Abstract

The antibiotic lactacystin was reported to covalently modify β-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [3H]lactacystin treatment modifies all the proteasome's catalytic β-subunits. Lactacystin and its more potent derivative β-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short- and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the β-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-γ-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all β-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition. The antibiotic lactacystin was reported to covalently modify β-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [3H]lactacystin treatment modifies all the proteasome's catalytic β-subunits. Lactacystin and its more potent derivative β-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short- and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the β-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-γ-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all β-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition. MHC 1The abbreviations used are: MHC, major histocompatibility complex; IFN-γ, interferon-γ; LLnL,N-acetyl-l-leucinyl-l-leucinal-l-norleucinal; FITC, fluorescein isothiocyanate; KLH, keyhole limpet hemocyanin; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-4-methylcoumarin. 1The abbreviations used are: MHC, major histocompatibility complex; IFN-γ, interferon-γ; LLnL,N-acetyl-l-leucinyl-l-leucinal-l-norleucinal; FITC, fluorescein isothiocyanate; KLH, keyhole limpet hemocyanin; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-4-methylcoumarin. class I molecules typically bind 8–9-residue peptides derived from cellular or viral proteins. Most of these peptides are generated by protein breakdown in the cytosol and are transported by the transporter associated with antigen presentation transporter into the endoplasmic reticulum (1York I.A. Rock K.L. Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar). Here the peptide, a MHC class I heavy chain, and a β2-microglobulin molecule associate, and the complex is then transported through the Golgi apparatus to the plasma membrane (1York I.A. Rock K.L. Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar). This process allows T lymphocytes to screen for cells that are synthesizing foreign or abnormal proteins. The mechanisms responsible for the generation of the class I-presented peptides had been unclear until recently. However, a variety of recent evidence has suggested that the proteasome plays a primary role in this process and that during the turnover of cytosolic and nuclear proteins a fraction of the peptides generated by the proteasome are utilized for MHC class I presentation (1York I.A. Rock K.L. Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar, 2Goldberg A.L. Rock K.L. Nature. 1992; 357: 375-379Crossref PubMed Scopus (503) Google Scholar, 3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar).Proteasomes are found in the nucleus and cytosol of all cells and are essential components of the ATP-ubiquitin-dependent pathway for protein degradation. The 20 S proteasome is a 700-kDa particle with multiple peptidase activities, including a chymotryptic-, tryptic-, and peptidylglutamyl-like activity (4Orlowski M. Biochemistry. 1990; 29: 10289-10297Crossref PubMed Scopus (412) Google Scholar, 5Rivett J. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (380) Google Scholar, 6Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2223) Google Scholar). It is a cylindrical-shaped structure composed of four rings, the outer two each contain seven α-subunits and the inner two each contain seven β-subunits (7Grziwa A. Baumeister W. Dahlmann B. Kopp F. FEBS Lett. 1991; 290: 186-192Crossref PubMed Scopus (110) Google Scholar, 8Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber A. Science. 