Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules

Cell, Vol. 78, 761-771, September9, 1994, Copyright© 1994 by Cell Press

Inhibitors of the Proteasome Block the Degradation of Most Cell Proteins and the Generation of Peptides Presented on MHC Class I Molecules Kenneth L. Rock,*t Colette Gramm,* Lisa Rothstein,* Karen Clark,* Ross Stein,$ Lawrence Dick,t Daniel Hwang,§ and Alfred L. Goldberg§ *Division of Lymphocyte Biology Dana-Farber Cancer Institute Boston, Massachusetts 02115 tDepartment of Pathology Harvard Medical School Boston, Massachusetts 02115 §Department of Cellular and Molecular Physiology Harvard Medical School Boston, Massachusetts 02115 ~Myogenics, Incorporated 1 Kendall Square, Building 200 Cambridge, Massachusetts 02139

Summary Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblaste, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules. Introduction Proteins in eukaryotic cells are continually being turned over at distinct rates, and this degradative process plays important roles. First, regulation of cell cycle, gene transcription, and metabolic pathways requires the rapid elimination of key regulatory proteins (e.g., cyclins) or ratelimiting enzymes. Second, cell proteins with abnormal conformation, whose accumulation could be damaging, are degraded particularly rapidly (Goldberg and St. John, 1976; Rechsteiner, 1987; Hershko and Ciechanover, 1992). However, the importance of protein degradation in many cellular responses has been difficult to study because of the lack of selective inhibitors of the major catabolic systems of the cell. Eukaryotic cells contain multiple proteolytic systems, including the lysosomal proteases, calpains, the ATP-

ubiquitin-proteasome-dependent pathway, and an ATPindependent nonlysosomal process (Gronostajski et al., 1985; Dice, 1987; Tawa et al., 1992). The major neutral proteolytic activity in the cytosol and nucleus is the proteasome, a 20S (700 kDa) particle with multiple peptidase activities (Orlowski, 1990; Rivett, 1993). The functions of the proteasome in vivo are still unclear. It is essential in the ATP-ubiquitin-dependent pathway, where it functions as the proteolytic core of the 26S (1500 kDa) complex that degrades ubiquitin-conjugated proteins (Goldberg, 1992; Hershko and Ciechanover, 1992; Rechsteiner et al., 1993). In this pathway, proteins are first modified by covalent conjugation to ubiquitin, which marks them for rapid hydrolysis by the 26S proteasome. Although this pathway is clearly important in the breakdown of abnormal or shortlived normal polypeptides, its role in the turnover of the bulk of cell proteins is uncertain (Ciechanover et al., 1984; Gropper et al., 1991). Another important function of intracellular proteolysis is to generate the small peptides that are presented to T lymphocytes to initiate immune responses (Goldberg and Rock, 1992; Germain and Margulies, 1993). As part of the continual surveillance by the immune system against autologous cells that are virally infected or have undergone oncogenic transformation, certain peptide fragments of cellular proteins are transported from the cytosol into the endoplasm ic reticulum by the TAP1/TAP2 transporter (Townsend and Trowsdale, 1993). These peptides then bind to newly synthesized major histocompatibility complex (MHC) class I molecules and are transported to the plasma membrane. If a peptide is displayed for which the immune system is not tolerant (e.g., a viral or mutated sequence), then cytolytic CD8 T lymphocytes will be stimulated and eliminate the offending cell. The precise origin of most of the peptides presented on MHC class I molecules is still not clear. Recently, evidence has accumulated that the proteasome may play a role in MHC class I presentation (Goldberg and Rock, 1992; Monaco, 1992). Ubiquitin conjugation, and presumably therefore the 26S proteasome, has been shown to be important for MHC class I presentation of a model antigen (Michalek et al., 1993). Moreover, two subunits of the proteasome (LMP2 and LMP7) are encoded in the MHC locus and induced by y-interferon (Monaco, 1992). Their incorporation into the proteasome has been shown to alter peptidase activities of this particle in a manner that should favor the generation of the types of peptides that are preferentially transported and bind to MHC class I molecules (Driscoll et al., 1993; Gaczynska et al., 1993). However, whether the proteasome plays a major or minor role in antigen processing is a key unresolved issue. In fact, evidence has been presented that proteolysis is not necessary for class I presentation, and that the antigenic peptides are instead generated directly (as so-called peptons) via the synthesis of short mRNA transcripts, the initiation of translation at aberrant start sites, or both (Boon and Van Pel, 1989). Reagents that could block the proteolytic

Cell 762

Table 1. Effect of Peptide-AldehydeInhibitorson 20S and 26S Proteasomes (K, in p.M) 20S with Suc-Leu- 26S with Suc-Leu- 26S with Z-LeuInhibitor Leu-VaI-Tyr-AMCa Leu-VaI-Tyr-AMCb Leu-Glu-I3NA~

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processing of antigens for M HC class I presentation could help resolve the importance of such a pathway. The roles of the lysosomal-endosomal proteases in MHC class II antigen presentation and in the degradation of endocytosed proteins has been established by the use of weak bases and inhibitors of the cysteine proteases of the lysosome (Germain and Margulies, 1993); however, these agents do not affect class I antigen presentation (Braciale et al., 1987) or the degradation of the bulk of cell proteins (Dice, 1987; Tawa et al., 1992). This article reports agents that inhibit proteasome function in intact cells, and we use them to demonstrate that this particle is required for the degradation of both longlived and short-lived cytosolic proteins and for the generation of most peptides presented on MHC class I molecules.

