Proteasomes Modulate Conjugation to the Ubiquitin-like Protein, ISG15*

Mingjuan LiuDagger , Xiao-Ling Li§, and Bret A. HasselDagger §||

From the § Greenebaum Cancer Center,  Department of Microbiology and Immunology, and Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 epsilon -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).

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 Ikappa 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.

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-/- mice indicating that ISG15 conjugation is a dynamic process and that UBP43 serves a critical function in regulating the cellular conjugate pool.

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reagents-- IFN-alpha 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.

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 beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha -induced 2fTGH cells were treated with the proteasome inhibitor MG132. We have determined previously that the simultaneous treatment with IFN-alpha 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-alpha 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 alpha -actin protein did not change in response to IFN-alpha 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-alpha 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).

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.


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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-alpha 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.

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.


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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-alpha 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.

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.


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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-alpha 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.

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 alpha -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-alpha 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.

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-/- 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-alpha 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.

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 alpha -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.

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 alpha -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-alpha 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).

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.


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Fig. 8.   ISG15 conjugates are not simultaneously modified by ubiquitin. A, 2fTGH cells were treated with 1000 units/ml IFN-alpha 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

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-/- 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.

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 Ikappa 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-gamma 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.

    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.

    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

    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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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