From the § Greenebaum Cancer Center, ¶ Department
of Microbiology and Immunology, and Program in
Molecular and Cell Biology, University of Maryland School of
Medicine, Baltimore, Maryland 21201
Received for publication, August 8, 2002, and in revised form, October 9, 2002
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ABSTRACT |
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ISG15 is a ubiquitin-like protein
that is induced by interferon and microbial challenge. Ubiquitin-like
proteins are covalently conjugated to cellular proteins and may
intersect the ubiquitin-proteasome system via common substrates or
reciprocal regulation. To investigate the relationship between ISG15
conjugation and proteasome function, we treated interferon-induced
cells with proteasome inhibitors. Surprisingly, inhibition of
proteasomal, but not lysosomal, proteases dramatically enhanced the
level of ISG15 conjugates. The stimulation of ISG15 conjugates occurred
rapidly in the absence of protein synthesis and was most dramatic in
the cytoskeletal protein fraction. Inhibition of ISG15 conjugation by
ATP depletion abrogated the proteasome inhibitor-dependent
increase in ISG15 conjugates, suggesting that the effect was mediated
by de novo conjugation, rather than protection from
proteasomal degradation or inhibition of ISG15 deconjugating activity.
The increase in ISG15 conjugates did not occur through a stabilization
of the ISG15 E1 enzyme, UBE1L. Furthermore, simultaneous modification
of proteins by both ISG15 and ubiquitin did not account for the
proteasome inhibitor-dependent increase in ISG15
conjugates. These findings provide the first evidence for a link
between ISG15 conjugation and proteasome function and support a model
in which proteins destined for ISG15 conjugation are
proteasome-regulated.
Ubiquitin (ub)1 is the
most highly conserved protein among eucaryotes and functions to
post-translationally modify cellular proteins by covalent conjugation.
Ub conjugation is carried out by the concerted activities of E1 (ub
activation), E2 (ub conjugation), and E3 (ub ligase) enzymes in an
ATP-dependent process (1). Sequential transfer of ub-thiol
ester intermediates between ubiquitin-conjugating enzymes results in
isopeptide bond formation between the Ubiquitin-like proteins (ubls) are a growing family of proteins that
function to post-translationally modify cellular targets in a pathway
parallel to, but distinct from, that of ub (5). Ubls are translated
with carboxyl-terminal extensions that are processed to expose residues
that function in conjugation; this conjugation sequence is composed of
invariant LRGG carboxyl-terminal residues in orthologues of ISG15,
Nedd8, and ub (5). Like ub, ubls form covalent conjugates with cellular
proteins that can be reversed by DUB-like enzymes. However, ubl
conjugates are not typically targeted for degradation in the
proteasome; rather, conjugation to the ubls studied to date modulates
the subcellular location, protein interaction, and biochemical activity
of substrate proteins (6). Recent reports have demonstrated reciprocal
regulation by ub and ubl conjugation pathways. For example, the ubl
SUMO competes with ub for conjugation to lysine residues on
I ISG15 was among the first interferon-stimulated genes (ISG) to be
cloned and is induced independently of IFN, as one of the earliest and
strongest responses to microbial challenge (10-12). Sequence analysis
revealed that ISG15 is a ubl composed of tandem ub domains (13). Like
other ubls, ISG15 is translated as a 17-kDa precursor, and the
carboxyl-terminal octapeptide extension is rapidly processed by a
100-kDa protease to expose the ISG15 conjugation domain (14, 15). Free
ISG15 is induced rapidly following IFN treatment, and high molecular
weight ISG15 conjugates appear following a lag of 12-24 h (16). The
ISG15 E1 enzyme, UBE1L, has been identified recently; induction of this
enzyme by IFN requires protein synthesis that may account for the lag
in conjugate formation (17). A candidate ISG15 E2 enzyme, 1-8U, has
been identified based on its homology to other E2 enzymes and is a
member of a family of interferon-induced genes that are also directly
induced by virus and double-stranded RNA (18, 19). An ISG15 E3 enzyme has not been identified. We and others (20-22) have recently cloned a
DUB, UBP43, that is coordinately induced with ISG15 in response to IFN,
double-stranded RNA, and lipopolysaccharide and functions to
deconjugate specifically ISG15 from target proteins. ISG15 conjugates
are dramatically increased in cells from UBP43 In contrast to the regulation of ISG15 expression and conjugation,
little is known about the identity of specific ISG15 conjugates or the
functional role of ISG15 conjugation. ISG15 conjugates were reported to
localize to intermediate filaments; however, the relationship of this
localization to ISG15 function is unclear (23). Very recently, the
protease inhibitor, serpin 2a, was identified as the first ISG15
conjugate; how ISG15 conjugation may modulate the properties of this
protein remains to be determined (24). In light of the interactions
reported between ub and other ubls, we investigated the potential role
of proteasome function in ISG15 conjugation. Surprisingly, proteasome
inhibitors dramatically increased the level of ISG15 conjugates in
IFN-treated cells. This activity did not reflect an increase in ISG15
expression or the stabilization of ISG15 conjugates from proteasome
degradation; rather, the increase in ISG15 conjugates was mediated
through de novo conjugation. In addition, the proteasome
inhibitor-dependent increase in ISG15 conjugates did not
occur through the modulation of UBE1L or UBP43. These findings provide
the first evidence of a link between ISG15 conjugation and proteasome
function, and support a model in which proteins destined for ISG15
conjugation are regulated in a proteasome-dependent manner.
Reagents--
IFN- Cell Culture and Transfection--
2fTGH cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 100 µg/ml penicillin/streptomycin at 37 °C in a
humidified incubator of 5% CO2 (all cell culture reagents
were from Invitrogen).
For transfections, cells were seeded at 6.3 × 104
cells/cm2 and transfected with UBE1L expression vector
(kindly provided by Ethan Dmitrovsky, Dartmouth Medical School,
Hanover, NH), or vector lacking insert, using LipofectAMINE reagent as
directed by the supplier (Invitrogen). Transfected cells were harvested
at the times indicated in figure legends.