Results Inhibiting Proteasome-Catalyzed Hydrolysis of Peptides and Ubiquitinated Protein The 20S proteasome can cleave peptides on the carboxyl side of hydrophobic, basic, and acidic residues (Orlowski, 1990; Rivett, 1993). These peptidase functions, commonly referred to as the chymotryptic, tryptic, and peptidylglutamyl peptide hydrolyzing activities, were measured by monitoring the hydrolysis of the fluorogenic substrates Suc-Leu-Leu-VaI-Tyr-AMC, Z-Leu-Leu-Arg-AMC, and Z-Leu-Leu-Glu-13NA, respectively. Certain peptide aldehydes have been reported to inhibit this chymotryptic activity (Vinitsky et al., 1992; Tsubuki et al., 1993). We therefore tested the ability of this class of compounds to inhibit 20S proteasomes pu rifled from rabbit skeletal muscle (Table 1; Figure 1). We confirmed that N-acetyI-L-leucinyI-L-leucinaI-L-norleucinal (LLnL) could inhibit the hydrolysis of Suc-Leu-Leu-VaI-Tyr-AMC (K,, 0.14 ~M), while a closely related analog, N-acetyI-L-leucinyI-L-leucinylmethional (LLM), was a significantly weaker inhibitor (K,, 1.0 pM). A structurally related compound, N-carbobenzoxyI-L-

leucinyI-L-leucinyI-L-norvalinal(MG115), was an even more potent inhibitor of the chymotryptic activity of the 20S proteasome (K,, 0.021 IIM). In vivo, the 20S proteasome exists predominantly as part of the 26S complex (Orino et al., 1991), which contains additional subunits that allow the ATP-dependent degradation of ubiquitinoconjugated proteins (Goldberg, 1992; Hershko and Ciechanover, 1992; Rechsteiner et al,, 1993). The catalytic properties of the 26S particle have not been studied in depth. We therefore purified this complex from rabbit muscle and tested the effects of the peptide aldehydes on the hydrolysis of the same substrates (Table 1). These aldehydes inhibit the 20S and 26S proteasomecatalyzed hydrolysis of Suc-Leu-Leu-VaI-Tyr-AMC with the same rank order of potency, although inhibition of the 26S structure required higher concentrations of these compounds. LLnL and MG115 also inhibited the hydrolysis of Z-Leu-Leu-Glu-13NA and, to a much lesser degree, Z-Leu-Leu-Arg-AMC. In vivo, the major function of the proteasome must be in digesting complete proteins and ubiquitin-conjugated proteins. Although most proteins are hydrolyzed by the 26S proteasome only after conjugation to multiple ubiquitin molecules, this particle can hydrolyze certain proteins, such as casein (Driscoll and Goldberg, 1990), without further modification. MG115 and LLnL inhibited the hydrolysis of [14C]methyl-casein (Figure 2) and did so by preventing the initial cleavages of the casein molecule (data not shown). Furthermore, MGl15 and LLnL reduced hydrolysis of a ubiquitin-conjugated protein by the purified 26S particle (Figure 2). The degradation of ubiquitinated 1251-iysozyme was an ATP-dependent process that was linear for 15 min and then proceeded at a slower rate (D. H. and A. L. G., unpublished data). A dose-dependent inhibition of the early (Figure 2) and late phases (data not shown) was seen with both MG115 and LLnL. In contrast, LLM did not significantly inhibit the catabolism of ubiquitin-conjugated lysozyme (data not shown). It is noteworthy that these

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three aldehydes showed the same relative potencies for inhibiting the degradation of casein, ubiquitin-conjugated lysozyme, and Suc-Leu-Leu-VaI-Tyr-AMC. Although the potency of MGl15 and LLnL appears much greater against the fluorogenic peptide, a rigorous kinetic analysis of their ability to inhibit degradation of ubiquitinated proteins is not yet possible for several reasons, and these data probably underestimate their inhibitory activity. The present methods for preparing ubiquitinated substrates yield only low amounts of heterogeneous conjugates, which vary widely in the number of ubiquitin moieties incorporated and in their susceptibilities to hydrolysis (Tamura et al., 1991). Moreover, because of the limited amounts of ubiquitin-conjugated proteins that can be generated, these assays are necessarily performed with concentrations of 26S proteasome that must be higher than that of the actual ubiquitinated substrate. The reaction progress curves for the hydrolysis of ubiquitinated ~251-1ysozymeare nonlinear probably because of the substrate heterogeneity. Consequently, the Kr, for the reaction (which must be very low) and the K, values for the inhibitors cannot be accurately measured and may not even be interpretable. Nevertheless, these experiments do indicate that at micromolar levels, the peptide aldehydes can be used to inhibit proteasomal degradation of ubiquitin-conjugated and free polypeptides. As reported previously, LLnL and LLM also inhibit calpains and lysosomal cysteine proteases (e.g., cathepsin B) (Sasaki et al., 1990), and we observed a similar inhibition with MGl15 (see Table 1). In contrast to their very different activities against the proteasome, the three peptide aldehydes inhibited calpain and cathepsin B with similar potencies (K, values, 5-12 nM). Thus, it is possible, by using different concentrations of these three agents, to implicate the proteasome in specific responses.

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Figure 2. Effectof PeptideAldehydeson ProteolyticActivitiesof the 26S ProteasomeComplex The percent of inhibition of the generationby the 26S proteasome of TCA-solublecpm from p4C]methyl-casem(A) and ubiquitinJ2Sllysozyme (B) are plotted versus the various concentrationsof each peptide aldehyde.Productionof acid-solublefragmentsoccurred at linearrates,whichwereproportionalto proteasomeconcentration.Up to 40% of caseinwas degradedin 60 min, and up to 10% of the total ubtquitin-conjugated~2Sl-lysozymewas degradedin 10 rain.

