YOUNG YANG, JAMES B. WATERS, KLAUS FROH, AND PER A. PETERSON. Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037.
Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4928-4932, June 1992 Immunology
Proteasomes are regulated by interferon y: Implications for antigen processing (major hbitocompatlbility complex/class I moecules/lymphokines)
YOUNG YANG, JAMES B. WATERS, KLAUS FROH, AND PER A. PETERSON Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
Communicated by Frank J. Dixon, February 27, 1992
ABSTRACT Class I major histocompatibility complex (MHC) molecules present antigenic peptides of cytoplasmic origin to T cells. As the lengths of these peptides seem stried to eight or nine amino acids, an unusual proteolytic system must play a role in antigen processing. Proteasomes, a major extralysosomal proteolytic system, are responsible for the degradation of cytoplasmic proteins. We demonstrate that several proteasomal subunits, including MHC-encoded subunits, are regulated by interferon y. These data and the finding that MHC-encoded and other interferon -regulated proteasomal subunits are uniquely associated with proteasomes strongly suggest that the immune system has recruited proteasomes for antigen processing.
strengthened by the findings, presented in this communication, that several proteasomal subunits, including MHCencoded subunits, are regulated by interferon y (IFN--y) and that the incorporation of several more subunits into proteasomes appears to depend on the expression of the MHCencoded proteasomal subunits. Moreover, the pattern of expression of IFN-y-regulated subunits suggests complexities in the regulation of proteasomes with respect to its subunit composition, subcellular localization, and its incorporation into larger ubiquitin-related proteolytic complexes. Possible functions for the MHC-encoded and IFN-yregulated proteasomal subunits in antigen processing are discussed.
Major histocompatibility complex (MHC) class I molecules are believed to primarily obtain antigenic peptides from proteins synthesized within the cell (1). The recent observation that a putative transmembrane peptide transporter (2-6) is essential for peptide loading onto class I molecules suggests that antigenic peptides, which are acquired by class I molecules in a pre-Golgi compartment (7-9), may arise in the cytoplasm. However, the proteolytic system responsible for generating class I MHC binding peptides has remained enigmatic. The majority of proteolytic activity in the cytoplasm can be attributed to the activity of proteasomes (10, 11). This major extralysosomal proteolytic system (10) relies on a complex series of enzymatic events where proteins become targeted for degradation by covalent conjugation to the polypeptide ubiquitin (12, 13). The ubiquitinated protein is subsequently degraded into small peptides and free amino acids in an ATP-dependent process by 26S ubiquitin-dedicated proteasome complexes. This 26S proteasome complex contains the 19S form of the proteasome as its major proteolytic component (14). The 19S proteasome also exists in free form in the cell and may represent an independent and separately regulated proteolytic system (14, 15). The proteasome displays several distinct peptidase activities, being able to cleave on the carboxyl side of basic, acidic, and neutral amino acids, which suggests it can generate a wide variety of peptides from diverse protein substrates (10, 11). In addition, the enzymatic activity of the proteasome is regulated by a number of different compounds, including nucleotides and polycations (16, 17). These observations make the proteasome an attractive candidate as a proteolytic generator of antigenic peptides of cytoplasmic origin for class I MHC molecules (18). This suggestion is further supported by the recent findings that two proteasomal subunits are encoded in the MHC region (19-23). The view that the proteasome is a proteolytic generator of antigenic peptides for class I MHC molecules is further
MATERIALS AND METHODS Cell Cultures. HeLa cells (ATCC CCL185) were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 8% fetal calf serum, 2 mM glutamine, penicillin (100 ,ug/ml), and streptomycin (100 pug/ml). Splenocytes of four different H-2 haplotypes (H-2k, H-2s, H-2d, and H-2q), T1 and T2 cells (24), and murine astrocytes were grown in RPMI 1640 medium. Metabolic Radiag and Immunoprecipitaton. Metabolic labeling of cells was carried out as described (25) with the following modifications. Unless otherwise stated, cells were treated with IFN-y (2500 units/ml) for 96 hr prior to the metabolic labeling. Pulse media contained 0.15 mCi of L-[35S]methionine per ml and 0.15 mCi of L-[35S]cystine per ml (Amersham; 1 Ci = 37 GBq) in methionine- and cystinedeficient DMEM. Cells were routinely labeled for 4 hr followed by chase periods of different lengths of time (up to 36 hr) in the presence of normal culture medium. Immunoprecipitations (25), SDS/PAGE (26), and fluorography (27) were carried out as described (25). First-dimension nonequilibrium pH gradient gel electrophoresis (using Ampholines pH 3.5-10) was done as described (28). Antisera and Materials. Rabbit anti-human and anti-rat proteasome sera were kindly provided by A. Ichihara (29). IFN-y from human T lymphocytes was obtained from Boehringer Mannheim. Mouse IFN-y was obtained from Genentech and Amgen Biologicals. Homogenization of HeLa Cells. Two milliliters of isotonic sucrose solution was added to HeLa cell monolayer. Cells were scraped from dishes and homogenized directly by 45 passages through a precision clearance between the wall of a metal chamber and a metal ball bearing of a Dounce homogenization vessel (30). During homogenization, the homogenates were monitored using phase-contrast microscopy to assess the number of broken cells, the number of intact and clean nuclei, and the absence of cytoplasmic clumps. Ammonium Sulfate Fractionation of HeLa Cell Homogenates. Homogenized HeLa cells, untreated and IFN-y-
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Abbreviations: MHC, major histocompatibility complex; IFN-y, interferon 'y; 2D, two-dimensional.