Western Blot Analysis--
Cells were treated as described in
figure legends, and total cell lysates were prepared using
radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium
chloride, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). The protein concentration in cell lysates
was determined by the Bradford microassay (Bio-Rad), and 100 µg of
lysate protein was separated on 7.5 and 10% SDS-polyacrylamide gels
for analysis of UBE1L and ISG15, respectively. Proteins were
electrotransferred to Immobilon-P membrane (Millipore). The membranes
were blocked in 5% nonfat milk for 30 min at room temperature and then
sequentially reacted to the primary antibody for 30 min in blocking
buffer and the horseradish peroxidase-conjugated secondary antibody
(1:10,000 dilution; Sigma). The immunoreactive complex was visualized
by the Pierce SuperSignal chemiluminescent substrate (Pierce) and
exposure to X-Omat AR film (Eastman Kodak Co.). The bound antibodies
were stripped from blots by incubation in 0.2 M NaOH for 5 min.
Differential Detergent Fractionation--
Cytosolic/membrane,
cytoskeleton-associated/nuclear, and cytoskeletal protein fractions
were prepared as described previously (25). Briefly, 2fTGH cells were
washed in PBS, resuspended in 5 pellet volumes of buffer A (10 mM PIPES, pH 6.8, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, 1.2 mM phenylmethylsulfonyl fluoride)
supplemented with 0.01% digitonin and 0.5% Triton X-100, and placed
on ice for 30 min. The extraction mix was centrifuged at 5000 × g for 10 min. The supernatant containing the
cytosolic/membrane fraction was collected, and the pellet was then
resuspended by vortexing in buffer A supplemented with 1% Tween 40 and
0.5% deoxycholate for 5 s. The resuspended pellet was centrifuged
at 14,000 × g for 20 min; the supernatant was
collected and represented the nuclear/cytoskeleton-associated protein
fraction. The insoluble pellet represented the cytoskeletal fraction
and was resuspended in 1× sample buffer (0.12 M Tris/HCl,
8% SDS, 40% glycerol, 0.36 M Immunoprecipitation--
Cells were lysed in RIPA buffer, and 1 mg of protein was incubated for 30 min on ice with 20 µl protein A/G
plus-agarose (Santa Cruz Biotechnology) in a total volume of 1 ml. The
mixture was centrifuged at 1000 × g for 5 min to
preclear the lysate of any proteins that nonspecifically bound to the
matrix. The supernatant was incubated with ISG15 monoclonal antibody at
2 µg/ml for 1 h on ice and then 20 µl of protein A/G
plus-agarose was added to the immune complexes for an additional 1 h on ice. The bound proteins were pelleted at 1000 × g
for 5 min and washed with RIPA buffer three times. The final pellets
were resuspended in 40 µl of 1× sample buffer and were boiled for 5 min prior to loading on a 10% SDS-PAGE gel.
ISG15 Conjugates Are Increased following Treatment with Proteasome
Inhibitor--
Post-translational modification by ubls including SUMO
and NEDD8 can intersect the ubiquitin pathway resulting in agonistic or
antagonistic effects on the activity of conjugating enzymes or
substrate half-life (7, 26, 27). Information on this reciprocal
regulation between ub and ubl pathways provides important insights into
the biologic functions of ubl conjugation. To determine whether the
ISG15 conjugation pathway was modulated by proteasome function,
IFN-
MG132 is a peptide aldehyde inhibitor of chymotrypsin-like proteasomal
proteases with nonspecific activity against calpains and certain
lysosomal cysteine proteases (30, 31). To demonstrate that the
proteasome was the MG132 target that mediated the increase in ISG15
conjugates, IFN-induced cells were treated with distinct proteasome
inhibitors, including the highly proteasome-specific inhibitor,
lactacystin, and inhibitors of lysosomal and cellular proteases. All
inhibitors employed were cell-permeable, thus access to intracellular
targets was not a limiting factor (32, 33). Western blot analysis of
ISG15 conjugates in cells treated with these inhibitors revealed that
compounds that blocked proteasome activity, but not inhibitors of other
proteolytic pathways, resulted in an increase in ISG15 conjugates (Fig.
2). An identical pattern of ISG15
conjugates and a similar magnitude of conjugate stimulation were
observed in response to distinct proteasome inhibitors, indicating that
proteasome inhibition by multiple independent mechanisms results in a
similar effect on ISG15 conjugates. These results provide the first
evidence of a link between proteasome function and ISG15
conjugation.
Cytoskeleton-associated ISG15 Conjugates Are Preferentially
Stimulated by Proteasome Inhibitors--
Analysis of ISG15 conjugates
in total cell lysates revealed that certain conjugate species are
increased more dramatically than others following treatment with
proteasome inhibitor. A subset of ISG15 conjugates is tightly
associated with the intermediate filament network of the cytoskeleton,
suggesting that distinct conjugate pools are localized to specific
cellular compartments (23). To determine whether proteasome-sensitive
ISG15 conjugates are associated with a particular subcellular fraction,
differential extraction was employed. Lysates containing cytoplasmic
and membrane proteins, nuclear and soluble cytoskeletal proteins, and
insoluble cytoskeletal proteins were prepared from 8 × 105 IFN-induced cells treated with or without MG132. The
differential detergent fractionation method employed here was
previously demonstrated to efficiently separate proteins found in these
subcellular compartments (25). Indeed, the cytoplasmic protein tubulin
was detected by Western blot in the cytosolic/membrane fraction with
little or no contaminating protein found in the other fractions (Fig.
3C). The most striking
MG132-stimulated increase in ISG15 conjugates was observed in the
insoluble cytoskeletal fraction, in which a predominantly high
molecular weight population of conjugates was detectable only following
MG132 treatment (Fig. 3A). Interestingly, no free ISG15 was
found in the insoluble cytoskeletal fraction; in contrast, ISG15 in the
soluble cytoskeletal and nuclear protein fraction was almost
exclusively free, with conjugates barely detectable. Both free ISG15
and ISG15 conjugates were previously found to associate with the
cytoskeleton by immunofluorescence staining (23); thus it is likely
that the free ISG15 in the soluble cytoskeletal and nuclear protein
fraction is derived from ISG15 that is loosely associated with the
cytoskeleton. The cytosolic and membrane protein fraction contained
both free ISG15 and an MG132-stimulated conjugate population (Fig.
3A). To compare the distribution of ub conjugates following
MG132 treatment with that observed for ISG15, the blot in Fig.
3A was reacted with anti-ub antibody. The monoclonal ISG15 antibody reacts specifically with free ISG15 and ISG15 conjugates; however, the polyclonal ub antibody exhibits cross-reactivity primarily
to free ISG15 and, to a lesser degree, with ISG15 conjugates (13, 34).
Thus, free ISG15 is detected by ub antibody in IFN-treated cells (Fig.
