolysis
In the first group of experiments, wild-type D2
protein (D2) was transiently expressed in a Chinese hamster cell line
(CHO-ts20), which has a temperature-sensitive E1, the Ub-activating
enzyme. These cells retain a functional ubiquitination pathway when
cultured under the permissive temperature (<35 C). At 40 C, the
heat-sensitive E1 has less than 10% of its normal activity (13). Ts20
cells grown at 30 C were transfected in pairs with D2-expressing
plasmid. This technique leads to transient expression of equal
quantities of protein as assessed by measuring GH in the media 24
h later (see Materials and Methods). After 48 h, one
plate of each pair was placed at 40 C while the other remained at 30 C.
At the indicated times, cells were processed for D2 activity (Fig. 1A
). In three independent experiments the
D2 activity increased an average of approximately 30% at the
restrictive temperature (Fig. 1B
), whereas no changes were detected in
similarly treated wild-type CHO cells (data not shown).

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Figure 1. Inhibition of the Ub-Activating Enzyme, E1, Blocks
D2 Degradation
A, Paired plates of ts20 expressing wild-type hD2 were kept at the
permissive temperature (30 C) for 48 h. B, At that time one plate
of each pair was transferred to the restrictive temperature (40 C), and
D2 activity was assayed at the indicated times and expressed relative
to D2 in cells kept at 30 C. The data shown are the mean ±
SD (n = 2) of three different experiments. C, Similar
protocol to evaluate effects of E1 inhibition on D2 degradation or
substrate-induced proteolysis. After 4 h, 100 µM CX
(D) or 30 nM rT3 (E) was added and D2
measured 1 h later and shown relative to control at 30 C. *,
P < 0.05 vs. 0 time-point. There were no
significant differences between the 4 and 5 h time points for
cells kept at the restrictive temperature (40 C) in experiments D and
E.
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We next determined whether inactivation of E1 would affect the
half-life of D2 or the effects of substrate to reduce its activity.
rT3 was chosen as D2 substrate because it is
metabolically inactive, its deiodination product is also inactive, and
the D2 Km(rT3) is similar
to that of T4. In fact, in a previous publication
(11) we showed that both T4 and
rT3 cause similar acceleration of D2
inactivation. Plates containing ts20 cells transiently expressing D2
were kept at 30 C or placed at 40 C for 4 h. Cycloheximide (100
µM) or rT3 (30 nM) was
added to one plate of each pair (Fig. 1C
). These treatments caused an
approximately 20% reduction in D2 activity in cells at 30 C
(P < 0.05). However, at 40 C, these agents had no
significant effect on D2 activity (P > 0.05
vs. time 0; P < 0.05 vs. 30 C)
(Fig. 1
, DE). The effects of CH or rT3 in
wild-type CHO cells transiently expressing D2 were not affected by
incubation at 40 C (data not shown). These results indicate that E1 is
rate limiting in the basal and substrate-induced changes in D2
activity.
To confirm that there was a correlation between enzyme activity
and D2 protein we evaluated changes of
75Se-labeled D2 under the same conditions. Paired
plates of ts20 cells transiently expressing wild D2 were labeled with
Na2[75Se]O3,
and 24 h later one plate was shifted to 40 C for 4 h (Fig. 2A
). The plate lysates were processed for
immunoprecipitation (IP) using D2 antiserum and the precipitates
analyzed by SDS-PAGE. The 31-kDa 75Se-D2 band
increased 1.8 ± 0.6 fold (P < 0.05) in cells at
40 C vs. those at 30 C, again consistent with impaired
ubiquitination (and subsequent proteolysis) of D2 at the restrictive
temperature (Fig. 2
, B and C) as the explanation for the increased D2
activity. However, no 75Se-labeled higher
molecular mass proteins were visualized in the control samples as
would be expected if ubiquitination of D2 was occurring under these
circumstances (Fig. 2B
).

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Figure 2. IP of hD2 Transiently Expressed in 75Se-Labeled
ts20 Cells
A,. Ts20 cells transfected in pairs expressing wild-type hD2 were
labeled with 75Se. After 1 day, one plate of each pair was
transferred to 40 C. After 4 h the D2 was IPd with an anti-D2
antiserum. B, IP pellets were resolved by 12% SDS-PAGE in three
separate experiments, nos. 1, 2, and 3. C, Autoradiograph densitometric
analysis of the gel in Fig. 2B , shown relative to control at 30 C. *,
P < 0.05 vs. cells kept at 30 C.