1995; 268: 533-539Crossref PubMed Scopus (1369) Google Scholar). Proteolysis occurs in the central chamber of this particle and is catalyzed through a nucleophilic attack on the peptide bond by the N-terminal threonine hydroxyl groups on certain β-subunits, named X (ε), Y(δ), Z (HCO), and their homologues LMP2, LMP7, and LMP10 (MECL-1) (9Zwickl P. Kleinz J. Baumeister W. Nat. Struct. Biol. 1994; 1: 765-771Crossref PubMed Scopus (170) Google Scholar, 10Seemüller E. Lupas A. Stock D. Löwe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (582) Google Scholar, 11Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1493) Google Scholar, 12Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (540) Google Scholar). The 20 S particle functions as the proteolytic core of a larger 26 S (2000 kDa) ATP-dependent proteasome complex which selectively degrades proteins that are modified by conjugation to multiple ubiquitin molecules (6Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2223) Google Scholar, 13Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1192) Google Scholar, 14Rechsteiner M. Hoffman L. Dubiel W. J. Biol. Chem. 1993; 268: 6065-6068Abstract Full Text PDF PubMed Google Scholar). Although the ubiquitin-proteasome pathway is clearly essential for the rapid degradation of short-lived or highly abnormal proteins and polypeptides in yeast (15Seufert W. Jentsch S. EMBO J. 1992; 11: 3077-3080Crossref PubMed Scopus (121) Google Scholar) and mammalian cells (16Gropper R. Brandt R.A. Elias S. Bearer C.F. Mayer A. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1991; 266: 3602-3610Abstract Full Text PDF PubMed Google Scholar, 17Ciechanover A. Finley D. Varshavsky A. Cell. 1984; 37: 57-66Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 18Kulka R.G. Raboy B. Schuster R. Parag H.A. Diamond G. Ciechanover A. Marcus M. J. Biol. Chem. 1988; 263: 15726-15731Abstract Full Text PDF PubMed Google Scholar, 19Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar), its role in degradation of the bulk of cell proteins, which are long-lived, is uncertain (16Gropper R. Brandt R.A. Elias S. Bearer C.F. Mayer A. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1991; 266: 3602-3610Abstract Full Text PDF PubMed Google Scholar, 20Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1587) Google Scholar).Several lines of evidence had suggested that the proteasome was also responsible for the generation of some class I-presented peptides. Two of the proteasome's catalytic β-subunits, LMP7 and LMP2, are encoded in the MHC region (21Monaco J.J. McDevitt H.O. Hum. Immunol. 1986; 15: 416-426Crossref PubMed Scopus (73) Google Scholar, 22Monaco J.J. Immunol. Today. 1992; 13: 173-179Abstract Full Text PDF PubMed Scopus (394) Google Scholar), and the absence of these subunits in mutant cells or mice decreases the efficiency of presentation of certain antigens (23Fehling H.J. Swat W. Laplace C. Kuhn R. Rajewsky K. Muller U. von Boehmer H. Science. 1994; 265: 1234-1237Crossref PubMed Scopus (441) Google Scholar, 24Cerundolo V. Kelly A. Elliott T. Trowsdale J. Townsend A. Eur. J. Immunol. 1995; 25: 554-562Crossref PubMed Scopus (109) Google Scholar, 25Van Kaer L. Ashton-Rickardt P.G. Eichelberger M. Gaczynska M. Nagashima K. Rock K.L. Goldberg A.L. Doherty P.C. Tonegawa S. Immunity. 1994; 1: 533-541Abstract Full Text PDF PubMed Scopus (364) Google Scholar, 26Sibille C. Gould K.G. Willard-Gallo K. Thomson S. Rivett A.J. Powis S. Butcher G.W. DeBaetselier P. Curr. Biol. 1995; 5: 923-930Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, inhibiting ubiquitin conjugation in a TS mutant decreased antigen presentation of a model protein (27Michalek M.T. Grant E.P. Gramm C. Goldberg A.L. Rock K.L. Nature. 1993; 363: 552-554Crossref PubMed Scopus (283) Google Scholar). On the other hand, modifications of a protein that stimulate its ubiquitinylation and degradation by 26 S proteasomes enhance its rate of MHC class I presentation (28Grant E.P. Michalek M.T. Goldberg A.L. Rock K.L. J. Immunol. 1995; 155: 3750-3758PubMed Google Scholar). Finally, when 20 S proteasomes are incubated with a protein for extended periods, they can generate some class I-binding peptides, although such experiments involve highly nonphysiological conditions (29Boes B. Hengel H. Ruppert T. Multhaup G. Koszinowski U.H. Kloetzel P.-M. J. Exp. Med. 1994; 179: 901-909Crossref PubMed Scopus (280) Google Scholar, 30Dick L.R. Aldrich C. Jameson S.C. Moomaw C.R. Pramanik B.C. Doyle C.K. DeMartino G.N. Bevan M.J. Forman J.M. Slaughter C.A. J. Immunol. 1994; 152: 3884-3894PubMed Google Scholar, 31Niedermann G. Butz S. Ihlenfeldt H.G. Grimm R. Lucchiari M. Hoschützky H. Jung G. Maier B. Eichmann K. Immunity. 