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Figure 3. Effect of Peptide Aldehydes on Protein Catabolism m Lymphoblasts Representativeexperimentsshowingthe effectof peptidealdehydes on the degradationof short-livedproteins(A), aminoacidanalog(canavanine)-containingproteins(B), long-livedproteinswithoutpreincubation withinhibltors(C),and long-livedproteinsaftera 2 hr preincubation with mhibitors(D). Closedcircles, LLnL; open circles, MGl15; open triangles, LLM. (E) displaysa comparisonof the mean inhibitionby MGl15 on the degradationon long-livedproteins (open triangles), short-lived normal proteins(opendiamonds),and abnormalproteins (opensquares)for multipleexperiments.(F) displaysa comparisonof the meaninhibitionby LLnLin multipleexperimentson the degradation of long-livedproteins(with preincubation)(opentriangles)and on the MHC class I presentationof ovalbumin(closedtriangles).LB27.4cells were labeledwith [aH]tyrosine,and the effects of LLnL, MGl15, or LLM on the catabolismof labeledproteinsfor 60 min was determined. The total incorporation(cpm) of pH]tyrosineinto TCA-insolublefractionswas as follows:44,106(A),393,373(B),778,006(C),and 335,353 (E). The percentagesof total radioactivitythat wereconvertedto TCAsoluble fractions were 220/0(A), 72°/0(B), 30/0(C), and 70/0(E).

an extralysosomal pathway that is dependent on ATP (Goldberg and St. John, 1976; Hershko and Ciechanover, 1992); however, it is uncertain which protease(s) degrades the bulk of cell proteins. To investigate whether proteasomes are involved, we examined the effects of the peptide aldehydes on degradation of short-lived and long-lived normal proteins, as well as highly abnormal proteins that have incorporated the amino acid analog canavanine. Although ubiquitination has been implicated in the hydrolysis of such short-lived and abnormal polypeptides (Ciecha-

Cell 764

nover et al., 1984; Gropper et al., 1991), the enzymes that degrade long-lived proteins have not been identified. To measure the degradation of short-lived proteins, B lymphoblastoid cells were labeled with [3H]tyrosine for 1 hr. Inhibitors were then added, and the release of acid-soluble radioactivity from prelabeled proteins was measured in the presence of excess nonradioactive tyrosine. The release of labeled amino acids occurs at varying rates that reflect the degradation of different classes of cell proteins (see, e.g., Gronostajski et al., 1985). Typically, about 20%-30% of the pulse-labeled proteins were degraded in 1 hr. This process was extralysosomal, because it was insensitive to chloroquine or leupeptin, as reported previously (Goldberg and St. John, 1976; Gronostajski et al., 1985). By contrast, the peptide aldehydes inhibited the degradation of these short-lived proteins in a dose-dependent manner (Figure 3A). MG 115 was 2- to 3-fold more potent than LLnL, which was more than 10-fold more potent than LLM. To measure the very rapid breakdown of abnormal proteins, cells were incubated with the arginine analog canavanine during the pulse labeling. During a chase period, the inhibitors were added and degradation of labeled proteins measured. As expected (Goldberg and St. John, 1976; Dice, 1987), the canavanine-containing proteins were degraded very rapidly (typically 60%-72% per hour) by a chloroquine-insensitive mechanism. The peptide aldehydes also markedly inhibited this process (Figure 3B), and their potencies against the degradation of the abnormal proteins and short-lived normal proteins were essentially identical. By contrast, the bulk of cell proteins are quite stable, and the pathway catalyzing their hydrolysis is unknown. To measure the degradation of these long-lived proteins, the cells were incubated with [3H]tyrosine for 18 hr. After a 1 hr chase to allow the degradation of short-lived proteins, inhibitors were added, and the degradation of radioactive proteins was measured. Under these conditions, their breakdown was extralysosomal and largely ATP dependent, as shown by the sensitivity to blockers of oxidative phosphorylation and glycolysis (Gronostajski et al., 1985) (Figure 3, legend). The labeled proteins were long lived (typically 3 % - 4 % degraded per hour), and the peptide aldehydes inhibited their degradation with potencies similar to those observed for short-lived and abnormal proteins (Figure 3C). In fact, when the degree of inhibition of longlived, short-lived, and abnormal proteins was plotted against the concentration for MG115 (Figure 3E) or LLnL (data not shown), very similar shaped curves were obtained (and identical slopes were observed on a log plot; data not shown). These observations indicate that longlived proteins are being degraded by the same inhibitorsensitive protease as the short-lived components. It is remarkable that even without preincubation the peptide aldehydes inhibited proteolysis in intact cells by >70% within 1 hr. Thus, these compounds readily penetrate cell membranes. With long-lived proteins, even greater inhibition of degradation (>98%) was achieved by preincubation with the inhibitors before measuring protein breakdown (Figure 3D). Some of the differences between the potencies of the three inhibitors could reflect differences in their

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A P C X 10-4 Figure 4. Peptide Aldehydes Inhibit MHC Class I Antigen Presentation LB27.4 cells were incubated with LLnL, MG115, or LLM for 1 hr, and then ovalbumm(A, C, E) or SIINFEKLpeptide(B, D, F) was introduced into the cytosol by electroporation. Subsequently, cells were either fixed immediately(BG),or after a 2 hr incubationat 37°C in the continued presence or absence of the inhibitors. In (E) and (F) inhibitors were used at 50 taM.The presence of peptide-MHC complexeson the cell surfacewas assayedas described in the methods. Datarepresent the mean of duplicatecultures. In the absence of antigen, background responseswere <1500 cpm. rates of accumulation by cells. In fact, the potency of MGl15 relative to LLnL increased about 2-fold after the preincubation with these inhibitors. Surprisingly, the degree of inhibition with MGl15 or LLnL appeared much greater for proteolysis in intact iymphoblasts than for in vitro hydrolysis of macromolecular substrates by the purified 26S proteasomes (Figure 3). Possibly, these inhibitors accumulate in cells, or the 26S proteasomes may have been altered during purification. However, as noted above, the degradation assays of ubiquitinated lysozyme probably underestimate the potency of these inhibitors. It is noteworthy that the rank order of potency of the three peptide aldehydes paralleled that observed in experiments with purified 20S and 26S proteasomes but did not correlate with their effects on calpain and cathepsin B (see Table 1).