3B and Figs. 5-7). A minor population of ub conjugates that
loosely associated with the cytoskeleton was detected; however, similar
to ISG15, the most dramatic MG132-stimulated increase in ub conjugates
was observed in a population that is tightly associated with the
cytoskeletal fraction. MG132 treatment also increased ub conjugates in
the cytosolic and membrane fraction. These results suggest that
cytoskeleton is an important site for proteasome function and ISG15
conjugation.
Proteasome Inhibitor-mediated Stimulation of ISG15 Conjugates
Occurs Rapidly and Does Not Require Protein Synthesis--
The
immediate effect of proteasome inhibition is the stabilization of
ubiquitylated proteins; however, depending on the function of the
target protein, downstream consequences of proteasome inhibition can
affect diverse cellular processes including signal transduction and
transcription (35, 36). To determine whether the increase in ISG15
conjugates by proteasome inhibitors occurred through an increase in
ISG15 gene expression, IFN-induced cells were treated with MG132 in the
presence or absence of the protein synthesis inhibitor cycloheximide.
Following just a 2-h treatment with MG132, ISG15 conjugates were
dramatically up-regulated and continued to increase through 6 h
post-treatment (Fig. 4). The increase in
conjugates occurred in the presence of cycloheximide, indicating that
protein synthesis was not required. Indeed, MG132 treatment did not
affect the level of free ISG15, consistent with a conjugate-targeted activity that is independent of any change in ISG15 gene expression. Changes in the level of ISG15 conjugates did not reflect different amounts of protein in the samples analyzed, as indicated by a similar
signal when the Western blot was reacted with an actin antibody (Fig.
4, lower panel). These findings indicate that proteasome inhibitors induce a rapid and specific increase in ISG15 conjugates that does not require protein synthesis.
The Proteasome Inhibitor-dependent Increase in ISG15
Conjugates Requires de Novo Conjugation--
The steady state level of
ISG15 conjugates in IFN-treated cells reflects a dynamic balance
between conjugating and deconjugating activities (17, 22). In addition,
the observation that proteasome inhibitors enhance ISG15 conjugates
suggests that the turnover of ISG15 substrates, prior to or following
ISG15 conjugation, may impact the conjugate population. Thus, three
possible mechanisms may account for the increase in ISG15 conjugates
upon proteasome inhibition: 1) the stabilization of ISG15 conjugates or
substrates by protection from proteasome degradation, 2) an increase in
de novo ISG15 conjugation activity, and 3) an inhibition of
ISG15 deconjugating activity by ubp43. Conjugation of ub and ubls is ATP-dependent, and ATP depletion is an established strategy
to inhibit de novo conjugation (37). To address the role of
de novo ISG15 conjugation as a mechanism for the proteasome
inhibitor-dependent increase in ISG15 conjugates,
IFN-induced 2fTGH cells were treated with MG132 in the presence or
absence of the ATP-depleting agents, 2-deoxyglucose and
2,4-dinitrophenol. 2-Deoxyglucose depletes ATP through the hexokinase
shunt resulting in the production of 6-phospho derivatives that cannot
be further metabolized, whereas 2,4-dinitrophenol acts to uncouple the
electron transport chain. Western blot analysis revealed that ATP
depletion inhibited de novo conjugation and reduced the
total population of both ISG15 and ub conjugates (Fig.
5, A and B, compare
lanes 2 and 4). However, whereas ATP depletion
completely abolished the MG132-dependent increase in ISG15
conjugates, no effect of ATP depletion on the stabilization of ub
conjugates in MG132-treated cells was observed (Fig. 5, A
and B, compare lanes 2 and 3 with
lanes 4 and 5). A quantitative representation of
this result is shown in Fig. 5, D and E. The
expression of NEM Antagonizes the Stimulation of ISG15 Conjugates by Proteasome
Inhibitor--
Ub and ubl deconjugating activity is not
ATP-dependent; therefore, the finding that ATP depletion
abrogated the proteasome inhibitor-dependent increase in
ISG15 conjugates suggested that an alteration of ISG15 deconjugation by
UBP43 was not involved in the stimulation of conjugation by proteasome
inhibitors. However, UBP43
To determine whether UBP43 inhibition contributed to the
MG132-dependent increase in ISG15 conjugates, IFN-induced
cells were first treated with NEM for 20 min; NEM was then removed, and
MG132 was added for 6 h. This treatment regimen did not result in
toxic effects or cell death (40). Surprisingly, NEM antagonized the increase in ISG15 conjugates by MG132 in a dose-dependent
manner, with a 4-fold reduction in conjugates observed at 50 µM NEM (Fig. 6A, compare 3rd and
11th lanes). The opposing effects of NEM and MG132 suggested
that these agents stimulate ISG15 conjugates by distinct mechanisms. In
contrast to ISG15, NEM pretreatment resulted in no significant change
in MG132-stabilized ub conjugates. This finding is consistent with the
idea that the effect of NEM on ub conjugates was mediated primarily
through the inhibition of proteasome activity and thus was not
augmented by MG132 treatment. (Fig. 6B, 3rd,
5th, 7th, 9th, and
11th lanes). However, the opposing effects of MG132 and NEM
on ISG15 conjugates suggested that an additional step(s) was involved.
Expression of The ISG15 E1 Enzyme, UBE1L, Is Not Regulated by the
Proteasome--
De novo ISG15 conjugation is required for
the proteasome inhibitor-mediated increase in ISG15 conjugates observed
in IFN-treated cells. This effect of proteasome inhibitor may occur
through the stabilization of proteins targeted for ISG15 conjugation or
the stabilization of ISG15-conjugating enzymes. Specific ISG15
substrates have not been characterized; however, the ISG15 E1 enzyme,
UBE1L, was recently discovered (17). To determine whether MG132
treatment stabilized UBE1L protein that, in turn, stimulated ISG15
conjugation, a UBE1L expression vector, or vector without a cDNA
insert, was transiently transfected into 2fTGH cells. The cells were
treated with or without MG132 for 6 h at 20 and 50 h
post-transfection. Expression of transfected UBE1L in total cell
lysates was determined by Western blot reacted to antibody specific for
UBE1L. MG132 treatment did not affect the steady state level of UBE1L,
suggesting that UBE1L is not proteasome-regulated (Fig.
