Jun 10, 1994 - antibodies, and A. Scott for secretarial assistance. These studies .... DeMartino, G. N., Orth, K., McCullough, M. L., Leel, W.,. Munn, T. Z. ...
Proc. Nati. Acad. Sci. USA
Vol. 91, pp. 9213-9217, September 1994 Biochemistry
Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7 (proteolysis/antigen presentation/major histocompatibility complex ca I molecule)
MARIA GACZYNSKA*, KENNETH L.
ROCKtt, THOMAS SPIES4, AND ALFRED L. GOLDBERG*
Departments of *Celi Biology and tPathology, Harvard Medical School, Boston, MA 02115; and tDana-Farber Cancer Institute, Boston, MA 02115
Communicated by Tom Maniatis, June 10, 1994
ABSTRACT Recent studies have implicated proteasomes in the generation ofthe antigenic peptides that are presented on major histocmpatibility complex class I molecules to T lymphocytes. Interferon y modifies the subunit composition of proteasomes and causes changes in their peptidase activities that should favor the production of peptides with hydrophobic or basic carboxyl -termini (i.e., the types found on major histocompatibility complex class I molecules). It has been proposed that these changesIn peptidase activity are due to incorporation into proteasomes of the major histocompatibility complex-encoded subunits.LM1P2 and -7, which are induced by interferon y. Here we show by gene transfection into lymphoblasts or HeLa cells that LMP7 Increases the capacity (V,) of 20S and 26S proteasomes to cleave peptides after hydrophobic and basic residues without, affecting hydrolysis after acidic residues. These changes depended on the amount of LMP7 subunits incorporated into proteasomes. Transfection of LMP2 reduced cleavage of peptides after acidic residues, increased hydrolysis after basic residues, and did not affect the hydrophobic aciity. Since the activity of the total proteasome population changed after incorporation of only small amounts of LMP2 or -7, these subunits must cause major alterations in peptidase activity. Thus, their expression can account for the changes in proteasome activity induced by interferon 7, and these findings lend further support to the proposed roles of LMPs in altering the nature of the peptides generated for antigen presentation.
degrade model proteins or ubiquitinated proteins but increases their capacity to cleave oligopeptides after hydrophobic and basic residues and reduces cleavage after acidic residues (5-7). These functional alterations should favor the degradation of proteins to oligopeptides that terminate in hydrophobic or basic residues (5). Such peptides bind selectively to MHC class I molecules (14). The 20S proteasome contains about 14 distinct, but related, subunits, whose precise functions in catalyzing proteolysis are unknown. Treatment of cells with IEN-y changes the subunit composition of 20S and 26S particles; it induces the addition of new subunits and the loss or modification of several others (15). Two of the new subunits added, LMP2 and -7 (15), are encoded in the MHC region and appear to replace subunits normally present in the 20S particle (16-19). Moreover, the changes in peptidase activity induced by IFN-y are not seen in a human lymphoblast deletion mutant (line 721.174) lacking 1 megabase of the MHC region (5, 20) that includes the LMP2 and -7 genes. The proteasomes of this deletion strain show a lower activity than those of control cells against hydrophobic and basic substrates and enhanced hydrolysis after acidic residues (5). These findings led to the suggestion (5-8) that incorporation of LMP2 and -7 are responsible for the alterations in peptidase activities, even though other changes in the proteasome subunits occur with IFN- yand many other genes are included within this MHC deletion. The present studies were undertaken to test whether expression of LMP2 and -7, the two MHC-encoded proteasome subunits (9, 10, 16-19), mediate these IFN-y-induced changes in peptidase activity. We therefore transfected each of these cDNAs into HeLa cells or 0.174 lymphoblasts. Proteasomes were then isolated from transfectant and control cells, the levels of the LMP2 and LMP7 subunits were assessed, and their several peptidase activities were compared (5). The present studies indicate that each of these MHC-encoded subunits alter the proteasome's peptidase activities in characteristic ways and strongly indicate that the different populations of proteasomes with distinct peptidase capacities can exist in vivo.