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Identification of Ub-D2
To enhance the possibility of detecting Ub-D2, HEK-293 cells
transiently expressing FLAG-NH2-cysD2 protein
(Fig. 3A
) were treated with 100
µM CX or 30 µM rT3,
and sonicates were processed for Western blotting with anti-FLAG
antibody (Fig. 4A
). The
32 kDa
FLAG-NH2-cysD2 band behaved similarly to
the
31 kDa 75Se-D2 band detected in the IP
of HEK-293 cells reported previously (11). It disappeared with a
half-life of approximately 2 h in the presence of CX and was also
decreased by treatment with rT3. Both effects
were blocked by concomitant exposure to 10 µM MG132, an
inhibitor of proteasomal proteolysis (Fig. 4B
). D2 activity paralleled
the level of FLAG-NH2-cysD2 protein, as
visualized by the Western blot (Fig. 4
, B and C). Most importantly,
five high molecular mass bands (100300 kDa) were observed in the
Western blot, consistent with the formation of polyubiquitinated D2
(Fig. 4D
). These bands constituted 1020% of the immunoreactive FLAG
protein in the cells.

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Figure 3. Schematic Diagram of FLAG-Tagged Deiodinases
Containing Sec-to-Cys Mutations in the Active Center.
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Figure 4. Western Blot of FLAG-NH2-cysD2 Fusion
Protein Transiently Expressed in HEK-293 Cells
A, HEK-293 cells transfected in pairs with
FLAG-NH2-cysD2-expressing plasmid were treated for 2 h
with 100 µM CX, 30 µM rT3,
and/or 10 µM MG132 as indicated. Controls (C) were
treated with vehicle (NaOH or DMSO). After 2 h, lysates were
resolved in a 12% SDS-PAGE or assayed for D2. B, Western blot using
anti-FLAG antibody and C. D2 activity in the same cells. D, Western
blot of a second experiment in which HEK-293 cells were prepared as in
panel A and processed for the blot. Cell sonicates were resolved by
7.5% SDS-PAGE. The FLAG-NH2-cysD2 is the 33-kDa protein.
Negative controls are indicated and included untransfected cells, cells
transfected with empty vector, or cysD2 without FLAG. E, Transiently
expressed FLAG-NH2-cysD2 was isolated using anti-FLAG
agarose matrix. After extensive washing the beads were mixed with
loading buffer and resolved in a 7.5% SDS-PAGE. Western blot using
anti-Ub antibody and Western blot using anti-FLAG antibody after
removal of the anti-Ub. The unmarked bands are nonspecific and
partially due to reactivity of the peroxidase-labeled second antibody
with the anti-FLAG IgG from the matrix. F, Western blot of particulate
fraction or cytosol of HEK-293 cells transiently expressing
FLAG-NH2-cysD2. Cell sonicates were centrifuged at
2,500 x g for 10 min and the supernatant was spun
at 100,000 x g for 1 h to separate microsomes
from cytosol. *, P < 0.05 vs.
control cells.
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To confirm that these bands were indeed ubiquitinated FLAG-D2,
sonicates of HEK-293 cells transiently expressing FLAG-D2 were affinity
purified on an anti-FLAG agarose matrix. The samples were then stained
with an anti-Ub antiserum (Fig. 4E
). Three 110- to 300-kDa bands,
similar in size to those seen with the anti-FLAG antibody in Fig. 4D
, reacted with the anti-Ub antiserum. Stripping the anti-Ub antibodies
from the blot and reprobing it with anti-FLAG antibody confirmed that
these proteins were ubiquitinated FLAG-cysD2 (Fig. 4E
). Microsomal and
cytosolic fractions were prepared from sonicates of HEK-293 cells, and
FLAG-cysD2 as well as Ub-FLAG-cysD2 were found to be localized
predominantly in the particulate fraction (Fig. 4F
).
If the high molecular mass FLAG-D2 proteins are ubiquitinated, then it
should be possible to block their formation by inactivating E1 in the
ts20 cells. Increasing the incubation temperature to 40 C for
2 h markedly reduced the amount of the high molecular mass
FLAG-NH2-cysD2 complexes in ts20 cells (Fig. 5A
). There was also an associated
approximately 20% increase in
32 kDa FLAG protein and D2 activity
(100 ± 13 vs. 126 ± 18%; P <
0.05) in the same cells.

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Figure 5. Inhibition of E1 Blocks the Formation of Ub-D2
Conjugates
A, One of each pair of ts20 cells transiently expressing
FLAG-NH2-cysD2 was transferred from 30 C to 40 C for 2
h. Cell sonicates were processed for Western analysis using anti-FLAG
antibody after 7.5% SDS-PAGE. B and C, HEK-293 cells transiently
expressing FLAG-NH2-cysD2 were treated with 30
µM rT3 (B) or 10 µM MG132 (C)
for 2 h. Cell sonicates were processed for Western analysis with
anti-FLAG antibody after 7.5% SDS-PAGE of controls (C) and treated
cells. Below each gel there is a detail of the
FLAG-NH2-cysD2 band in a film properly exposed for that
band.