7A). To confirm that endogenous UBE1L was similarly not affected by proteasome inhibition, 2fTGH cells were treated with IFN for 24 h, and then MG132 was added for an additional 6 h prior to harvesting cell lysates. Western blot analysis revealed a strong induction of UBE1L by IFN;
however, the protein level was not increased following MG132 treatment
(Fig. 7B). Expression of Modification of ISG15 Substrates by ub--
In the absence of
de novo conjugation, ISG15 conjugates do not accumulate in
MG132-treated cells suggesting that existing ISG15 conjugates are not
directly degraded in the proteasome (Fig. 5). However, ub conjugates
that are stabilized by proteasome inhibitors may be subsequently
conjugated to ISG15 at a distinct site. To directly examine the
possibility that ISG15 conjugates are simultaneously modified by ub,
ISG15 was first immunoprecipitated from IFN-induced cells that had been
treated, or not, with MG132. ISG15 immunoprecipitates were then
analyzed by Western blot reacted to ISG15 and ub antibodies. Both free
and conjugated ISG15 were efficiently immunoprecipitated, as similar
patterns of ISG15 immunoreactive signals were observed in whole cell
lysates and immunoprecipitates (Fig.
8A). Although Western blot
analysis of whole cell lysates revealed an increased ub-immunoreactive
signal in lysates from MG132-treated cells, little or no immunoreactive
signal was observed in ISG15 immunoprecipitates (Fig. 8B).
However, a faint, high molecular weight ub signal was detected in the
ISG15 immunoprecipitates from MG132-treated cells, raising the
possibility that certain ISG15 substrates may be simultaneously modified by ub and ISG15. These potentially co-modified proteins represent a minor fraction of the ISG15 conjugates increased in proteasome inhibitor-treated cells, suggesting that co-modification is
not the primary mechanism by which proteasome inhibition leads to an
increase in ISG15 conjugates. Indeed smaller molecular mass ISG15
conjugate species that were markedly increased in response to
proteasome inhibitor (Fig. 1) did not appear as potentially co-modified
proteins. The identification of specific ISG15/ub immunoreactive
species is required to definitively address co-modification by ISG15
and ub.
The ubiquitin-proteasome pathway accounts for 80-90% of protein
turnover in cells and affects nearly every cellular process (4). Ubls
are a growing class of post-translational modifiers that are conjugated
to target proteins through an analogous enzymatic pathway to that of
ub. Recent studies have determined that ub and ubl conjugation pathways
can intersect through common substrates (7) and reciprocal regulation
(8, 9). ISG15 is an IFN-induced ubl; however, a relationship between
ISG15 conjugation and proteasome function has not been investigated.
Therefore, we examined the effect of proteasome inhibitors on the
population of ISG15 conjugates present in IFN-induced cells. Proteasome
inhibition resulted in a dramatic increase in ISG15 conjugates
providing the first evidence of a link between proteasome function and
ISG15 conjugation.
The steady state level of ubl conjugates is influenced by the
availability of free ubl modifier and substrate and the activity of
conjugating and deconjugating enzymes. To dissect the contribution of
these parameters to the MG132-dependent increase of ISG15
conjugates, we first employed ATP-depleting agents to distinguish
between ATP-dependent conjugation and ATP-independent
deconjugation activities. Inhibition of conjugation is predicted to
reduce the level of conjugates, and this was observed for both ub
(compare lanes 2 and 4 in Fig. 5B) and
ISG15 (compare lanes 2 and 4 in Fig.
5A) in ATP-depleted cells. Inhibition of proteasome activity
is predicted to increase the level of ub and ISG15 conjugates if they
are normally degraded in the proteasome, although DUB activity may
partially mask this effect. Thus, proteasome inhibition should result
in higher levels of conjugates in both the presence and absence of ATP
depletion. This was observed for ub conjugates but not for ISG15
conjugates; indeed, ATP depletion abrogated the effect of MG132 on
ISG15 conjugates. Thus, in the absence of de novo
conjugation, ISG15 conjugates did not accumulate in proteasome
inhibitor-treated cells suggesting that the MG132-dependent
increase in ISG15 conjugates was not due to a simple protection from
proteasomal degradation, and required de novo conjugation.
It is important to note that nothing is known about the ATP
requirements for UBE1L activity. A previous study in reticulocytes
demonstrated that a 6-h 2-deoxyglucose/2,4-dinitrophenol treatment
depleted ATP levels by 88% (37). The similar conditions employed here
reduced the level of both ub and ISG15 conjugates; however, we did not
determine the extent to which E1 or UBE1L was inhibited. Accordingly,
it is possible that ATP depletion altered ISG15 conjugation by a
mechanism distinct from, or in addition to, an effect on UBE1L. For
example, as ISG15 conjugation is an induced response to microbial
challenge, one or more of the ISG15-conjugating enzymes may be
regulated by kinases that would also be affected by ATP depletion. A
better understanding of the components and biochemistry of the
ISG15-conjugating enzymes is required to address this possibility.
Finally, ATP depletion may modulate ISG15 conjugation by inhibiting
proteasome activity; however, the Km value of the
proteasome for ATP (17 µM) (41) is about half that of the
ub E1 (36 µM) (42). The predominant effect of ATP
depletion is thus predicted to be an inhibition of ub E1 activity;
indeed, an effect of MG132 on ub conjugates was maintained in
ATP-depleted cells (Fig. 5) suggesting that the direct effect on
proteasomes was minimal.
ATP depletion was not predicted to alter DUB activity, yet it abrogated
the MG132-dependent increase in ISG15 conjugates, Therefore, the modulation of UBP43 activity did not appear to be a
primary mechanism by which proteasome inhibitors stimulate ISG15
conjugates. However, a striking increase in ISG15 conjugates was
observed in cells from UBP43 Conjugation of ub and ubls requires the free protein modifier,
conjugating enzymes, and the protein substrates of conjugation; accordingly, the proteasome inhibitor-dependent regulation
of one or more of these components may mediate the
MG132-dependent increase ISG15 conjugates. The increase in
conjugates occurred in the presence of protein synthesis inhibitor,
indicating that an alteration of ISG15 gene expression is not involved.
This finding points toward a modulation of the ISG15 conjugation
pathway enzymes, or the conjugation substrates themselves, as a
mechanism by which proteasome inhibitors increase conjugates. A
requirement for de novo conjugation suggested that the ISG15
E1 enzyme may be a target of proteasomal modulation; for example, the
stabilization of UBE1L by proteasome inhibitors may result in enhanced
ISG15 conjugation. However, we found no evidence for proteasome
regulation of transfected or endogenous UBE1L. An IFN-regulated gene,
1-8U, was recently identified as a putative ISG15 E2
(18). The extent to which 1-8U or yet to be
identified ISG15 E3 enzymes may be stabilized by proteasome inhibitors
and contribute to the stimulation of ISG15 conjugates by proteasome
inhibitors remains to be determined.