Effect of Protease Inhibitors on MHC Class I Presentation Since LLnL and MGl15 inhibited the degradation of the

Proteolysis, Proteasomes,and Antigen Presentation 765

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%% Figure 5. Inhibitionby Peptide Aldehyde Is Reversible (A) Reversibleinhibition of ovalbummpresentation.One group of LB27.4 cells was premcubatedwith (open triangles) or without LLnL (closed circles) (50 gM) for 1 hr, electroporatedwith ovalbumin(30 mg/ml)with or without LLnL (50 gM), incubatedin the continuedpresenceor absence of LLnL (50 p.M)for an additional 1 hr, and then fixed. Another group of LB27.4 cells (open triangles)was exposed to LLnL (50 p.M)under the same conditions and for the same length of time (2 hr), but the inhibitor was subsequentlyremovedby washing. Subsequently,ovalbuminwas introducedinto these later cells by electroporat~on,and they were incubatedfor an additional1 hr withoutLLnL and then fixed. Antigen presentation was assayedas described in Figure 4. In the absenceof antigen, backgroundresponseswere <1,400 cpm. (B) Reversibleinhibitionof MHC class I assembly.Autoradiogramsof immunoprecip~tatedKb heterodimersfrom RMA cells treatedwithout (contrN) or wrth LLnL (100 i.tM)and then [3SS]methioninelabeled and solubilizedimmediately(as described in [A]) or washed and allowed to recoverfor 2 hr at 37°C beforemetaboliclabelingand detergentlysls. Detergentlysateswere preclearedand immunoprecipitatedwith Y3 monoclonalantibody (anti-Kb heterodimers)and analyzedon SDS-PAGE gels. The positions of the class I heavy chains and I~a-microglobulinon the autoradiograms are indicated by H and [32, respectively

great majority of cell proteins, we used these compounds to determine the importance of the proteasome in generating peptides for MHC class I presentation. We tested their effects on the presentation of ovalbumin that was introduced by electroporation into the cytosol of murine B lymphoblastoid cells. As shown previously, introduction of ovalbumin into the cytosol leads to its proteolytic processing (Moore et al., 1988) and presentation on MHC class I molecules. K bcomplexes containing the ovalbuminderived peptide SIINFEKL were detected on the cell surface2 hr later by an antigen-specificT-T hybridoma(Figure 4A). However, when the antigen-presenting cells were exposed to LLnL, the presentation of ovalbumin was inhibited in a dose-dependent manner up to 100% (Figure 4A). MG115 was approximately 5-fold more potent than LLnL (Figure 4C) and caused a 50% inhibition (2-fold shift) at 0.4 I~M. In contrast, at concentrations up to 100 ~tM, LLM did not affect ovalbumin presentation (Figure 4E). Therefore, the rank order of potency of the inhibitors was similar in assays with purified proteasomes, intracellular proteolysis, and antigen presentation. Moreover, when the degrees of inhibition of protein breakdown (e.g., of the long-lived component) and of antigen presentation were compared at different concentrations of LLnL (see Figure 3F) or MG115 (data not shown), the shapes of the curves were very similar. These observations indicate that the

inhibitors affect these two processes by the same mechanism. To test whether these aldehydes blocked the generation of the antigenic peptides and not another step(s) in the class I pathway, the naturally presented SlINFEKL was synthesized and introduced into the cytosol by electroporation (Figures 4B, 4D, 4F). Under these conditions, neither LLnL, MGl15, nor LLM blocked presentation. In these experiments, SIINFEKL was not binding directly to MHC class I molecules on the cell surface, because presentation was not detected when cells were inactivated with paraformaldehyde after the introduction of the peptide (Figures 4B, 4D, and 4F). Moreover, presentation of the electroporated peptide required the TAP1/TAP2 transporter and was inhibited by brefeldin A (A. Criau and K. L. R., unpublished data). The peptide aldehydes also do not inhibit the presentation of SIINFEKL in cells infected with a vaccinia viral construct containing a minigene encoding this peptide (M. Michalek and K. L. R., unpublished data). Therefore, LLn L and MG 115 selectively blocked the production of the immunogenic peptide and not any subsequent step in the MHC class I pathway. Moreover, when LLnL was removed, the cells fully recovered their ability to present ovalbu min within 1 h r (Figu re 5A). This reversibility indicates that the cells were not damaged. In these experiments, the inhibitors were added to cells permeabilized

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Inhibitor [~M] Figure 6. LLnL and MGl15 Inhibit the Assemblyof K" Class I Molecules Autoradiogramsof immunoprecipitatedKb heterodimers(H plus ~2), free heavychains (H), or free light chains (~2) from [~S]methioninelabeledliMA cellstreatedwith (plus)or without(minus)inhibitors.(A), LLnL-treatedcells; (B), LLnL, LLM, or E644reatedcells; (C), MG115treatedcells, lIMA cells(15 x 106)weretreatedwithor withoutpeptlde aldheydes(100 I~M, unless indicatedotherwise)or E64 (25 ~M) for 2.5 hr with [sSS]methionine(0.7 mCi/ml) addedfor the last 30 min of incubation. Detergentlysates were preclearedand immunoprecipitated sequentiallywith Y3 (anti-Kb H plus 62 chain), rabbit anti-exon 8 (anti-Kb H chain), B22.249 (anti-De H plus 132 chains), 28.14.8S (anti-Db H chain), and $19.8.503 (anti-132)antibodiesand analyzed on SDS-PAGE gels. The positionsof the class I heavy chains and 13~-microglobulinon the autoradiograrnsare indicatedby H and 1~2, respectively.