An initial step in the presentation of intracellular and viral proteins to the immune system is their proteolytic degradation in the cytosol to 8- or 9-residue peptides (1, 2). The antigenic fragments are then taken up into the endoplasmic reticulum, where they associate with the major histocompatibility complex (MHC) class I molecules (3). These peptideprotein complexes are then transported to the cell surface for presentation to cytotoxic T cells (1, 3). Recently, strong evidence has been obtained implicating the proteasome (multicatalytic proteinase complex) and the ATP-ubiquitindependent pathway (4) in the process of the generation of
antigenic fragments (5-10). The 20S (700 kDa) proteasome particle functions as the proteolytic core of the 26S (1500 kDa) complex that degrades ubiquitin-conjugated proteins (10-13). This particle exhibits multiple peptidase activities, including three well-characterized activities that preferentially hydrolyze small peptides on the carboxyl side of hydrophobic, basic, or acidic residues (10-13). Moreover, these three activities are regulated by the cytokine interferon y (IFN-y), which is a potent stimulator of MHC class I antigen presentation. Treatment of cells with IFN-y does not affect the ability of 20S or 26S particles to
cell line HeLa.S3 was obtained from the American Tissue Type Collection. Cell lines were transfected by electroporation with LMP2 and LMP7 cDNAs in pRSV.5 (21) and then selected for resistance to G418. The LMP7-transfected 0.174
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Abbreviations: MHC, major histocompatibility complex; IFN-y, interferon y; Suc, succinyl; MCA, methylcoumarin; Boc, butoxycarbonyl; Cbz, carbobenzoxy; fBNA, 13-naphthylamide.
MATERIALS AND METHODS Cell Lines. The human B-lymphoblastoid cell lines 721 and 0.174 (20) were kindly provided by Robert DeMars (University of Wisconsin, Madison). The human cervical carcinoma
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Biochemistry: Gaczynska et al.
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FIG. 1. Transfection of lymphoblasts and HeLa cells with LMP2 and LMP7. Typical results of a Western blot analysis of proteasomes are shown. (Left) Lymphoblasts. Lanes: 1, 0.174 mutants that contain no LMP2 or LMP7; 2, LMP2-transfected 0.174 cells where most antigen is expressed as a large LMP2 precursor (22), unlike in HeLa transfectant; 3, wild-type 721 cells (20); 4, 0.174; 5, LMP7transfected 0.174; 6, wild-type 721. (Right) HeLa cells. Lanes: 7, control cells that contain no LMP2; 8, LMP2 transfectant; 9, control; 10, LMP7 transfectant.
cell line has been described (22). The production of LMP2 and LMP7 proteins was verified by Western blot analysis. Proteasome Isolation and Peptidase Assays. Parent and transfectant cells were homogenized in a Dounce homogenizer and by vortex mixing with glass beads, in 50 mM Tris HCl/5 mM MgCl2/2 mM ATP/250 mM sucrose, pH 7.4 (5). Fractions containing total 20S and 26S proteasomes were isolated by differential centrifugation of homogenates: for 20 min at 10,000 X g, then for 1 h at 100,000 x g and for 5 h at 100,000 x g. Pellets were solubilized in 50 mM Tris.HCl/5 mM MgCl2/2 mM ATP/20%o (vol/vol) glycerol, pH 7.4. Resulting "proteasome fractions" were used for peptidase assays and Western blot analysis. Degradation of the fluorogenic peptides, N-succinyl-Leu-Leu-Val-Tyr-7-amino-4methylcoumarin (Suc-LLVY-MCA), N-tert-butoxycarbonyl-Leu-Arg-Arg-7-amido-4-methylcoumarin (Boc-LRRMCA) and N-carbobenzoxy-Leu-Leu-Glu-f-naphthylamide (Cbz-LLE-3NA) was assayed at 37°C, for 40 min or 1 h in the presence of apyrase (5 units/ml), as described (5). Western Blot Analysis. Proteasomes or crude cell lysates (10 ug per lane) were electrophoresed by SDS/PAGE on 12.5% gels and transferred to nitrocellulose. Filters were incubated with anti-LMP2 (1:200 dilution) or anti-LMP7 (1:200 dilution) antibodies (kindly provided by K. Tanaka, University of Tokushima, Tokushima, Japan) followed by 1251-labeled protein A. The levels of the mature LMP2 and LMP7 subunits were determined with a Phosphorlmager (5). The amount of radioactive protein A bound to antigen was linearly dependent (regression coefficient, 0.99) on the amount of proteasomal protein loaded on the SDS/PAGE gel at least in the range of 2-20 pg of protein per lane; 10 pg per lane was typically used in our experiments. We have verified that there were equal amounts of proteasome loaded by U~~~
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densitometry of proteasome bands on Coomassie-stained 4% polyacrylamide gels.
