By
From the * Max-Planck-Institut für Immunbiologie, 79108 Freiburg, Germany; Hewlett-Packard
GmbH, 76337 Waldbronn, Germany; § Department of Biochemistry, University of Utah, Salt Lake
City, Utah 84123;
Botanisches Institut, Universität Kiel, 24089 Kiel, Germany; ¶ The Kitasato
Institute, Tokyo 108, Japan; and ** Max-Planck-Institut für Biochemie, 82152 Martiensried,
Germany
To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes,
phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have
structural features in common with peptides eluted from MHC class I molecules, suggesting
that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report
that the processing of polypeptides by proteasomes is conserved in evolution, not only among
vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length
and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes
to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon induction in vivo or addition of recombinant proteasome activator 28
in vitro alters proteasomal proteolysis in such a
way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the
evolution of MHC class I molecules.
Tlymphocytes recognize peptide fragments of protein
antigens presented on the cell surface by the class I
and class II molecules of the MHC.The peptide fragments
are generated proteolytically inside the cell. MHC class II
molecules are loaded in a secretory compartment with peptides generated in endosomes. MHC class I molecules are
loaded with peptides mainly generated in the cytoplasm and transported into the ER/cis-Golgi by the peptide transporter associated with antigen processing (TAP1; 1, 2). The
vast majority of peptides presented by MHC molecules are
derived from self-proteins. The peptide-binding grooves of class II molecules are open at both ends and bind peptides
of heterogenous length (usually 12-25 amino acids [aa]);
the peptide-binding grooves of class I molecules are closed
at both ends and usually bind peptides of closely defined
length (8-10, mostly 9 aa). In the latter case, the peptide is
usually fixed by two allele-specific anchor residues that are
complementary to allele-specific pockets in the MHC class I
peptide-binding groove (3, 4). In addition, H bonds are
formed between relatively invariant polar aa at the ends of
the binding groove and the NH2 and COOH termini of
the peptide (5). Typically, one of the allele-specific pockets, the COOH-terminal F pocket, accommodates an aliphatic, aromatic, or positively charged aa at the COOH terminus of an octa/nonamer peptide. The second anchor may
reside at the second, the third, or the fifth position from the
NH2 terminus of the peptide, and is more variable (3).
For the proteolytic generation of MHC class I epitopes,
the immune system of vertebrates appears to have recruited
the proteasomes. These ubiquitous multi-subunit endoproteases are phylogenetically ancient, as they occur in archea
and bacteria, as well in eukarya. In eukaryotic cells, proteasomes appear to be the major proteolytic system of the nucleus and the cytosol. Three main forms are observed. The
20S proteasome, by itself capable of degrading misfolded or damaged polypeptides, represents the proteolytic core of the
larger and more complex 26S proteasomes and the 20S-
proteasome activator (PA)28 complexes (6). It has a barrel-shaped hollow structure with four layers of rings, each
composed of seven subunits. The outer rings consist of proteolytically inactive The assembly of the class I heavy chain with The presently available data on the substrate/ligand specificities of proteasomes, TAP, and MHC class I, in addition
to suggesting coordinated function of the three systems, argue for some degree of coevolution. It has been suggested
that peptide binding to MHC class I and class II may have
been determined by proteolytic pathways available in ancestors before emergence of the vertebrate immune system
(15). Here, we examine the hypothesis that proteasome-mediated proteolysis may have influenced the evolution of
MHC class I. This hypothesis requires that the capacity of
proteasomes to generate fragments with the general structural features of MHC class I binding peptides is conserved
and extends back in evolution to before the emergence of
MHC and T cell recognition. Although structural homologies would anticipate a high degree of conservation in proteasomal functions, differences in cleavage site usage between proteasomes at different stages of evolution have been
reported (16, 17). Furthermore, it has been suggested that
the IFN- Reagents, Cell Lines, and Antibodies.