Taken together, our data suggest that free ISG15 and its known
conjugating and deconjugating enzymes are not the targets of proteasome
regulation responsible for the proteasome
inhibitor-dependent increase in ISG15 conjugates. However,
the proteasome regulation of ISG15 protein substrates could mediate
this effect. Indeed, our findings are consistent with a model in which
proteins destined for ISG15 conjugation are normally degraded in the
proteasome. Treatment with IFN induces ISG15 and its conjugating
enzymes, and proteasome inhibitors stabilize a subset of its substrates resulting in an increase in ISG15 conjugates (Fig.
9). In agreement with a requisite DUB
step prior to ISG15 conjugation in this model, only two minor signals
that represented potential ub-modified ISG15 conjugates were detected
(Fig. 8). Thus co-modification by ub and ISG15 could not account for
the dramatic increase in ISG15 conjugates observed in proteasome
inhibitor-treated cells. The role of ubiquitylation in
proteasome-dependent processes can be examined in rodent
cell lines that express a temperature-sensitive E1 (45); however, the
lack of antisera that cross-react with rodent ISG15 precluded this
approach in our study. The question of whether ISG15 competes with ub
for the same lysine residue on target proteins requires the
identification of specific ISG15 conjugates. In this scenario, ISG15
may have a stabilizing effect on its conjugates. Indeed, SUMO
modification of I
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino group of a substrate
lysine residue and the carboxyl-terminal glycine of ub. The conjugated
ub substrate can be targeted for further ubiquitylation in which
polyubiquitin chains linked through lysine 48 are formed. Importantly,
ubiquitylation is reversible, as ub can be removed from conjugates
through the action of deconjugating enzymes (DUBs) (2). DUBs share
highly conserved catalytic domains but exhibit great sequence diversity
outside of these regions; the heterogeneity of these noncatalytic
domains is thought to reflect substrate-specific activity. Thus,
ubiquitin conjugation is a dynamic balance between conjugation and
deconjugation. In the best characterized outcome of ubiquitylation,
proteins conjugated to poly-ub chains of four or greater are targeted
for degradation in the 26 S proteasome (3). The proteasome is composed
of two 19 S "cap" complexes that bind, de-ubiquitylate, and unfold
substrates to facilitate entry into the barrel-shaped 20 S proteolytic
core. Identification of the protease activities of the proteasome as chymotrypsin-like, trypsin-like, and caspase-like permitted the development of peptide inhibitors of proteasome function. Studies using
cell-permeable proteasome inhibitors revealed that the ub-proteasome pathway is responsible for 80-90% of protein turnover in cells and is
essential for the regulation of virtually all cellular processes
(4).
B resulting in a stabilizing effect of SUMO conjugation (7). In two
other examples of an intersection between ub and ubl pathways, the ubl NEDD8 and its conjugates are degraded in the proteasome via the adaptor
protein NUB1, and SUMO modification of promonocyte leukemia, itself a ub E3 enzyme, recruits the 19 S proteasome subunit to nuclear
bodies (8, 9). Protein modification by ub and ubls is thus emerging as
a complex network of post-translational regulation with the capacity to
rapidly modulate protein function.
/
mice indicating
that ISG15 conjugation is a dynamic process and that UBP43 serves a
critical function in regulating the cellular conjugate pool.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2b was obtained from Schering and was used
at 1000 units/ml. Proteasome inhibitors were from Calbiochem and used
at the following concentrations: MG132 (Z-Leu-Leu-Leu-CHO), 20 µM; proteasome inhibitor I (Z-Ile-Glu(OtBu)-Ala-Leu-CHO),
50 µM; proteasome inhibitor II (Z-Leu-Leu-Phe-CHO), 20 µM; ALLN (N-acetyl-Leu-Leu-Nle-CHO), 50 µM; and lactacystin, 20 µM. Protease
inhibitors (4-amidino-phenyl)methanesulfonyl fluoride and
N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatin), leupeptin (Ac-Leu-Leu-argininal), and the protease inhibitor mixture were from Roche Molecular Biochemicals. 2-Deoxy-D-glucose,
2,4-dinitrophenol, N-ethylmaleimide (NEM), and detergents
(digitonin, SDS, Triton X-100, Tween 40, and deoxycholate) were from
Sigma. Monoclonal antibody to ISG15 (used at 1 µg/ml) was a kind gift
from Ernest Borden (Taussig Cancer Center, The Cleveland Clinic
Foundation, Cleveland, OH). The polyclonal rabbit anti-ubiquitin
antibody (used at 2 µg/ml) was generously provided by Arthur L. Haas
(Medical College of Wisconsin, Milwaukee). Rabbit anti-actin polyclonal antibody (used at 1:500 dilution) was from Sigma, and the rabbit polyclonal UBE1L antibody (used at 1 µg/ml) was kindly provided by
Ethan Dmitrovsky (Dartmouth Medical School, Hanover, NH). The protein
molecular mass marker set was from Invitrogen.
-mercaptoethanol). 4×
sample buffer was added to all samples to give a 1× final
concentration and was incubated in a boiling water bath for 5 min prior
to loading on the gel.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced 2fTGH cells were treated with the proteasome inhibitor
MG132. We have determined previously that the simultaneous treatment
with IFN-
and proteasome inhibitor blocks IFN-stimulated signal
transduction and the induction of ISGs (28). In this study, we wanted
to examine specifically the role of proteasome function on existing
ISG15 conjugates; therefore, a regimen in which cells were first
treated with IFN-
to induce ISG15 conjugates and then treated with
proteasome inhibitor was employed. All proteasome inhibitor treatments
were for 6 h or less and did not result in cell toxicity (Refs. 4,
28, and data not shown). IFN treatment induced a high level of ISG15
conjugates in 2fTGH cells as detected by Western blot. Remarkably,
addition of MG132 resulted in a further 16-fold increase in ISG15
conjugates (Fig. 1). The stimulation of
ISG15 conjugates by proteasome inhibitor was also observed in
fibrosarcoma (HT1080), lung carcinoma (A549), and promonocytic (THP-1)
cell lines indicating that it was a general response of IFN-treated
cells to proteasome inhibitor (data not shown). 2fTGH cells, an
HT1080-derived cell line (29), exhibited a high level of ISG15
conjugation in response to IFN, and ISG15 conjugates were further
increased by MG132 treatment; therefore, subsequent analyses employed
this cell line. The increase in ISG15 conjugates occurred in the
absence of any significant change in free ISG15, suggesting that an
alteration of ISG15 gene expression was not involved (also see Fig. 4
below). Levels of the constitutively expressed
-actin protein did
not change in response to IFN-
and MG132 indicating that this
treatment did not result in a global change in protein expression (Fig.