1995; 2: 289-299Abstract Full Text PDF PubMed Scopus (210) Google Scholar).More definitive evidence for the proteasome's general importance in antigen presentation in vivo requires methods to specifically inhibit proteasome function in intact cells. Recently, certain peptide aldehydes (such asN-acetyl-l-leucinyl-l-leucinal-l-norleucinal, LLnL, andN-carbobenzoxyl-l-leucinyl-l-leucinyl-l-norvalinal, MG115) have been shown to strongly inhibit multiple peptidase activities of proteasomes and to reduce protein hydrolysis (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Moreover, these agents can enter cells and block the degradation of most cellular proteins and the generation of the majority of class I-presented peptides (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Although these peptide aldehydes can also inhibit the cysteine proteases found in lysosomes and calpains (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar), several findings argued that the inhibition of protein degradation and antigen presentation was due to effects on the proteasome. For example, the rank order of potencies of different peptide aldehydes in inhibiting the proteasome was the same as for blocking protein degradation and antigen presentation and did not correlate with efficacy against cysteine proteases (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Also, inhibition of these cysteine proteases did not affect protein breakdown or antigen presentation. Nevertheless, more selective proteasome inhibitors are needed to establish definitively a major role for the proteasome in these processes.A chemically distinct type of proteasome inhibitor is the antibiotic lactacystin which was isolated from Streptomyces by Omura and colleagues (32Omura S. Matsuzaki K. Fujimoto T. Kosuge K. Fuzuya T. Fujita S. Nakagawa A. J. Antibiot. ( Tokyo ). 1991; 44: 113-118Crossref PubMed Scopus (530) Google Scholar) and synthesized by Corey and colleagues (33Fenteany G. Standaert R.F. Reichard G.A. Corey E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3358-3362Crossref PubMed Scopus (227) Google Scholar). Fenteany et al. (11Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1493) Google Scholar) have shown that [3H]lactacystin bound covalently to a polypeptide identified as proteasome β-subunit X in bovine brain cells and that lactacystin could irreversibly inhibit the chymotryptic- and tryptic-like peptidase activity and reversibly inhibit the post-acidic activity of purified proteasomes. Lactacystin spontaneously hydrolyzes into clasto-lactacystin β-lactone, which appears to be the active inhibitor that reacts with the N-terminal threonine of subunit X (34Dick L.R. Cruikshank A.A. Grenier L. Melandri F.D. Nunes S.L. Stein R.L. J. Biol. Chem. 1996; 271: 7273-7276Abstract Full Text PDF PubMed Scopus (355) Google Scholar). Because lactacystin and the β-lactone appeared to be highly specific inhibitors that do not affect cysteine or serine proteases, they are potentially very useful research tools. We therefore examined whether they inhibit proteasomes that contain β-type subunits not expressed in neurons (e.g. LMP7, LMP2, and MECL-1), whether they reduce protein hydrolysis in vitro and in cells, and whether they can block MHC class I antigen presentation. MHC 1The abbreviations used are: MHC, major histocompatibility complex; IFN-γ, interferon-γ; LLnL,N-acetyl-l-leucinyl-l-leucinal-l-norleucinal; FITC, fluorescein isothiocyanate; KLH, keyhole limpet hemocyanin; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-4-methylcoumarin. 1The abbreviations used are: MHC, major histocompatibility complex; IFN-γ, interferon-γ; LLnL,N-acetyl-l-leucinyl-l-leucinal-l-norleucinal; FITC, fluorescein isothiocyanate; KLH, keyhole limpet hemocyanin; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-4-methylcoumarin. class I molecules typically bind 8–9-residue peptides derived from cellular or viral proteins. Most of these peptides are generated by protein breakdown in the cytosol and are transported by the transporter associated with antigen presentation transporter into the endoplasmic reticulum (1York I.A. Rock K.L. Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar). Here the peptide, a MHC class I heavy chain, and a β2-microglobulin molecule associate, and the complex is then transported through the Golgi apparatus to the plasma membrane (1York I.A. Rock K.L. Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar). This process allows T lymphocytes to screen for cells that are synthesizing foreign or abnormal proteins. The mechanisms responsible for the generation of the class I-presented peptides had been unclear until recently. However, a variety of recent evidence has suggested that the proteasome plays a primary role in this process and that during the turnover of cytosolic and nuclear proteins a fraction of the peptides generated by the proteasome are utilized for MHC class I presentation (1York I.A. Rock K.L. Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar, 2Goldberg A.L. Rock K.L. Nature. 