fore, if LLnL and MG115 inhibit generally the production of peptides for MHC class I presentation, then they should reduce the assembly of class I molecules. To test this prediction, cells were labeled with [3sS]methionine, and class I molecules were immunoprecipitated with antibodies that detect a conformational determinant present only On intact heterodimers. A T lymphoblastoid cell line (RMA) was chosen for this analysis because of superior labeling and immunoprecipitation of class I molecules, and also because its assembly of class I heterodimers had been shown to depend on the supply of peptides (Townsend et al., 1990). Moreover, in these cells, LLnL and MG1 15 markedly inhibited the presentation of ovalbumin and the nonlysosomal degradation of cellular proteins (data not shown). In the presence of LLnL or MG115, there was a marked reduction in the generation of K b (Figures 6A and 6C) and Db (see Figure 7B, below) heterodimers. In contrast, the synthesis of unassembled heavy and light chains was not reduced (Figures 6A and 6C). Thus, treatment with LLnL and MGl15 prevented formation of class I heterodimers. Since protein synthesis was not impaired, these compounds were not depleting intracellular pools of ATP, nor were they generally toxic to the cells. Moreover, when LLnL was removed, the cells fully recovered their ability to assemble MHC class I molecules within 2 hr (Figure 5B). As found in our studies of ovalbumin presentation, MGl15 was >5-fold more potent than LLnL in blocking the assembly of class I molecules (data not shown) and LLM was much less potent (Figure 6B). Lysosomal cysteine proteases and calpains do not play a significant role in the generation of most peptides for MHC class I presentation, because LLM and L-transepoxysuccinic acid (E64), both of which are potent inhibitors of these enzymes, had no effect (Figure 6B). To confirm that LLnL prevented the generation of antigenic peptides, we examined whether supplying the peptides exogenously could reconstitute the assembly of class I heterodimers. In the presence of the appropriate MHC-binding peptides, SIINFEKL and ASNENMETM, K ~ (Figure 7A) and Db (Figure 7B) heterodimers were assembled and could be immunoprecipitated from detergent lysates of LLnL-treated cells in amounts similar to those from untreated cells. Since the formation of peptide-MHC complexes and peptide transport into the endoplasmic reticulum (Figures 4B, 4D, and 4F) were not blocked, LLnL must have prevented the generation of peptides.

Discussion The Proteasome and the Degradation of Most Cell Proteins

by electroporation; however, they caused a similar inhibition of antigen presentation in intact cells (data not shown). We used these inhibitors to determine whether most peptides presented on MHC class I molecules are generated by the proteasome. The stable assembly of MHC class I heterodimers in the endoplasmic reticulum requires peptide binding (Townsend and Trowsdale, 1993). There-

Despite their potential importance, the physiological functions of the ubiquitin and proteasome-dependent proteolyric pathways are still unclear and have been difficult to study, because there have been no selective inhibitors that block proteasome function in intact cells. The degradation of most cellular proteins is an extralysosomal process that can be prevented with metabolic poisons that

Proteolysis, Proteasomes, and Antigen Presentation 767

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+

Inhibitor Figure 7. Exogenous Peptides Reconstitute Class I Assembly in LLnL-Treated Cell Lysates Autoradiogramof immunoprecipitatedclass I heterodimersfrom detergent lysatesof [~S]methionine-labeledRMAcellstreated with (plus)or without (minus) LLnL (100 p.M)to which SIINFEKLand ASNENMETM peptides (plus) or no peptides (minus) were added. Treatment with inhibutors, and immunoprecipitationswith Y3 (anti-Kb heterodimers) (A) and B22.249(anti-Dbheterodimers)(B), were performed and analyzed as described in Figure 6. Peptides were present in the lysates during the preclearingsteps (approximately20 hr) before the specific immunoprecipitationswere performed. The positions of the class I heavy chains and l~2-microglobulinon the autoradiograms are indicated by H and I]2, respectively.

deplete cells of ATP (Gronostajski et al., 1985) and consequently affect many cell processes. Most of our knowledge of the ubiquitin-proteasome pathway has been obtained through studies of cell extracts, and its role in vivo has only been evaluated using indi rect or genetic approaches. For example, degradation of short-lived and abnormal pro-