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We next investigated whether exposure to substrate would increase the
conjugation of Ub to D2. Exposure of HEK-293 cells transiently
expressing FLAG-NH2-cysD2 to 30 µM
rT3 for 2 h caused a 2- to 3-fold increase
in the Ub-D2 conjugate bands (Fig. 5B
), establishing that
substrate-enzyme interaction accelerates the rate of D2 ubiquitination.
As expected, the FLAG-NH2-cysD2 band decreased by
4050% as did D2 activity (100 ± 20 vs. 45 ±
12%; P < 0.05). In a complementary experiment,
addition of 10 µM MG132 not only increased the
32 kDa FLAG-NH2-cysD2 band by 5060% but
also increased the high molecular mass
FLAG-NH2-cysD2 bands, as would be predicted if
MG132 blocked their proteasomal uptake (Fig. 5C
). The D2 activity of
these cells also increased parallel to the
FLAG-NH2-cysD2 bands (100 ± 17
vs. 155 ± 25%; P < 0.05).
The COOH Terminus Is Critical for the Proteolysis of Ub-D2
According to the current topological model for the deiodinases,
the NH2 terminus of D2 is located in the lumen of
the ER with its catalytic portion in the cytosol. As mentioned
previously, fusion of the FLAG sequence to the
NH2-terminus of D2 does not change its
degradation rate (Fig. 4B
). Surprisingly, the levels of D2 activity
(Fig. 6A
) and protein (Fig. 6B
) were
4-fold higher in cells transiently expressing cysD2 with the FLAG
epitope placed at the COOH terminus (Fig. 3B
). The amount of
Ub-FLAG-COOH-cysD2 was massively increased, about 20- to 30-fold,
suggesting that the degradation of Ub-D2 was blocked by the presence of
the COOH-terminal FLAG epitope (Fig. 6B
). Studies with CX confirmed
that the half-life of the FLAG-COOH-cysD2 protein was longer than that
of the wild-type D2 or the FLAG-NH2-cysD2 (Fig. 6
, C and D). While CX exposure caused an approximately 50% decrease in
the FLAG-NH2-cysD2 protein over 2 h,
FLAG-COOH-cysD2 protein disappeared much more slowly, with a predicted
half-life of 45 h. Parallel effects occurred in D2 activity.

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Figure 6. The COOH-Terminus FLAG Inhibits Proteasomal Uptake
of Ub-D2
A, D2 activity quantified from HEK-293 cells transiently expressing
either FLAG-NH2-cysD2 or FLAG-COOH-cysD2. Medium GH used as
internal control showed equal transfection efficiency for cells
transfected with the FLAG constructs. B, Western blot of the same
sonicates with anti-FLAG antibody after 7.5% SDS-PAGE. Below the gel
is shown a detail of a shorter exposure. Because of the construct
strategy the FLAG-COOH-cysD2 protein is approximately 1 kDa larger than
the FLAG-NH2-cysD2 protein (see Materials and
Methods). C, HEK-293 cells transiently expressing either
FLAG-NH2-cysD2 or FLAG-COOH-cysD2 were treated with vehicle
or CX for 2 h and D2 activity was measured. D, A
Western blot of the same lysate was probed with anti-FLAG antibody. The
two images are from the same immunoblot exposed for different time
periods. CysD2 was used as a negative control. The Ub-D2 conjugate
bands <100 kDa in size are only detected in the film exposed for
longer time. *, P < 0.05 vs.
vehicle-treated cells.
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The longer half-life of the COOH-terminal modified protein could be
explained by an impairment of D2 ubiquitination or interference with
the further processing of Ub-D2 in the proteasomes. It appears that the
latter is the case since the ratio of Ub-FLAG-COOH-cysD2 to
unconjugated D2 is several fold higher than that of the
FLAG-NH2-cysD2. It is also notable that
additional bands of intermediary molecular masses (40100 kDa) can be
visualized when the film is overexposed. Given the fact that these
bands are not present in the negative controls and that they are more
abundant in the sonicates from cells transfected with FLAG-COOH-cysD2
than with FLAG- NH2-cysD2, it is likely that they
are lower molecular mass Ub-cysD2 conjugates.