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peared completely (spots 2, 10, 15, and 16) following IFN-y treatment. In addition, the intensity of spot 9 increased considerably as a consequence of the IFN-y treatment. Experiments identical to those described above were also carried out with a murine astrocyte cell line. The subunit pattern of murine proteasomes was very similar to that observed for human proteasomes and the effect of IFN-y on the astrocyte proteasomes was analogous to that of the HeLa cell proteasomes (not shown). Thus, the subunit composition of proteasomes from two species is regulated by IFN-y. If the IFN-y-regulated proteasomal subunits are involved in the generation of antigenic peptides, one might assume that cells that express high levels of class I MHC molecules may also constitutively express high levels of the IFN-y-regulated proteasomal subunits. For this reason we chose to examine proteasomes from murine splenocytes. In addition, we could also determine if proteasomal subunits displayed structural polymorphisms indicative of many MHC-encoded proteins. Proteasomes from biosynthetically labeled splenocytes derived from four mouse strains representing the H-2 haplotypes d, k, s, and q were immunoprecipitated with an antiserum specific for rat proteasomes and subjected to 2D PAGE. The typical patterns of the proteasomal subunits are depicted in Fig. 2. All but one of the proteasomal polypeptide spots occurred at corresponding positions for all four haplotypes. The exception was spot 9, which is likely to correspond to one of the MHC-encoded subunits (19-23) and which obviously occurs in at least two allelic forms (21). The splenocyte proteasomal subunit pattern was intermediate between those of untreated and IFN-y-treated HeLa cells inasmuch as spots 2, 10, 15, and 16 were weak or absent, whereas spots b, d, and 9 were prominent. Thus, we conclude that only one proteasomal subunit, which is likely to be encoded in the MHC, displayed structural polymorphism and that some IFN-y-regulated proteasomal polypeptides are constitutively expressed in splenocytes. The latter finding is consistent with splenocytes being excellent antigenpresenting cells. Incorporation of MHC-Encoded and IFN-r-Regulated Subunits into Proteasomes. The data described above show that IFN-'y regulates more proteasomal subunits than the two that are known to be encoded by the MHC region (19-23). To examine if the MHC-encoded and IFN-y-regulated proteasomal subunits control the recruitment of other polypeptides into the proteasome, we examined the proteasomal subunit compositions of two human lymphoblastoid cell lines, T1 and T2, which differ with regard to their MHC regions (24) such that T2 cells lack the MHC-encoded proteasomal subunits. Biosynthetically labeled proteasomes from untreated and IFN-y-treated T1 and T2 cells were immunoprecipitated and analyzed by 2D PAGE. The typical proteasomal subunit patterns are depicted in Fig. 3. Although the overall subunit compositions were very similar to those observed for un-
treated, were centrifuged to remove nuclei and cell debris, and the resultant supernatants were subjected to ammonium sulfate fractionation as described (31). 26S proteasomes present in the supernatant were selectively precipitated with ammonium sulfate at 38% saturation; 19S proteasomes present in the supernatant after the 38% fractionation step were precipitated at 60%o ammonium sulfate (32). Fractionation of Microsomes and Cytosol. Untreated and IFN--treated HeLa cells were labeled for 4 hr, homogenized, and subjected to differential centrifugation. The homogenates were first spun at 13,000 rpm for 30 min to remove nuclei and cell debris. The resulting supernatants were then spun at 100,000 x g for 30 min to pellet microsomes. Proteasomes in the supernatant fractions (cytosolic fraction) and in the pellets (crude microsomal fraction) were separately immunoprecipitated and analyzed by two-dimensional (2D) gel electrophoresis.