teins is inhibited in yeast (Seufert and Jentsch, 1990) and mammalian cells (Ciechanover et al., 1984; Gropper et al., 1991) with mutations affecting ubiquitin conjugation and in yeast with mutated proteasome subunits (Seufert and Jentsch, 1992). We have identified low molecular weight inhibitors that block degradation of these processes in intact cells and that should facilitate further analysis of the role of the proteasome in other biological processes. Both LLnL and MG115 are competitive inhibitors of the hydrolysis of Suc-Leu-Leu-VaI-Tyr-AMC by both the 20S and 26S proteasomes. Although peptide aldehydes were most effective against the chymotryptic activity, these agents also inhibit the peptidylglutamic peptidase activity and, to a lesser degree, the tryptic activity of the proteasome. Interestingly, these inhibitors are less potent against the 26S proteasome, possibly because the active sites of the 20S particles are altered or less accessible when it is incorporated into the 26S complex. LLnL and MG115 also reduce the ability of the 26S proteasomes to degrade protein and ubiquitin-conjugated protein substrates. This blocking of protein degradation by peptidase inhibitors may involve a mechanism in which different peptidases function processively within the aqueous channel of the 20S proteasome. A number of independent findings presented here indicate that inhibition of the 26S proteasome by the peptide aldehydes accounts for most proteolysis in vivo. First, these agents inhibit function of purified proteasomes and the ubiquitin-dependent pathway in cell extracts. Second, they block the degradation of abnormal and short-lived normal proteins, which is known to be ubiquitin-proteasome dependent. Third, they also block the class I presentation of ovalbumin, which we have shown to require ubiquitin conjugation (Michalek et al., 1993). Therefore, these agents clearly inhibit 26S proteasome function in cells. Fourth, inhibition of this pathway in vivo is also consistent with the recent report that LLnL inhibits cyclin B degradation (Sherwood et al., 1993). The authors of that report assumed that LLnL acted by inhibiting calpains, although it is well established that cyclin degradation requires ubiquitin conjugation (Hershko et al., 1991). Fifth, the concentration dependence for inhibiting this breakdown of abnormal and short-lived proteins closely resembles that for long-lived cellular proteins and antigen presentation. These observations argue for a common underlying molecular mechanism (see below). Sixth, moreover, the active site(s) of the proteasome has specificity for the N-blocking group and side chains at the P1 position of the peptide aldehydes, and these inhibitors have the same relative rank order of potency against the proteasome as against protein degradation in intact cells. This again argues for a common underlying mechanism of inhibition. Seventh, in contrast, the potency of LLnL, LLM, and MGl15 against the other known cellular proteases that they affect (cysteine proteases) does not correlate with their effects in vivo. Eighth, furthermore, other very active inhibitors of cysteine proteases do not block degradation of most cell proteins or antigen presentation. It is extremely unlikely that there is some undiscovered

Cell 768

intracellular protease that has the identical sensitivity to the peptide aldehydes as the proteasome. A major new finding was that the proteasome is also involved in the degradation of long-lived proteins that constitute the bulk of cell proteins. Since the relative potency of the aldehydes against the degradation of tong-lived proteins correlated with their activity against the proteasome, this particle also appears responsible for the turnover of this class of proteins. Moreover, when the degrees of inhibition of the breakdown of abnormal and short-lived proteins were plotted against the log of the inhibitor concentration (LLnL, LLM, or MG115), three parallel straight lines were obtained with nearly identical slopes (data not shown). Consequently, these three degradative processes appear to be occurring by the same inhibitorsensitive step. It had been unclear whether degradation of long-lived proteins involved the proteasome, since the partial inactivation of ubiquitin conjugation failed to block their degradation, as it does with short-lived proteins (Ciechanover et al., 1984; Gropper et al., 1991). It remains to be established whether this proteasome-dependent breakdown of long-lived proteins also requires ubiquitination. Of special interest is the finding that LLnL and MG115 can inhibit intracellular proteolysis for several hours without obvious toxicity to the cells, which remain intact, exclude vital dyes, and show normal rates of protein synthesis. Identification of compounds that penetrate cells and inhibit proteasome function in a reversible manner should prove to be of appreciable value for cell biology and biochemistry. Since the proteasome is a highly conserved structure, these agents are likely to function in most cell types; in fact, these compounds reduce proteolysis in adult rat muscle (N. Tawa, L. D., R. Odessey, and A. L. G., unpublished data) and in several mouse and human cell lines (data not shown). Role of the Proteasome in Antigen Presentation A primary motivation for undertaking these studies was to determine the proteolytic pathway responsible for generating the majority of class I presented peptides. Our earlier work (Michalek et al., 1993) indicated a requirement for ubiquitination, and therefore also the 26S proteasome, in the presentation of injected ovalbumin. Beyond this example, the evidence that the proteasome plays a general role in antigen presentation was circumstantial and rested on the discovery that two of its alternate subunits were encoded in the MHC. Although these two subunits clearly alter its peptidase activities, their importance in antigen presentation has been controversial. In fact, analysis of mutant cells lacking LMP2 and LMP7 indicated that these subunits were not essential for class I antigen presentation (Arnold et al., 1992), and by extrapolation, the involvement of the proteasome was questioned. Since LLnL and MGl15 inhibit almost completely the supply of peptides for class I, the proteasome is not only the primary site for complete destruction of cell proteins but also for the generation of the hundreds of thousands of peptides presented on M HC class I molecules. Although these inhibitors also affect some lysosomal proteases and calpains, these enzymes are unlikely to be important for