Substrate Causes D1 Inactivation but Not Ubiquitination
As mentioned, the selenoenzyme D1 also catalyzes
T4 to T3 conversion
although the Km(T4) for
this enzyme (
2 µM) is approximately 1000-fold higher
than is that for D2 (12 nM) (14). While D1 has sequence
and, presumably, structural similarities to D2, it differs from the
latter in having a relatively long half-life (>12 h; Fig. 7A
). Accordingly, we prepared a
FLAG-NH2-cysD1 protein (Fig. 3C
) to allow
comparative studies of the degradation pathways of D1 and D2. The
FLAG-NH2-cysD1 was again catalytically active,
and the half-life of D1 activity of transiently expressed FLAG-cysD1
was >12 h, 6 times longer than that of FLAG-cysD2 (Fig. 7A
). Despite
much higher transient expression of D1, there was no detectable
Ub-FLAG-NH2-cysD1 conjugate in the HEK-293 cells
(Fig. 7B
). However, exposure to rT3 for 24 h
caused a 7080% reduction in D1 activity but no change in the level
of FLAG NH2-cysD1 protein (Fig. 7
, C and D).
These results indicate that, despite the fact that substrate causes
decreases in both D2 and D1 activities, the mechanisms by which those
changes occur are quite distinct.

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Figure 7. Transiently Expressed cysD1 Is Not Ubiquitinated in
HEK-293 Cells
A, HEK-293 cells transiently expressing either
FLAG-NH2-cysD1 or FLAG-NH2-cysD2 were treated
with 100 µM CX, harvested at the indicated times,
and processed for D1 or D2 activities. B, Similarly transfected cells
processed for Western blot with anti-FLAG antibody after 12% SDS-PAGE.
Cells transiently expressing cysD2 were used as a negative control.
C, HEK-293 cells transiently expressing
FLAG-NH2-cysD1 were treated with 5
µM rT3. Twenty four hours
later the cells were processed by Western blotting with anti-FLAG
antibody (C) or for D2 activity (D). Cells transiently expressing
FLAG-NH2-cysD2 or cysD2 were used as controls. *,
P < 0.05 vs. vehicle-treated cells.
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DISCUSSION
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The signal(s) that target D2 for ubiquitination are poorly
understood. No clear destabilizing sequences such as PEST elements,
N-end rule, or destruction box (15) are present in the D2 molecule even
though its half-life is relatively short. This is especially notable
since D2 is an integral membrane protein. Our preliminary topological
analysis suggests that D2 is an ER integral membrane protein with its
NH2 terminus within the ER (data not shown).
Recent studies suggest that both intralumenal and resident ER
transmembrane proteins are predominantly degraded by the cytosolic
proteasome system after they are dislocated to the cytosol. This
includes not only improperly folded proteins, which are subjected to
rapid proteasomal degradation, but also resident (properly folded) ER
proteins (Ref. 16 for review). One well known example of the latter is
the yeast Sec62 protein (17). This protein spans the ER membrane two
times and has both termini facing the cytosol. It is a major component
of the heptameric Sec complex, a protein-conducting channel of the ER
membrane. Like D2, Sec62 is heavily ubiquitinated and degraded by
proteasomes. Membrane extraction and proteolysis of Sec62 are coupled
mechanisms initiated through the proteins amino terminus and mediated
by the proteasomes (17).
Two results suggest that ubiquitination and proteasomal degradation of
D2 initiate at the COOH terminus. First we can identify Ub-D2 in
particulate fraction of cell sonicates (Fig. 4F
). The accumulation of
large amounts of Ub-D2 associated with the ER (2050% of total FLAG
protein) indicates that the rate of D2 ubiquitination exceeds that of
Ub-D2 proteolysis. This agrees with the hypothesis that
proteolysis of ER proteins requires extraction of Ub-conjugated protein
from the ER (16). The alternative possibility that the COOH-terminal
portion of Ub-D2 is clipped off by an ER-bound proteasome, or by
regulated intramembrane proteolysis (18), is unlikely because small
FLAG-containing protein fragments (<32 kDa) were not detected (Fig. 6
). Second, a comparison of the fate of NH2- and
COOH-FLAG-tagged D2 proteins allowed us to gain further insight into
the mechanistic aspects of D2 proteolysis. On one hand, conjugation of
FLAG to the NH2 terminus does not alter the
half-life of cysD2 (Fig. 4
) (11). In contrast, the fusion of the FLAG
sequence to the COOH terminus of D2 not only prolonged its half-life
but also increased the size of the Ub-D2 pool 20- to 30-fold (Fig. 6
).
Because both D2 activity and the
32 kDa protein were increased 3- to
4-fold in cells transfected with FLAG-COOH-cysD2, the increase in the
Ub-D2 pool is not due to increased ubiquitination. Rather, it is
probably caused by impaired proteasomal extraction/proteolysis of
Ub-D2. The accumulated Ub-D2 can then be recycled through Ub
isopeptidases to D2. Consequently, D2 half-life, protein levels, and
activity are all increased. This is supported by the data in Fig. 5
.
Likewise, moving ts20 cells to the restrictive temperature decreases
Ub-D2 conjugates while it increases D2 protein and activity (Fig. 5
).