RESULTS Proteasomal Subunit Composition Is Regulated by IFN-y. The assembly of class I MHC molecules in the endoplasmic reticulum in transfected cells that overexpress class I heavy chains and f32-microglobulin is limited by the availability of peptides that can bind to class I MHC molecules. This restriction can be overcome by treating the cells with IFN-y (E. Song, Y.Y., M. R. Jackson, and P.A.P., unpublished work). IFN-y induces changes in the level of expression of a number of gene products affecting various cellular responses that appear to be aimed at defending the cell against viral infections (33). We reasoned that it was likely that some ofthe components responsible for generating class I antigenic peptides might also be regulated by IFN-y. We focused our interest on the proteasome and examined whether its subcellular distribution and subunit composition were noticeably affected by IFN-,y treatment. Proteasomes from IFN-y-treated and untreated HeLa cells were immunoprecipitated with an antiserum specific for human proteasomes and subjected to 2D PAGE. Typical autoradiograms are depicted in Fig. 1. The proteasome consists of several individual subunits whose apparent molecular masses range from =18,000 to -35,000 daltons (10, 11). However, the number of proteasomal subunits and their stoichiometry have not been unambiguously determined. The schematic diagram shown in Fig. 1C shows that by immunoprecipitation we could isolate proteasomes, which resolved into a total of 25 polypeptide spots by 2D gel electrophoresis. Spots 1-20 were reproducibly obtained with immunoprecipitated proteasomes from untreated HeLa cells. The proteasomal subunit pattern obtained after IFN--y treatment of the HeLa cells was clearly different from the pattern observed for proteasomes from untreated cells. Five novel spots were apparent, denoted a-e in Fig. 1, and four spots were greatly diminished or disapA
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FIG. 1. Some subunits of human proteasomes are regulated by IFN-y. Proteasomes of HeLa cells, untreated (A) or IFN-y-treated (B) before labeling, were immunoprecipitated with antibodies against human proteasomes and subjected to 2D PAGE. The solid arrows denote proteasomal subunits whose intensities were increased to a minor (thin arrows) or major (bold arrows) extent after treatment with IFN-'y. Open arrows denote proteasomal subunits whose intensities were reduced after treatment with IFN-y. (C) The schematic diagram is a composite of A and B. Filled spots indicate proteasomal subunits whose intensities increased; hatched spots indicate those whose intensities decreased following the IFN-y treatment.
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metabolically labeled for 4 hr. Proteasomes were immunoprecipitated with antibodies against rat proteasomes and analyzed by 2D gel electrophoresis. The proteasomal subunit that displayed allelic variation is denoted by the arrows. were
treated and IFN-y-treated HeLa cells (see Fig. 1), several differences were also apparent. Several additional polypeptides, denoted p, q, r, and s, were present in T1 cells. In addition, the IFN--induced polypeptides a and e were constitutively expressed in untreated T1 cells. After IFN-y treatment, the T1 proteasomes also displayed differences from those of IFN-y-treated HeLa cells. The spots denoted 2, 10, 15, and 16 were more prominent in T1 than in HeLa cells and polypeptide d was not induced in T1 cells. Moreover, polypeptide c disappeared from the T1 cells but was induced in the HeLa cells. The proteasomal subunit patterns of untreated and IFNy-treated T1 cells displayed multiple differences. Polypeptides 14, c, p, r, and s disappeared, whereas increased amounts of 9, 13, 15, 16, a, b, and e were observed. Some of these polypeptides also differed between proteasomes of untreated T1 and T2 cells. Thus, polypeptides 9, 15, a, and c were absent from T2 cells. With IFN-y treatment T2 proteasomes displayed the expected differences with the exception that polypeptide b disappeared. Thus, it can be concluded that in qualitative terms T1 and T2 proteasomes differed with regard to polypeptides 9, 15, a, b, and c. These data demonstrate that the incorporation of all but one of the IFN-regulated subunits into proteasomes was dependent on one or more genes, which is (are) likely to be MHC-encoded proteasomal subunits. Since polypeptide 9 is IFN-y-regulated, structurally polymorphic, and absent from untreated and IFN--treated T2 cells, we concluded that subunit 9 is encoded within the MHC. This conclusion is supported by the finding that the polypeptide, detected from HeLa cells transfected with the MHC-encoded proteasomal subunit RING10 (23) cDNA, is identical to polypeptide 9 as revealed by 2D PAGE (unpublished data). Furthermore, the observation that IFN-'y-regulated subunit b is absent from untreated and IFN-y-treated T2 cells in conjunction with the finding that subunit b is identical to the polypeptide detected from HeLa cells transfected with the other MHC-encoded proteasomal subunit RING12 (22) cDNA (unpublished data) strongly suggests that subunit b is encoded in the MHC. If the MHC-encoded subunits facilitate the generation of antigenic peptides, then the other IFN-y-regulated polypeptides might have contributing but independent effects. This suggestion is supported by the observation the IFN-y had virtually identical effects on T1 and T2 proteasomes despite the lack of the MHC-encoded subunits in T2 cells. It is,