MHC class I presentation, since potent inhibitors of these proteases (E64, LLM, or leupeptin) did not block the generation of antigenic peptides. By contrast, with LLnL and MG115, the dose-response curve for inhibiting the degradation of endogenously synthesized proteins resembled that for the inhibition of antigen presentation, implying a common mechanism of action. Moreover, the relative potency of the peptide aldehydes against purified proteasome is very similar to their relative potency in inhibiting antigen presentation. It remains to be established whether proteasome function in antigen presentation generally involves ubiquitination. A very different hypothesis has been proposed to explain how endogenous peptides presented on MHC class I molecules are generated. Instead of a proteolytic mechanism, the pepton hypothesis proposes that class I-presented antigens are generated by the synthesis of short transcripts, or by initiation and termination at nonclassical sites, or both (Boon and Van Pel, 1989). This model was proposed to account for several examples where antigenic epitopes were presented from genes that lacked promoters or translational start sites (Chomez et al., 1992), or where the antigenic sequence was out of the translational reading frame (Fetten et al., 1991), or followed a termination codon (Chomez et al., 1992). Because the peptide aldheydes blocked almost completely the generation of antigenic peptides (without affecting peptide transport or the delivery of peptide-MHC complexes to the cell surface), proteolysis must be the major mechanism by which peptides are generated for MHC class I presentation. Peptons, if they exist, are either a minor source of presented peptides or must require further proteolytic processing. In fact, one possible reason that the presentation of peptons has been detected may be that incomplete polypeptides resulting from mistakes in transcription or translation are preferentially degraded (Goldberg and St. John, 1976; Hershko and Ciechanover, 1992) and therefore might be especially effective in generating peptides for MHC class I presentation. It will therefore be of interest to examine whether LLnL and MG115 also inhibit the presentation of antigenic epitopes that have been suggested to arise from peptons. These peptide aldehydes represent the first compounds available that can penetrate living cells, inhibit selectively the bulk of intracellular proteolysis, and reversibly block the generation of antigenic peptides without affecting other steps in the class I pathway. Consequently, these proteasome inhibitors should be useful tools in the analysis of antigen presentation and the turnover of specific cell proteins, as well as many other problems in cell biology. Experimental Procedures Antigens and Reagents Chicken ovalbuminand E64 were purchasedfrom Sigma (St. Louis, MO). Peptideswere synthesizedby the molecularbiology core of the Dana-Farber CancerInstitute.LLnL, MG115,and LLM were provtded by MyoGentcs(Cambridge, MA). Pepttde-AMCsubstrates were purchasedfrom Calbiochem(La Jolla, CA). 13-Casein(Sigma)was labeled with [14C]methylby reductivealkylation.12Sl-lysozymewas conjugated with ubiquitinby a modificationof the procedureof Tamuraet al. (1991) and was provided by Dr. Olivier Coux (Harvard Medical School).Ap-

Proteolysis, Proteasomes, and Antigen Presentation 769

proximately 90% of this ~251-1ysozymeis tn the form of ubiquitin conjugates, which contamed from 3 to >10 ubiquitin moieties.

Cell Lines and Antibodies LB27.4 B lymphoblasts (Kappler et al., 1982), RF33.70 (OVA plus Kbspecific) T-T hybridomas (Rock et al., 1990), and RMA T lymphoblasts (K~irre et al., 1986) have been previously described. For antigen presentation experiments, LB27.4 cells were grown in Optimem (GIBCO, Grand Island, NY) supplemented with normal mouse serum (1%). In all other experiments, cells were grown in DMEM or RPM11640 (Irving Scientific, Santa Ana, CA) supplemented with fetal calf serum (10%) and antibiotics. Monoclonal anttbodies were prepared from hybridomas Y3 (anti-class I Kb heterodimers) (Jones and Janeway, 1981), B22 (anti-D b heterodimers) (H~immerling et aL, 1979), and 28.14.8S (anti-D b heavy chains or heterodimers) (Ozato and Sachs, 1980) and $19.8.503 (anti-~2-microglobulin monoclonal antibody). Rabbtt antiserum specific for sequences encoded by exon 8 of the Kb gene and reactive with free or ~2-microglobulin-assoctated K b heavy chains (Smith and Barber, 1990)was a gift from Dr. Brian Barber (University of Toronto, Toronto, Ontario, Canada)

Enzyme Assays Proteasome fractions were prepared from rabbit psoas muscle homogenates by differential centrifugation and were resolved into 26S and 20S particles by precipitation with ammonium sulfate (0%-38% and 40%-80%, respectively) followed by chromatography sequentially on a Mono Q and a Superose 6 (Pharmacia) column (Driscoll and Goldberg, 1990). The ammonium sulfate precipitation was omitted in preparing 26S proteasomes for assays of protein or ubiquitinated protein degradation. Cathepsm B from bovine spleen and the 80 kDa catalybc subunit of calpain from rabbit muscle were purchased from Sigma. Peptlde-AMC substrates and inhibitor (1-10 p.I) in DMSO were added to 2 ml of assay buffer that for the 20S proteasome contained 20 mM Tris-HCI, 0.5 mM EDTA, and 0 035% SDS (pH 8.0); for the 26S proteasome, contamed 20 mM Trts-HCI, 1 mM ATP, and 2 mM MgCI2 (pH 8.0); for cathepsm B, contamed 100 mM NaOAc, 5 mM EDTA, and 2 mM DTT; and for calpain, contained 20 mM Tris-HCI, 1 mM CaCIz, and 2 mM DI-I (pH 8.0). After equilibration at 37°C (proteasomes and cathepsin B) or 20°C (calpain), enzyme (1-5 p.I) was added, and reaction progress was monitored by the increase in fluorescence emission at 440 nm (Iox,380 nm). Velocities were calculated from linear, steady-state regions of reaction progress curves. Apparent inhibition constants (K,,,pp) were determined by nonhnear least-squares analysts of the dependence of steady-state veloctty (v,,) on inhibttor concentration ([I]), according to the followmg equation: v~s = v,,m,t/(1 + ([I]/K,.app)) These reactions were conducted at[S[ < Kin/5, so that K,.,~p = K,, the dissociation constant for the enzyme-inhibitor complex, and v,,m,t = (KJK~,)[E][S]. Protein degradatton was assayed as described (Driscoll and Goldberg, 1990). 26S proteasome (12-27 nM) was preincubated with or without mhibitors in a buffer containing 50 mM Tris (pH 7.25), 5 mM MgCI2, 1 mM DTT, 2 mM ATP, and 1% DMSO for 30 min on ice, and then radtoactive protein substrates (etther 50,000 cpm of [14C]methylcasem [2.5 p.g] or 5,000 cpm of ubiquttinJ251-1ysozyme [about 5 nM]) were added in a final volume of 20 ~1. For measurement of ubiquitin conjugate degradation, 2 p,g of nonradioactive lysozyme was added to inhtbit competitively the degradation of any free 1251-1ysozymepresent at the outset or generated by ~sopeptidases during the incubation. After incubation at 37°C for 10 min (ubiquitin-12Sl-lysozyme assays) or 60 min (caseinase assays), 20 p.g of BSA and 500 I~1of 10% trichloroacetic acid (-ICA) were added. The production of TCA-soluble radtoacttve peptides was determined.