The dynamic equilibrium between Ub-D2 and D2 also explains why D2
activity is stabilized for several hours by exposing CX-treated
pituitary tumor cells (10) or HEK-293 cells transiently expressing D2
(11) to MG132. The data presented in Fig. 5
also indicate that D2
activity parallels the levels of D2 protein and not Ub-D2 conjugates,
which, therefore, must be catalytically inactive. This indicates that
the D2 and Ub-D2 pools are in dynamic equilibrium that shifts toward D2
in the presence of MG132 or toward the formation of Ub-D2 when cells
are exposed to substrate. The immediate implication of these findings
is that proteasomal uptake can be a limiting step in D2 proteolysis, as
has been suggested for Sec62 (17).
Exposure to the substrate rT3 decreases D2
protein and activity by approximately 50%. The present results reveal
that this is due to an increase in D2 ubiquitination since both this
and the loss of D2 activity are blocked by E1 inactivation (Figs. 2
and 3
). Because D2-substrate interaction is required to increase D2
proteolysis (11), it is possible that postcatalytic structural changes
in the D2 protein accelerate the ubiquitination cascade. Alternatively,
the redox state of the molecule might play a role. Reducing agents,
e.g. dithiothreitol, act as cofactors for D2 catalysis
in vitro by reducing the Se in the enzymes active center
after it is oxidized during the monodeiodination process (19).
Oxidation of the Se- in the native enzyme or SH
in the Cys mutant could be the primary signal that accelerates
ubiquitination. This would explain why an oxidizing agent such as
diamide irreversibly inactivates D2 (20). Indeed, such a
redox-sensitive mechanism has been described for the ubiquitination and
proteasomal degradation of hypoxia-inducible factor 1
, involved in
the activation of the erythropoietin gene (21). However, a
catalytically inactive D2 mutant, in which alanine was substituted for
the Sec in the active center of the enzyme (alaD2), retains the typical
D2 short half-life while it is refractory to the substrate-induced
acceleration of its proteolysis (11). If the redox state of the active
center plays a role in accelerating ubiquitination it must be limited
to the latter process.
The results with D1 provide an important contrast suggesting that
simply deiodinating an iodothyronine does not accelerate ubiquitination
of a deiodinase. The present results confirm previous data that D1
activity is also decreased by exposure to substrate (20), but our
results show that this does not involve D1 ubiquitination (Fig. 7
).
Rather, the inactivation process is most likely the consequence of
oxidation of the active center Se that requires time until the
intracellular environment returns it to the reduced state, as
originally proposed (22). The finding that D1 is not ubiquitinated can
also explain its much longer half-life (Fig. 7
). Taken together, these
results indicate that T4 to
T3 conversion by D2 is more tightly regulated
than that by D1. Whether this is due to differences in protein
structure or in subcellular localization between the two
selenodeiodinases remains to be determined.
In summary, we propose that newly synthesized D2
(D2-Se-) exists in the ER membrane in a dynamic
equilibrium with its ubiquitinated (inactive) derivative
Ub-D2-Se- (Fig. 8
). The latter may be deubiquitinated by
isopeptidases, thus reactivating it, or it may enter proteasomes for
irreversible degradation. T4, the principal substrate of D2
[or rT3 (10, 11)], induces poorly understood changes
in the enzyme forming D2-Se, thus accelerating the ubiquitination
process, shifting the equilibrium toward Ub-D2-Se. Whether Ub-D2-Se can
be reactivated in vivo is not clear. The critical change
induced in D2-Se- by T4
constitutes a posttranslational control process regulating the rate of
T4 activation. Such a mechanism is especially
well suited to T4 since its 7-day plasma
half-life in humans precludes minute-to-minute variations due to
changes in secretion via feedback at the hypothalamic-pituitary level.
However, given the advantages of local intracellular control of hormone
activation, it would not be surprising if other hormone systems are
regulated in a similar fashion.

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Figure 8. Proposed Model of D2 Ubiquitination and Degradation
by Proteasomes
D2 (dark circle) is synthesized (1 ) and remains as a
resident protein in the ER. During its normal turnover it is
ubiquitinated (2 ). Deubiquitination by isopeptidases (3 ) is possible
particularly under conditions where the proteasomal degradation (4 ) is
impaired. Catalysis (5 ) results in oxidation of the Se in the active
center (hexagon). This oxidation or another structural
change caused by catalysis (6 ) accelerates ubiquitination (2 ) and
eventual degradation (4 ). Alternatively, an intracellular reducing
agent may regenerate the active form of D2 (7 ) after deubiquitination
(3 ), although there is currently no evidence to establish the presence
of this pathway.
|
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 |
MATERIAL AND METHODS
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Reagents
MG132, CX, rT3, and
T4 were obtained from Calbiochem (La
Jolla, CA). The iodothyronines (Sigma , St. Louis MO) were
dissolved in 40 mM NaOH and the other drugs were dissolved
in dimethylsulfoxide (DMSO). Pansorbin was from
Calbiochem. Outer ring-labeled T4
(specific activity: 4400 Ci/mmol) was from NEN Life Science Products, Inc. (Boston, MA).