RESULTS Transfection of Cells with LMP7 cDNA. To determine whether individual LMP subunits can incorporate into the proteasome and affect its activity, we transfected LMPdeficient cells with LMP cDNAs under the control of constitutive viral promoters. The 0.174 cells lack both LMP2 and LMP7 subunits (Fig. 1) due to a homozygous deletion in the MHC. In 0.174 lymphoblasts transfected with LMP7 cDNA, proteasomes contained up to 25% of the amount of LMP7 present in the wild-type 721 cells (Figs. 1 and 2). HeLa cells, in the absence of IFN-y, express no LMP2 molecules and low levels ofthe LMP7 subunit (Fig. 1). The proteasomes isolated from three LMP7-transfected HeLa lines expressed 20 ± 5% (n = 5 experiments) more LMP7 than control cells. However, the presence of even these low amounts of transfected subunits consistently altered the overall ability of the proteasome fraction to degrade specific fluorogenic substrates. In a typical experiment, proteasomes from 0.174 cells transfected with LMP7 cDNA showed a 93% greater maximal capacity (Vm,,) to hydrolyze the hydrophobic substrate, Suc-LLVY-MCA, and a 68% increase in V~n for the basic substrate, Boc-LRR-MCA (Fig. 3). Similarly, in HeLa cells, transfection with LMP7 consistently increased the maximal rates of cleavage of hydrophobic and basic substrates (P < 0.01; n = 5 or 6) (Table 1). Moreover, the magnitude of these increases in activity depended on the amount of LMP7 incorporated into proteasomes (Fig. 2). LMP7 transfection, however, did not affect proteasomal activity against the acidic peptide substrate, Cbz-LLE-PNA (Table 1 and Fig. 3). Transfection of HeLa Cells with LMP2. After LMP2 transfection of HeLa cells, proteasomes contained the LMP2 subunit at 28 ± 2% of the levels observed after 3 days of treatment with IFN-y (Fig. 1). Interestingly, proteasomes from the LMP2-transfected cells also showed a small but significant reduction in the amount of LMP7 subunit (-18 ± 5%; P < 0.05, n = 5). In the single LMP2-transfected 0.174 clone analyzed, unlike in HeLa, most LMP2 antigen was present in the proteasome fraction primarily as a larger polypeptide (Fig. 1). These observations suggest that the normal proteolytic processing (23) of the LMP2 protein requires LMP7 or another MHC-encoded gene but that this processing is not essential for LMP2 incorporation into particles. Incorporation of these low amounts of LMP2 altered proteasomal properties in a different fashion from LMP7. 160-
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FIG. 2. Increase in V.. for the hydrophobic substrate cleavage depends on the amount of LMP7 incorporated into proteasomes. V. values expressed relative to control 0.174 cells (Vm = 100%) (Left) or control HeLa (V,, = 100%) (Right). To normalize the amounts of LMP7 incorporated into proteasomes after transfection, the amount of LMP7 in wild-type 721 lymphoblasts was taken as 100%, and the amount of LMP7 in control (parent) HeLa cells was taken as 100%o. are
Biochemistry: Gaczynska et al.
Proc. Natl. Acad. Sci. USA 91 (1994)
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