therefore, conceivable that IFN-y controls several discrete steps in the proteasomal degradation of polypeptides (see below). IFN-r-Regulated Protesomal Subunits Are Uniquely Distributed Between the 19S and the 26S Proteasones. The proteasome exists in two molecular forms (the 19S proteasome and the 26S proteasome). As the 26S proteasome seems to degrade proteins extensively to yield very small peptides, which cannot bind to class I molecules, or free amino acids, elimination of functional 26S proteasomes might make more peptides available for the class I molecules. If the MHCencoded and IFN--regulated subunits facilitate the generation of antigenic peptides, the IFN--regulated subunits might impede the formation of 26S proteasomes or may inactivate their proteolytic activity. Therefore, to examine whether the IFN-y-regulated proteasomal subunits were distributed differently between the 19S and 26S proteasomes, biosynthetically labeled proteasomes from untreated and IFN-y-treated HeLa cells were fractionated into those that were precipitated at 38% ammonium sulfate saturation (26S proteasomes) and those that were precipitated between 38% and 60% saturation (19S proteasomes). The ammonium sulfate-precipitated materials were dissolved, dialyzed, immunoprecipitated with an antiserum specific for human proteasomes, and analyzed by 2D PAGE. Fig. 4 summarizes the results. The subunit pattern of the 26S proteasome from untreated HeLa cells lacked components 2, 9, 10, and 11, which were present in the 19S proteasome. Differences between the subunit patterns of the 19S and 26S proteasomes were more pronounced after IFN-y treatment. Fig. 4 C and D shows that IFN--induced subunits a and e were associated with the 26S proteasome, whereas those denoted b, c, and d were exclusively confined to the 19S proteasome. In addition, the 19S proteasome isolated from IFN-y-treated HeLa cells lacked subunits 2, 3, and 10. The 26S proteasome did not seem to lack any additional subunits following IFN-y treatment. Thus, the subunit compositions of the 19S and 26S proteasomes differ from each other prior to and after IFN-y treatment. Moreover, the presence of IFN--regulated proteasomal subunits seems mutually exclusive, as they are either incorporated into the 19S or the 26S proteasome. Suelular Distribution of Proteasomial Subunits. Since peptides derived from cytoplasmic proteins must enter the endoplasmic reticulum to become accessible to class I molecules, we examined whether proteasomes present in a microsomal fraction had a different subunit pattern than those existing in the cytoplasm, which would be consistent with targeting of proteasomes to the MHC-encoded peptide transporter. Biosynthetically labeled proteasomes from a crude microsomal fraction and a cytosolic fraction of HeLa cells were immunoprecipitated and subjected to 2D PAGE. Fig. 5, which displays typical autoradiograms, shows that the proteasomes present in the microsomal fraction were enriched for subunits 5 and 14 and lacked subunits 6, 7, 13, and 20. Following IFN-y treatment, proteasomes present in the crude microsomal fraction contained more of the IFN-yregulated subunits b and c than did proteasomes present in the cytosolic fraction. In contrast, the IFN--regulated subunits a and e were predominantly confined to proteasomes present in the cytosolic fraction. The difference in the subunit compositions of the 19S and 26S proteasomes and the difference in the subunit compositions of cytoplasmic and microsomal proteasomes reveal that multiple types of proteasomes must exist.
DISCUSSION The fact that the proteasome is essential for the homeostasis of cells in conjunction with its presence in lower eukaryotes
Immunology: Yang et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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argue for the proteasome having evolutionarily predated the immune system. It is not surprising that the immune system might have recruited the proteasome for the purpose of generating antigenic peptides that can be presented by class I molecules. The findings that the proteasomal subunit pattern was greatly affected by IFN-y treatment and that MHCencoded and other IFN-y-regulated proteasomal subunits are uniquely associated with proteasomes are, therefore, fully consistent with the proteasome being involved in antigen presentation. The view that proteasomes play a role in antigen processing is further strengthened by our preliminary experiments showing that proteasome-treated ovalbumin stabilized empty MHC Kb molecules and that the major product of the proteasome-processed ovalbumin was an octapeptide (unpublished data).
It has been shown that disruption of two proteasomal subunit genes in Saccharomyces cerevisiae profoundly affected the growth characteristics of cells (34-36). Since the growth characteristics of the T1 and T2 cells were not noticeably different, it can be assumed that the housekeeping functions of proteasomes were not adversely affected by the lack of IFN-y-regulated and the MHC-encoded subunits. It may, therefore, be assumed that the latter subunits alter the activity ofthe proteasomal system such that the generation of antigenic peptides becomes facilitated. The MHC-encoded subunit 9 was distributed among cytosolic and microsomal as well as 19S and 26S proteasomes. However, the MHCencoded subunit b was only present in 19S microsomal proteasomes. This unique distribution of subunit b among proteasomes may imply that subunit b-containing proteasomes are dedicated to the production of antigenic peptides.
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