Na2[75Se]O3
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 D1- and D2-Expressing Plasmids and
Mutagenesis
All constructs were cloned into the D10 mammalian expression
vector (23). Wild-type D2 constructs contained the SelP SECIS element
(24). Overlap-extension PCR was used to produce D2 mutants where the
Sec 133 was replaced by Cys (cys-D2), as described previously (11), and
subcloned into the same vector. A D1 mutant in which Cys was replaced
by Sec (G5; cysD1) was described previously (12).
Epitope-tagged D2 proteins were created using the eight-amino acid FLAG
sequence (Sigma). The NH2-FLAG-cysD2
(Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys Leu-Ala-Met) was
generated by the Bp84-Bp85 oligonucleotides [the former contained an
NcoI site, while the latter contained a SalI site
(lower case)] by Vent PCR on a hD2 Cys template. (Bp84
sense; 5'-catgccATGG GCATCCTCAG CGTAGACTTG CTGA; Bp85 antisense:
5'-ttccgcggcc gctatggccg acgtcgacTT AACCAGCTAA TCTAGTTTTC TTTCATCT).
The resulting fragment was cut by NcoI/SalI and
cloned into the corresponding sites of the D10 vector, which also
contained a 5'-EcoRI site and a strong Kozak consensus (-3A)
sequence with an in-frame NcoI site at the 3'-end of the
FLAG sequence (Fig. 3A
). This construct does not contain a SECIS
element so that translation of the NH2-FLAG-cysD2
terminates at the second UGA codon, the eighth codon from the COOH end
of the native D2 protein. Previous studies showed no effect of this
truncation on enzyme activity (25). The COOH-FLAG-cysD2 Cys mutant was
generated by the Bp97-Bp95 oligonucleotides [the former contained an
EcoRI site, while the latter contained an XbaI
site (lower case)] by Vent PCR on a hD2 Cys template. (Bp97
sense: 5'-ggaattcatt ATGGGCATCC TCAGCGTAGA CTTGCTGATC A; Bp95
antisense: GCTCTAGAtt acttgtcgtc atcgtccttg tagtcACCAG CTAATCTAGT
TTTCTTaCAT CTCTTGCT). The sense oligo contained the same Kozak
consensus as the NH2-FLAG-cysD2 construct, to ensure the
same rate of translational initiation. We also replaced the second Sec
codon (UGA) with Cys (UGU) using the Bp95 antisense oligo, to ensure
uniform translation (Fig. 3B
). Both FLAG-cysD2 constructs were
catalytically active.
The NH2-FLAG-cysD1 mutant was generated by the Bp92-Bp93
hD1 oligonucleotides on a rat Cys mutant template (NOREF>G5), incorporating
NheI and SalI restriction sites (lower
case) at the 5'- and 3'-ends of cysD2, respectively (Bp92 sense;
ctagctagcc ATGGGGCTGC CCCAGCCAGG GCTGTGGCTG A; Bp93 antisense:
ttccgcggcc gctatggccg acgtcgacTT AACTGTGGAG CTTTTCCAGA ACAGCACGA). The
fragment was cut by these enzymes and cloned between the corresponding
sites of a version of the above described NH2-FLAG D10
fusion vector, containing a unique NheI site 3' to the FLAG
sequence (Fig. 3C
). The resulting protein is a rat
NH2-FLAGcysD1 containing Pro, Pro, and Gly in positions 4,
6, and 7, respectively, as in the human wild-type D1. Its COOH terminus
contains His and Ser in positions 248249 and is followed by a stop
codon, making it a 29-kDa protein. The protein was catalytically
active. None of the FLAG-tagged Cys mutant deiodinase fusion proteins
contained a SECIS element. The accuracy of the construction was
confirmed by sequencing.
Procedures for Transfections
D1 and D2 were transiently expressed by introducing expression
vectors containing the respective cDNAs into human embryonic kidney
epithelial cells (HEK-293) or Chinese hamster ovary (CHO) ts20 cells.
To obtain uniform expression in all plates in an experiment, we used
the following pair-plating approach to the transfection. Cells were
initially plated in 60-mm dishes and grown until confluence in DMEM
supplemented with 10% FBS. Plasmid DNA was precipitated in ethanol and
then redissolved in 0.25 M CaCl2 in
HEPES buffer and equal amounts of suspension added to the pair of
plates. Ten micrograms of D10 vector containing D1 or D2 cDNAs were
combined with 4 µg of a D15 vector and 3 µg of TKGH plasmid per
plate. Cells and plasmid DNA were allowed to stand for 2030 min at
room temperature and subsequently incubated at 37 C. hGH was measured
in the media 48 h later as a monitor for transfection efficiency.