1, lower panel). Interestingly, a decrease in free ISG15
corresponding to the increase in ISG15 conjugates was not observed;
this finding likely reflects the limitations of signal quantification
by the Western blot analysis employed here. Specifically, an increase
in ISG15 conjugates is readily detectable due to the fact that the
substrates are heterogeneous in size and the signal is spread over a
range of molecular masses; in contrast, the small decrease in the
induced pool of free ISG15 that gives rise to these conjugates may not
be discernible as a reduction in the signal from the single 15-kDa
ISG15 band. Some conjugate species were more dramatically enhanced by
proteasome inhibitor treatment (see bands marked with arrows
in Fig. 1); however, all of the conjugate species observed in
MG132-treated cells could also be detected in cells treated with IFN
alone following a longer, 30 min, exposure of the Western blot (data
not shown). Thus, proteasome inhibition appeared to effect a
quantitative rather than a qualitative increase in ISG15
conjugates.
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Fig. 1.
MG132 increases the level of IFN-induced
ISG15 conjugates. 2fTGH cells were treated with 1000 units/ml
IFN- for 24 h, and then MG132 (20 µM) was added
for an additional 6 h as indicated. Free ISG15 and ISG15
conjugates in 100 µg of whole cell lysate were detected by Western
blot (upper panel). The migration of molecular mass markers,
M, is shown. The ISG15 conjugates that are most dramatically
increased by MG132 are indicated with arrows. The blot was
stripped and reacted with anti-actin antibody to show equivalent
protein loading (lower panel).
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Fig. 2.
The increase in ISG15 conjugates is a
specific response to the inhibition of proteasomal proteases.
2fTGH cells were treated with 1000 units/ml IFN- for 24 h, and
then proteasome inhibitors (A) or inhibitors of cellular,
nonproteasomal, proteases (B) were added at concentrations
and times described under "Materials and Methods." Free ISG15 and
ISG15 conjugates in 100 µg of whole cell lysate were detected by
Western blot. M, molecular mass markers.
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Fig. 3.
The proteasome inhibitor-stimulated ISG15
conjugate population localizes to the cytosolic and insoluble
cytoskeletal compartments. 2fTGH cells were treated with 1000 units/ml IFN- for 24 h, and then MG132 (20 µM)
was added for an additional 6 h as indicated. Differential
detergent fractionation of 106 cells/treatment was employed
to generate the indicated subcellular fractions as described under
"Materials and Methods." A, free ISG15 and
conjugates were detected by Western blot. B, the blot
in A was stripped and reacted with anti-ubiquitin antibody.
The arrowhead indicates the cross-reactive free ISG15
detected by anti-ubiquitin antibody. C, the blot in
A was stripped and reacted with anti-tubulin antibody.
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Fig. 4.
The proteasome
inhibitor-dependent increase in ISG15 conjugates is rapid
and does not require protein synthesis. 2fTGH cells were treated
with 1000 units/ml IFN- for 24 h, followed by the addition of
cycloheximide (CHX) (50 µg/ml) in the presence or absence
of MG132 (20 µM) for the indicated times. Free ISG15 and
ISG15 conjugates in 100 µg of whole cell lysate were detected by
Western blot (upper panel). The blot was stripped and
reacted with anti-actin antibody to show equivalent protein loading
(lower panel). The migration of molecular mass markers,
M, is shown.
-actin was not altered by the treatments in this
experiment and served as a control for protein quantitation and
efficiency of Western blot transfer (Fig. 5C). The increase in ub conjugates following proteasome inhibitor treatment, in both the
presence and absence of ATP depletion, is due to the protection of
conjugates from proteasomal degradation (4). However, in the absence of
de novo conjugation, ISG15 conjugates do not accumulate in
the presence of MG132 in ATP-depleted cells suggesting that they are
not directly targeted for degradation in the proteasome. Taken
together, these findings indicate that de novo conjugation,
rather than stabilization of existing ISG15 conjugates, is required for
the increase in ISG15 conjugates by proteasome inhibitors.
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Fig. 5.
De novo ISG15 conjugation is
required for the proteasome inhibitor-stimulated increase in
conjugates. 2fTGH cells were treated with 1000 units/ml IFN-
for 24 h, followed by the addition of 20 µM MG132
alone or in combination with the ATP-depleting agents (20 mM 2-deoxyglucose (2-DG), 0.2 mM
2,4-dinitrophenol (DNP)) for an additional 6 h.
A, free ISG15 and conjugates were detected by Western
blot. B, the blot in A was stripped and
reacted with anti-ubiquitin antibody; the arrowhead
indicates the cross-reactive free ISG15 detected by anti-ubiquitin
antibody. C, the blot in A was stripped and
reacted with anti-actin antibody to show equivalent protein loading.
The migration of molecular mass markers, M, is shown. The
signals from ISG15 and ubiquitin conjugates were quantified by
densitometry and are shown in D and E,
respectively.
/
mice exhibit an increased population of
ISG15 conjugates (22) that mimics the effect of MG132; this finding
raised the possibility that UBP43 may play an indirect role in the
stimulation of ISG15 conjugates by MG132. DUBs and ubl-deconjugating
enzymes exert their enzymatic activities via the cysteine SH group in their catalytic site (2). N-Ethylmaleimide, a
membrane-permeable thiol-blocking agent that irreversibly replaces the
hydrogen atom in SH groups, can thus be employed to examine the role of
DUBs in the stimulation of ISG15 conjugates by MG132 (38). Accordingly, IFN-induced 2fTGH cells were first treated with increasing doses of NEM
alone. Western blot analysis of ISG15 conjugates demonstrated a NEM
dose-dependent increase in ISG15 conjugates, with an 8- and
135-fold increase observed at 20 and 50 µM NEM,
respectively (Fig. 6A,
2nd, 4th, and 10th lanes). The
apparent low level of ISG15 conjugates in cells treated with IFN alone
reflects a short film exposure of the Western blot to facilitate
detection of changes in the ISG15 conjugate population (Fig.