Assays of Intracellular Protein Degradation To measure the degradation of short-lived proteins, LB27.4 cells were incubated with [aH]tyrosine (5 iiCi/ml) for 1 hr at 37°C. Labeled cells (10r) were incubated in triplicate m the presence or absence of mhibitors in a final volume of 200 ~1 of culture media (10% FCS) containing unlabeled tyroeine (50 pg/ml). After 1 hr of incubatton at 37°C, TCA (10%) was added, and the amount of TCA-soluble radtoacttvity in clad-

fled supernatants (15,000 x g) was determined in a liquid scintillation counter. The degradation of analog-containing proteins was measured in the same way as for short-hved proteins, except that arginme m the labeling media was replaced with canavanine (0.2 mg/ml). The degradation of short-lived normal and abnormal proteins was also largely resistant to chloroqume (<7°/0 inhibition at 20 p,M). To measure the degradation of long-lived proteins, LB27.4 cells were first incubated with [3H]tyrosme for 18 hr at 37°C in media containing dialyzed FCS (100/0) and then chased for 1 hr at 37°C with media containing unlabeled tyrosine (50 p.g/ml), and chloroquine (20 pM) was added for the last 30 rain of the chase. The catabolism of labeled proteins in the presence or absence of inhibitors was then determined as described for short-lived protems, except that chloroquine (20 p.M) was added to all groups to block lysosomal functton. In some experiments, labeled cells were preincubated with inhibitors for 2 hr at 37°C before measuring protein breakdown. Under these conditions, catabolism of long-lived proteins was resistant to the lysosomal mhtbitor leupeptin (<10% inhibition at 25 p.M).

Permeabilization of Cells by Electroporation Cells washed serum-free were resuspended in 0.5 ml of cold electroporation buffer (1 mM HEPES, 0.4 M mannttol, 0.15 M NaCI, 0.01 M PO4 [pH 7.4]) and electroporated using a Bethesda Research Laboratories CelI-Porator (400 v, 1180 I~F, high ohm setting). After electroporatien, cells were incubated for 10 min on ice and then washed. To introduce the antigen into the cytosol of cells, ovalbumin (30 mg/ml) or SIINFEKL peptide (1 p.g/ml) was added to the electroporation buffer

Antigen Presentation Assays LB27.4 cells were incubated for 1 hr at 37°C in Opttmem media with or without the protease inhibitors. Antigen with or without protease inhibitors was then introduced into the cytosol by electroporation. Subsequently cells were either fixed immediately with paraformaldehyde (1%) for 10 rain at 25°C or were incubated for 1-2 hr at 37°C in Optimem in the continued presence or absence of inhibitors and then fixed with paraformaldehyde (unless otherwise indicated). The presence of peptide-MHC complexes on the surface of LB27 4 cells was assayed by measuring the amount of interleukin-2 (IL-2) produced by the ovalbumin-Kb-specific T-T hybridoma RF33.70 after stimulation with LB27.4 cells in duplicate cultures, as previously described (Rock and Benacerraf, 1983).

Radiolabeling and Immunoprecipitation RMA cells (1.5 x 107) were washed with media lacking serum, and protease inhibitors were introduced by electroporation. The cells were subsequently incubated at 37°C for 1-3 hr in the labeling media (rnethionine-free RPMI or DMEM, 10% dialyzed fetal calf serum)in the continued presence or absence of the protease inhibitors, unless otherwise mdicated, and for the last 30 rain of incubation, [3sS]methionine (0.7 mCi/ml) was added. Immunoprecipitations were performed essentially as described (Townsend et al., 1990; Rock et al., 1991). Briefly, detergent lysates (0.5% Nontdet P-40, 0.5% Mega 9) were clarified by ultracentrifugatien, BSA (1% w/v) was added, and the sample volumes were adjusted to 0.5 ml. In some experiments, peptides (50 p.g/ml) were added to the lysls buffer as described (Townsend et al., 1990). Samples were precleared four times wtth 100 ml of IgSorb (10% w/v) for >45 rain at 4°C. Monoclonal antibody (15 ~g/ml) or rabbit antiserum (2 ~tl) was added, and the samples were incubated for 90 rain at 4°C, after which 100 :~1 of protein A-Sepharose (50 mg/ml) was added. After rctatmg for 45 rain at 4°C, the beads were washed five times. For sequential immunoprecipitations, the lysates were further precleared with the previous antibody and protein A-Sepharose or with protein A-Sepharose alone before a further round of immunoprecipitation with another anttbody. Immunoprecipitated proteins were resolved by SDS-PAGE (14%) under reducing conditions. Gels were ~ncubated in Autofluor (National Diagnostics, Atlanta, GA) and dried, and the labeled protems were visualized by autoradiography.

Acknowledgments We thank Dr. Olivier Coux for providing ubiquttm-conjugated lysosome, Fracesco Melandn for provtding 20S and 26S proteasomes used tn certain experiments, and Dr. Baruj Benacerraf for critical re-

Cell 770

view of the manuscript. This work was supported by grants from the National Institutes of Health (AI20248 and GM46147), the Muscular Dystrophy Association, and the National Aeronautics and Space Administration. Received April 11, 1994; revised July 13, 1994. References

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