Differences in hGH expression in cells transfected with the same DNA
precipitate were <5%.
75Se incorporation and D2 IP
These procedures were performed as previously described (11).
Briefly, transfected ts20 cells were labeled in vivo with
46 uCi 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 X100, 1% bovine hemoglobin, 1
mM iodoacetamide, 0.2 U aprotinin/ml, 1
mM phenylmethylsulfonyl fluoride (PMSF) in TSA
buffer (0.01 M Tris-HCl, pH 8.0, 0.14
M NaCl, 0.025% NaN3)] 1
ml/dish. After centrifugation of the lysate at 1,000 rpm for 5 min,
each 1 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 a D2 rabbit polyclonal antibody [No. 85254 (11)] to a final
dilution of 1:100. This antibody was generated against a synthetic
peptide SRSKSTRGEWRRMLTSEGLRC (residues 5272) selected from the human
D2 protein. Immunoprecipitates were obtained after 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) and then washed
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 and spun at top speed for 5 min, and 30 µl of the supernatants
were used for analysis by SDS-PAGE.
Western Blots of Epitope-Tagged D2
HEK-293 or ts20 cells transiently expressing the various
constructs were scraped, washed in PBS, suspended in lysis buffer (0.01
M Tris-HCl, pH 8.0, 0.14 M NaCl, 0.25
M sucrose, 1 mM PMSF, 2 µg/ml aprotinin, 2
µg/ml leupeptin), and sonicated for 34 sec. In one experiment cell
sonicates were centrifuged at 2,500 x g for 10 min,
and the supernatant was spun at 100,000 g for 1 h to
separate microsomes from cytosol. Protein concentration was measured as
described by Bradford (26), and 2040 µg were analyzed by 7.5% or
12% SDS-PAGE and electrotransferred to a polyvinylidene fluoride
membrane (Immobilon, Millipore Corp.,
Bedford, MA). The blots were probed with an anti-FLAG M2 antibody
(1:3333, Sigma) or with a polyclonal anti-Ub antibody
(1:1000, Chemicon, Temecula CA), or both. In the latter case, Ub
detection was followed by stripping and exposure to the same FLAG
detection. Samples processed for the Ub antibody were previously
purified on M2-anti-FLAG affinity agarose (Sigma). The
Western blot was carried out using the Chemiluminescence Kit of
Roche Molecular Biochemicals (Indianapolis IN), according
the instructions of the manufacturer.
Statistical Analysis
Data are presented as mean ± SD
throughout the studies. Students t test was used for
comparative analysis. Five percent was the level of significance
required to reject the null hypothesis.
 |
ACKNOWLEDGMENTS
|
---|
Dr. Kenneth Rock from University of Massachusetts (Worcester,
MA) kindly provided the ts20 cells. Human D2 cDNA was a Genethon clone
kindly provided by Drs. V. Galton and D. St. Germain.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Womens Hospital, HIM Building Room 550, 77 Avenue Louis Pasteur, Boston Massachusetts 02115. E-mail:
abianco{at}rics.bwh.harvard.edu
This work was supported by NIH Grant RO1-DK-36256. A.C.B. was partially
supported by the University of Sao Paulo (Sao Paulo, Brazil).
Received for publication June 15, 2000.
Revision received August 8, 2000.
Accepted for publication August 10, 2000.
 |
REFERENCES
|
---|
-
Murakami Y, Matsufuji S, Hayashi S, Tanahashi N, Tanaka K 2000 Degradation of ornithine decarboxylase by the 26S proteasome.
Biochem Biophys Res Commun 267:16[CrossRef][Medline]
-
McGee TP, Cheng HH, Kumagai H, Omura S, Simoni RD 1996 Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in
endoplasmic reticulum membranes is accelerated as a result of increased
susceptibility to proteolysis. J Biol Chem 271:2563025638[Abstract/Free Full Text]
-
Schork SM, Thumm M, Wolf DH 1995 Catabolite inactivation of
fructose-1,6-bisphosphatase of Saccharomyces cerevisiae.
Degradation occurs via the ubiquitin pathway. J Biol Chem 270:2644626450[Abstract/Free Full Text]
-
Hammerle M, Bauer J, Rose M, Szallies A, Thumm M, Dusterhus
S, Mecke D, Entian KD, Wolf DH 1998 Proteins of newly isolated mutants
and the amino-terminal proline are essential for
ubiquitin-proteasome-catalyzed catabolite degradation of
fructose-1,6-bisphosphatase of Saccharomyces cerevisiae.