6A, 2nd lane). Although NEM can also inhibit ub
and ubl conjugation by blocking the catalytic cysteines of E1, E2, and
some E3 enzymes, the increase of ISG15 conjugates by NEM suggests that
UBP43, rather than an ISG15-conjugating enzyme, was the major target of
NEM inhibition under the conditions employed. Similar to its effects on
ISG15 conjugates, treatment with NEM alone also resulted in an increase
in ub conjugates, suggesting that it inhibited ub-DUB activity (Fig.
6B, 2nd, 4th, 6th,
8th, and 10th lanes). Importantly, we cannot
distinguish between the effect of NEM on ub-DUBs that act upstream of
the proteasome to remove ub from the poly-ubiquitylated substrates, and
its effect on ub-DUBs that act downstream of the proteasome to recycle
ub from the poly-ub chain. Blocking the activity of upstream ub-DUBs
would directly prevent deconjugation leading to increased degradation;
whereas inhibition of downstream ub-DUBs would accumulate poly-ub
chains that, in turn, inhibit proteasome activity and mimic the effect
of proteasome inhibitors (39). Indeed, the effect of NEM on ISG15
conjugates may be due to proteasome inhibition by accumulated ub
chains.
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Fig. 6.
NEM antagonizes the increase in ISG15
conjugates by proteasome inhibitor. 2fTGH cells were treated with
1000 units/ml IFN- for 24 h, followed by the addition of NEM at
the indicated concentration for 20 min; specified samples were further
treated with 20 µM MG132 for 6 h. A,
free ISG15 and conjugates were detected by Western blot.
B, the blot in A was stripped and reacted
with anti-ubiquitin antibody; the arrowhead indicates the
cross-reactive free ISG15 detected by anti-ubiquitin antibody.
C, the blot in A was stripped and reacted
with anti-actin antibody to show equivalent protein loading. The
migration of molecular mass markers, M, is shown. The
signals from ISG15 and ubiquitin conjugates were quantified by
densitometry and are shown in D and E,
respectively.
-actin was not altered by NEM or MG132 treatment and
served as a control for protein quantitation (Fig. 6C). The
results from these experiments were quantified by densitometry and are
depicted in Fig. 6, D and E. Taken together,
these data support a model in which the increase in ISG15 conjugates by
MG132 requires de novo ISG15 conjugation and is independent
of ISG15 deconjugating activity.
-actin served as a control for
protein quantitation in these blots (Fig. 7, A and
B, lower panel). These findings indicate that
stabilization of UBE1L is not the mechanism by which proteasome
inhibitors stimulate ISG15 conjugation.
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Fig. 7.
The steady state level of UBE1L is not
regulated by the proteasome. A, 2fTGH cells were transfected
with a UBE1L expression plasmid or empty vector; at 20 and 50 h
post-transfection, cells were treated with 20 µM MG132
for 6 h prior to harvesting the cells. UBE1L protein in 100 µg
of whole cell lysate was detected by Western blot. B, 2fTGH
cells were treated with 1000 units/ml IFN- for 24 h, and 20 µM MG132 was added for 6 h prior to harvesting the
cells. Endogenous UBE1L protein in 100 µg of whole cell lysate was
detected by Western blot. The blots in A and B
were stripped and reacted with anti-actin antibody to demonstrate
equivalent protein loading (lower panels).
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Fig. 8.
ISG15 conjugates are not simultaneously
modified by ubiquitin. A, 2fTGH cells were treated with
1000 units/ml IFN- for 24 h, and 20 µM MG132 was
added for 6 h as indicated prior to harvesting the cells. ISG15
and conjugates were immunoprecipitated (i.p.) from whole
cell lysates (1 mg of protein). ISG15 and conjugates in
immunoprecipitates (lanes 1-3) and total cell lysates
(lanes 4-6, 100 µg/lane) were detected by Western
blot. B, the blot in A was stripped and
reacted with anti-ubiquitin antibody. Heavy and light chains of the
antibody are indicated with open arrowheads; the
asterisks indicate nonspecific bands, and the closed
arrowhead indicates the free ISG15 detected by anti-ubiquitin
antibody. ctrl, control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice, which mimicked the effect of
proteasome inhibitor treatment (22). This finding indicated that the
ISG15 conjugate pool is dynamically regulated and demonstrated the role
of UBP43 in this process. Therefore, we employed the thiol-alkylating
agent, NEM, to investigate a potential contribution of UBP43 to the
MG132-dependent increase in ISG15 conjugates. NEM
treatment, which inhibits ub- and ubl-conjugating and -deconjugating enzymes, resulted in a dose-dependent stimulation of ub and
ISG15 conjugates, reflecting the inhibition of ub- and
ISG15-deconjugating activity by NEM. However, NEM antagonized
MG132-dependent increase of ISG15 conjugates but not the
increase of ub conjugates. The opposing effects of NEM and MG132 on
ISG15 conjugation suggested that these agents stimulate ISG15
conjugates by distinct mechanisms. The inhibition of ub-DUB activity by
NEM may result in the accumulation of poly-ub chains that, in turn,
inhibit proteasome activity (39). In addition, a high dose of NEM (5 mM) can directly inhibit the trypsin-like activity of the
proteasome (43). These activities of NEM are consistent with the
absence of a modulation of MG132-stabilized ub conjugates by NEM.
Indeed, the stimulation of ISG15 conjugates by NEM alone may result
from its direct inhibition of the proteasome. Alternatively, NEM may
influence ISG15 conjugation via effects on thiol-sensitive proteins
that are distinct from DUBs. Thus, a definitive analysis of the role of
UBP43 in the stimulation of ISG15 conjugates by proteasome inhibitors
requires a direct assay for UBP43 activity in intact cells. In this
regard, a vinyl sulfone ub derivative has been developed that
irreversibly modifies the DUB active site thiol group; isotopic
labeling of this derivative permits the direct labeling of active DUBs
in cell lysates (44). An analogous ISG15 derivative would permit an
analysis of UBP43 in cell lysates.
B occurs at the same lysine that is conjugated by
ubiquitin; SUMO modification of these substrates thus antagonizes
ub-mediated proteasome targeting resulting in protein stabilization
(7).
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Fig. 9.