1992; 357: 375-379Crossref PubMed Scopus (503) Google Scholar, 3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Proteasomes are found in the nucleus and cytosol of all cells and are essential components of the ATP-ubiquitin-dependent pathway for protein degradation. The 20 S proteasome is a 700-kDa particle with multiple peptidase activities, including a chymotryptic-, tryptic-, and peptidylglutamyl-like activity (4Orlowski M. Biochemistry. 1990; 29: 10289-10297Crossref PubMed Scopus (412) Google Scholar, 5Rivett J. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (380) Google Scholar, 6Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2223) Google Scholar). It is a cylindrical-shaped structure composed of four rings, the outer two each contain seven α-subunits and the inner two each contain seven β-subunits (7Grziwa A. Baumeister W. Dahlmann B. Kopp F. FEBS Lett. 1991; 290: 186-192Crossref PubMed Scopus (110) Google Scholar, 8Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber A. Science. 1995; 268: 533-539Crossref PubMed Scopus (1369) Google Scholar). Proteolysis occurs in the central chamber of this particle and is catalyzed through a nucleophilic attack on the peptide bond by the N-terminal threonine hydroxyl groups on certain β-subunits, named X (ε), Y(δ), Z (HCO), and their homologues LMP2, LMP7, and LMP10 (MECL-1) (9Zwickl P. Kleinz J. Baumeister W. Nat. Struct. Biol. 1994; 1: 765-771Crossref PubMed Scopus (170) Google Scholar, 10Seemüller E. Lupas A. Stock D. Löwe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (582) Google Scholar, 11Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1493) Google Scholar, 12Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (540) Google Scholar). The 20 S particle functions as the proteolytic core of a larger 26 S (2000 kDa) ATP-dependent proteasome complex which selectively degrades proteins that are modified by conjugation to multiple ubiquitin molecules (6Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2223) Google Scholar, 13Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1192) Google Scholar, 14Rechsteiner M. Hoffman L. Dubiel W. J. Biol. Chem. 1993; 268: 6065-6068Abstract Full Text PDF PubMed Google Scholar). Although the ubiquitin-proteasome pathway is clearly essential for the rapid degradation of short-lived or highly abnormal proteins and polypeptides in yeast (15Seufert W. Jentsch S. EMBO J. 1992; 11: 3077-3080Crossref PubMed Scopus (121) Google Scholar) and mammalian cells (16Gropper R. Brandt R.A. Elias S. Bearer C.F. Mayer A. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1991; 266: 3602-3610Abstract Full Text PDF PubMed Google Scholar, 17Ciechanover A. Finley D. Varshavsky A. Cell. 1984; 37: 57-66Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 18Kulka R.G. Raboy B. Schuster R. Parag H.A. Diamond G. Ciechanover A. Marcus M. J. Biol. Chem. 1988; 263: 15726-15731Abstract Full Text PDF PubMed Google Scholar, 19Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar), its role in degradation of the bulk of cell proteins, which are long-lived, is uncertain (16Gropper R. Brandt R.A. Elias S. Bearer C.F. Mayer A. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1991; 266: 3602-3610Abstract Full Text PDF PubMed Google Scholar, 20Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1587) Google Scholar). Several lines of evidence had suggested that the proteasome was also responsible for the generation of some class I-presented peptides. Two of the proteasome's catalytic β-subunits, LMP7 and LMP2, are encoded in the MHC region (21Monaco J.J. McDevitt H.O. Hum. Immunol. 1986; 15: 416-426Crossref PubMed Scopus (73) Google Scholar, 22Monaco J.J. Immunol. Today. 1992; 13: 173-179Abstract Full Text PDF PubMed Scopus (394) Google Scholar), and the absence of these subunits in mutant cells or mice decreases the efficiency of presentation of certain antigens (23Fehling H.J. Swat W. Laplace C. Kuhn R. Rajewsky K. Muller U. von Boehmer H. Science. 1994; 265: 1234-1237Crossref PubMed Scopus (441) Google Scholar, 24Cerundolo V. Kelly A. Elliott T. Trowsdale J. Townsend A. Eur. J. Immunol. 1995; 25: 554-562Crossref PubMed Scopus (109) Google Scholar, 25Van Kaer L. Ashton-Rickardt P.G. Eichelberger M. Gaczynska M. Nagashima K. Rock K.L. Goldberg A.L. Doherty P.C. Tonegawa S. Immunity. 