J Biol Chem 273:2500025005[Abstract/Free Full Text]
-
Larsen PR, Silva JE, Kaplan MM 1981 Relationships between
circulating and intracellular thyroid hormones: physiological and
clinical implications. Endocr Rev 2:87102[Medline]
-
St. Germain DL 1988 The effects and interactions of
substrates, inhibitors, and the cellular thiol-disulfide balance on the
regulation of type II iodothyronine 5'-deiodinase. Endocrinology 122:18601868[Abstract]
-
Leonard JL, Kaplan MM, Visser TJ, Silva JE, Larsen PR 1981 Cerebral cortex responds rapidly to thyroid hormones. Science 214:571573[Medline]
-
Silva JE, Leonard JL 1985 Regulation of rat cerebrocortical
and adenohypophyseal type II 5'-deiodinase by thyroxine,
triiodothyronine, and reverse triiodothyronine. Endocrinology 116:16271635[Abstract]
-
St. Germain DL 1986 Hormonal control of a low Km (type II)
iodothyronine 5'-deiodinase in cultured NB41A3 mouse neuroblastoma
cells. Endocrinology 119:840846[Abstract]
-
Steinsapir J, Harney J, Larsen PR 1998 Type 2 iodothy-ronine
deiodinase in rat pituitary tumor cells is inactivated in proteasomes.
J Clin Invest 102:18951899[Abstract/Free Full Text]
-
Steinsapir J, Bianco AC, Buettner C, Harney J, Larsen PR 2000 Substrate-induced down-regulation of human type 2 deiodinase (hD2) is
mediated through proteasomal degradation, requires interaction with the
enzymes active center. Endocrinology 141:11271135[Abstract/Free Full Text]
-
Berry MJ, Maia AL, Kieffer JD, Harney JW, Larsen PR 1992 Substitution of cysteine for selenocysteine in type I iodothyronine
deiodinase reduces the catalytic efficiency of the protein but enhances
its translation. Endocrinology 131:18481852[Abstract]
-
Chowdary DR, Dermody JJ, Jha KK, Ozer HL 1994 Accumulation of
p53 in a mutant cell line defective in the ubiquitin pathway. Mol Cell
Biol 14:19972003[Abstract]
-
Silva JE, Mellen S, Larsen PR 1987 Comparison of kidney and
brown adipose tissue iodothyronine 5'-deiodinases. Endocrinology 121:650656[Abstract]
-
Hershko A, Ciechanover A 1998 The ubiquitin system. Annu Rev
Biochem 67:425479[CrossRef][Medline]
-
Bonifacino JS, Weissman AM 1998 Ubiquitin and the control of
protein fate in the secretory and endocytic pathways. Annu Rev Cell Dev
Biol 14:1957[CrossRef][Medline]
-
Mayer TU, Braun T, Jentsch S 1998 Role of the proteasome in
membrane extraction of a short-lived ER-transmembrane protein. EMBO J 17:32513257[Abstract/Free Full Text]
-
Brown MS, Ye J, Rawson RB, Goldstein JL 2000 Regulated
intramembrane proteolysis: a control mechanism conserved from bacteria
to humans. Cell 100:391398[Medline]
-
Berry MJ, Larsen PR 1994 Selenocysteine and the
structure, function, and regulation of iodothyronine deiodination:
update 1994. Endocr Rev 3:265269
-
St. Germain DL 1988 Dual mechanisms of regulation of type I
iodothyronine 5'-deiodinase in the rat kidney, liver, and thyroid
gland. Implications for the treatment of hyperthyroidism with
radiographic contrast agents. J Clin Invest 81:14761484[Medline]
-
Salceda S, Caro J 1997 Hypoxia-inducible factor 1
(HIF-1
) protein is rapidly degraded by the ubiquitin-proteasome
system under normoxic conditions. Its stabilization by hypoxia depends
on redox-induced changes. J Biol Chem 272:2264222647[Abstract/Free Full Text]
-
St. Germain DL, Croteau W 1989 Ligand-induced inactivation of
type I iodothyronine 5'-deiodinase: protection by propylthiouracil
in vivo and reversibility in vitro. Endocrinology 125:27352744[Abstract]
-
Gossen M, Bujard H 1992 Tight control of gene expression in
mammalian cells by tetracycline-responsive promoters. Proc Natl Acad
Sci USA 89:55475551[Abstract]
-
Salvatore D, Bartha T, Harney JW, Larsen PR 1996 Molecular
biological and biochemical characterization of the human type 2
selenodeiodinase. Endocrinology 137:33083315[Abstract]
-
Salvatore D, Harney JW, Larsen PR 1999 Mutation of the Secys
residue 266 in human type 2 selenodeiodinase alters 75Se incorporation
without affecting its biochemical properties. Biochimie 81:14
-
Bradford MM 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]