A model of the relationship between
proteasome activity and ISG15 conjugation. A subset of proteins
destined for ISG15 conjugation is ubiquitylated and targeted for
degradation in the proteasome (ubiquitin conjugation). IFN treatment
induces ISG15 and ISG15-conjugating enzymes (ISG15 conjugation). MG132
treatment of IFN-induced cells results in the stabilization of ub
conjugates; de-ubiquitylation of these conjugates increases the ISG15
substrate pool, leading to increased levels of ISG15 conjugates.
S, protein substrate of ub and ISG15 conjugation;
asterisk, ub in poly-ub chain; open circle,
ISG15; barrel structure, 26 S proteasome.
Importantly, all conjugates detected on Western blots of IFN-induced, proteasome inhibitor-treated cells were also observed, albeit at lower levels, in cells treated with IFN alone. In addition, distinct ISG15 conjugate species were stimulated to different levels in response to proteasome inhibition, suggesting that certain conjugate substrates are more rapidly turned over. This quantitative and selective change in ISG15 conjugates suggests that authentic ISG15 conjugates are directly or indirectly proteasome-regulated and that the proteasome inhibitor-dependent increase in ISG15 conjugates is biologically relevant, and not due to conjugation to non-substrate proteins stabilized by MG132.
In addition to modulating protein stability by antagonizing ub conjugation, an established function of ubls involves targeting their conjugates to specific subcellular compartments. For example, the first SUMO conjugate identified, RanGAP1, requires SUMO modification for its localization to the nuclear pore (46, 47). More recently, SUMO modification was determined to be critical for targeting proteins for subnuclear structures known as nuclear bodies (48). Similarly, immunofluorescence staining revealed that a subpopulation of free ISG15 and its conjugates are associated with the intermediate filament network (23). By employing differential detergent fractionation, we confirmed this localization, and we determined that a significant fraction of free ISG15, but only a very small fraction of conjugates, is loosely associated with the cytoskeleton. Strikingly, a substantial portion of the MG132-stimulated ISG15 conjugates was resistant to detergent extraction and thus cofractionated with cytoskeletal proteins. MG132 treatment reduced the level of free ISG15 loosely associated with the cytoskeleton, suggesting that free ISG15 is conjugated to cytoskeletal components or tightly associated proteins upon MG132 treatment. The dramatic effect of proteasome inhibitors on the cytoskeletal fraction of ISG15 conjugates suggests that this compartment is an important site of proteasome function and ISG15 conjugation.
ISG15 is induced as one of the earliest and strongest responses to IFN,
and to microbial challenge independent of IFN, suggesting that it
serves an important function in host defense. Consistent with this
role, the NS1 proteins of influenza virus A and B inhibit ISG15
expression and conjugation, respectively, suggesting that ISG15
mediates an antiviral activity that must be circumvented by the virus
(17). However, the specific functions of ISG15 in the host response to
microbial challenge are not known. For example, ISG15 is released from
cells where it exhibits immunomodulatory activities including IFN-
induction and NK cell activation (49), yet the in vivo role
of extracellular ISG15 in host defense has not been examined. Within
cells, our data indicate that the cytoskeleton is an active site of
ISG15 conjugation following IFN treatment. The cytoskeleton functions
to provide mechanical strength, maintain cell shape, and regulate
vesicle trafficking and cell movement (50) through the cooperative
interaction of microfilaments, microtubules, and intermediate filaments
(51). The cytoskeleton is also important for viral trafficking and
replication (52, 53). For both herpes simplex virus 1 (HSX1) and
adenovirus serotype 2, transport from the cell periphery to the nucleus
after host entry is dependent on the interaction of specific viral
proteins with the microtubule network (54, 55). Accordingly, disruption of microtubules results in a 40% reduction in HSX1 nucleocapsid transport to the nucleus (56). In the export of assembled viral progeny, vaccinia viral capsids sequentially interact with microtubule and actin networks to translocate from the site of replication (57).
For viruses that assemble at the plasma membrane including influenza
and retroviruses, viral proteins have also been found to be associated
with cytoskeleton (58, 59). Furthermore, actin filaments play a central
role in the replication of human parainfluenza virus type 3;
accordingly, cytochalasin D, a potent actin-depolymerizing agent,
reduced the viral RNA to 30% that in untreated control cells (53).
ISG15 may interfere with the viral activities that are localized to
this compartment via the modification of cytoskeletal proteins or
tightly associated components to alter virus-host protein interactions.
Alternatively, ISG15 may directly conjugate to viral proteins; this
possibility is currently under investigation.
In summary, these findings provide the first evidence for a link
between ISG15 conjugation and proteasome function and suggest that
ISG15 conjugation may modulate protein stability. A complete understanding of this relationship, and how proteasomal regulation relates to the biologic functions of ISG15, will require the
identification of specific ISG15 conjugates. The first ISG15 conjugate,
serpin2a, was recently identified (24). Interestingly, ISG15-conjugated serpin2a was neither proteasome-regulated nor localized to the cytoskeleton. This is in agreement with our finding that certain ISG15
conjugates are increased to a greater extent by proteasome inhibitors
than others and that a subset of the ISG15 conjugate population
localizes to the cytoskeleton. ISG15 conjugation is thus likely to
exert diverse, possibly substrate-specific functions to mediate
antimicrobial activities.
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ACKNOWLEDGEMENTS |
---|
We thank Ernest Borden (The Cleveland Clinic Foundation), Arthur Haas (Medical College of Wisconsin), and Ethan Dmitrovsky (Dartmouth Medical School) for kindly providing reagents. We thank B. Timothy Hummer for critical discussion of this manuscript.
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FOOTNOTES |
---|
* This work was supported by Grant RPG-99-195-01 from the American Cancer Society (to B. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Greenebaum Cancer
Center, University of Maryland School of Medicine, 655 West Baltimore
St., 9th Floor BRB, Baltimore, MD 21210. Tel.: 410-328-2344; Fax:
410-328-6559; E-mail: bhassel@som.umaryland.edu.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M208123200
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ABBREVIATIONS |
---|
The abbreviations used are: ub, ubiquitin; ubl, ubiquitin-like; IFN, interferon; DUB, de-ubiquitylating enzyme; NEM, N-ethylmaleimide; ISG, interferon-stimulated gene; E1, ubiquitin-activating enzyme; E2, ubiquitin conjugating enzyme; E3, ubiquitin-ligase; Z, benzyloxycarbonyl; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); SUMO, small ubiquitin-like modifier.
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