1994; 1: 533-541Abstract Full Text PDF PubMed Scopus (364) Google Scholar, 26Sibille C. Gould K.G. Willard-Gallo K. Thomson S. Rivett A.J. Powis S. Butcher G.W. DeBaetselier P. Curr. Biol. 1995; 5: 923-930Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, inhibiting ubiquitin conjugation in a TS mutant decreased antigen presentation of a model protein (27Michalek M.T. Grant E.P. Gramm C. Goldberg A.L. Rock K.L. Nature. 1993; 363: 552-554Crossref PubMed Scopus (283) Google Scholar). On the other hand, modifications of a protein that stimulate its ubiquitinylation and degradation by 26 S proteasomes enhance its rate of MHC class I presentation (28Grant E.P. Michalek M.T. Goldberg A.L. Rock K.L. J. Immunol. 1995; 155: 3750-3758PubMed Google Scholar). Finally, when 20 S proteasomes are incubated with a protein for extended periods, they can generate some class I-binding peptides, although such experiments involve highly nonphysiological conditions (29Boes B. Hengel H. Ruppert T. Multhaup G. Koszinowski U.H. Kloetzel P.-M. J. Exp. Med. 1994; 179: 901-909Crossref PubMed Scopus (280) Google Scholar, 30Dick L.R. Aldrich C. Jameson S.C. Moomaw C.R. Pramanik B.C. Doyle C.K. DeMartino G.N. Bevan M.J. Forman J.M. Slaughter C.A. J. Immunol. 1994; 152: 3884-3894PubMed Google Scholar, 31Niedermann G. Butz S. Ihlenfeldt H.G. Grimm R. Lucchiari M. Hoschützky H. Jung G. Maier B. Eichmann K. Immunity. 1995; 2: 289-299Abstract Full Text PDF PubMed Scopus (210) Google Scholar). More definitive evidence for the proteasome's general importance in antigen presentation in vivo requires methods to specifically inhibit proteasome function in intact cells. Recently, certain peptide aldehydes (such asN-acetyl-l-leucinyl-l-leucinal-l-norleucinal, LLnL, andN-carbobenzoxyl-l-leucinyl-l-leucinyl-l-norvalinal, MG115) have been shown to strongly inhibit multiple peptidase activities of proteasomes and to reduce protein hydrolysis (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Moreover, these agents can enter cells and block the degradation of most cellular proteins and the generation of the majority of class I-presented peptides (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Although these peptide aldehydes can also inhibit the cysteine proteases found in lysosomes and calpains (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar), several findings argued that the inhibition of protein degradation and antigen presentation was due to effects on the proteasome. For example, the rank order of potencies of different peptide aldehydes in inhibiting the proteasome was the same as for blocking protein degradation and antigen presentation and did not correlate with efficacy against cysteine proteases (3Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). Also, inhibition of these cysteine proteases did not affect protein breakdown or antigen presentation. Nevertheless, more selective proteasome inhibitors are needed to establish definitively a major role for the proteasome in these processes. A chemically distinct type of proteasome inhibitor is the antibiotic lactacystin which was isolated from Streptomyces by Omura and colleagues (32Omura S. Matsuzaki K. Fujimoto T. Kosuge K. Fuzuya T. Fujita S. Nakagawa A. J. Antibiot. ( Tokyo ). 1991; 44: 113-118Crossref PubMed Scopus (530) Google Scholar) and synthesized by Corey and colleagues (33Fenteany G. Standaert R.F. Reichard G.A. Corey E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3358-3362Crossref PubMed Scopus (227) Google Scholar). Fenteany et al. (11Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1493) Google Scholar) have shown that [3H]lactacystin bound covalently to a polypeptide identified as proteasome β-subunit X in bovine brain cells and that lactacystin could irreversibly inhibit the chymotryptic- and tryptic-like peptidase activity and reversibly inhibit the post-acidic activity of purified proteasomes. Lactacystin spontaneously hydrolyzes into clasto-lactacystin β-lactone, which appears to be the active inhibitor that reacts with the N-terminal threonine of subunit X (34Dick L.R. Cruikshank A.A. Grenier L. Melandri F.D. Nunes S.L. Stein R.L. J. Biol. Chem. 1996; 271: 7273-7276Abstract Full Text PDF PubMed Scopus (355) Google Scholar). Because lactacystin and the β-lactone appeared to be highly specific inhibitors that do not affect cysteine or serine proteases, they are potentially very useful research tools. We therefore examined whether they inhibit proteasomes that contain β-type subunits not expressed in neurons (e.g. LMP7, LMP2, and MECL-1), whether they reduce protein hydrolysis in vitro and in cells, and whether they can block MHC class I antigen presentation.