The protease inhibitor
N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) was purchased from Sigma Chemical Co. (St. Louis, MO); IFN- Immunoprecipitation Experiments.
EL4 cells (107 cells/ml) were
incubated for 2 h at 37°C in the presence or absence of proteasome inhibitors in cysteine and methionine-free medium, and for
the last 45 min of incubation, (35S) cysteine/methionine (700 µCi/
ml) was added. After metabolic labeling, cells were lysed in 0.5%
Nonidet P-40 (ICN Biomedicals Inc., Plainview, New York) and
0.5% Mega 9 (Sigma Chemical Co.). Samples were precleared for
60 min at 4°C with protein A-Sepharose (Pharmacia, Uppsala,
Sweden) pretreated with 1 mg/ml bovine serum albumin. For
immunoprecipitation, either mAb Y3 (15 µg) or 4 µl rabbit Peptides and Protein Substrates.
Peptides were synthesized by
using solid-phase 9-fluorenylmethoxycarbonyl chemistry in a peptide synthesizer (431A; Applied Biosystems, Foster City, CA) and
subsequently purified by reverse phase HPLC. The identity of peptides was established by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-Tof-MS) and aa
sequence analysis on Hewlett-Packard Co. (Palo Alto, CA) instruments. Ribulose 1,5 bisphosphate carboxylase small subunit was purified from intact pea chloroplasts by denaturing continuous electrophoresis (Grimm, R., manuscript submitted).
Purification of 20S Proteasomes and of Recombinant PA28 Proteasome Digests and Analyses.
Digestions of synthetic peptides (6 µg) and of the small subunit of ribulose 1,5 bisphosphate
carboxylase (10 µg) with isolated proteasomes (1 µg) were performed at 37°C, except in the case of Thermoplasma proteasomes
at 60°C, in a total volume of 300 µl buffer (20 mM Hepes/KOH
[pH 7.0] 1 mM EGTA, 0.5 mM EDTA, 5 mM MgCl2, 0.5 mM
2-mercaptoethanol, 0.02% sodium azide). Digestions of ovalbumin
fragments Ova37-77 and Ova239-281, and of the protein substrate
were done in the presence of 0.004% SDS. In the experiments shown in Fig. 7, recombinant PA28
-type subunits, the inner rings of
-type
subunits. Archaebacterial proteasomes possess one type of
proteolytically active
subunit, i.e., all seven members in a
ring have a NH2-terminal threonine acting as the nucleophile in peptide-bond hydrolysis. In eukaryotes, three
out of seven different
-type subunits contain such a site. Proteolysis takes places inside the central cavity, between
the two
rings (7, 8). In vertebrates, the three proteolytically active
-type subunits (X, Y, and Z) have IFN-
-inducible
homologues (LMP7, LMP2, and MECL1) replacing their
constitutive counterparts when induced. The PA28 proteasome activator, giving rise to the 20S-PA28 complexes, is
IFN-
-inducible as well. LMP2 and LMP7, but not
MECL1 and PA28, are encoded by genes in the MHC (9).
2 microglobulin (
2m) can be substantially inhibited by peptide aldehydes, potent but not absolutely specific proteasome inhibitors (10). This seminal information, together with the
discovery of the MHC-encoded proteasome subunits (11),
have stimulated a host of investigations into the role of vertebrate 20S proteasomes in the processing of antigens presented by MHC class I (for review see references 6 and 9).
Although most of these studies converged in suggesting a
major role for proteasomes in the generation of MHC class
I epitopes, it is also clear that the evidence in support of this notion remains, to some extent, circumstantial. We have
recently reported that the length distribution of dual cleavage proteolytic fragments produced by mouse 20S proteasomes centers around 8-11 mer. The frequencies of individual aa at the COOH termini of proteolytic fragments
generated by proteasomes correlated strikingly with that at
the corresponding positions of so far eluted MHC class I
ligands. For the NH2 termini too, a significant enrichment
of small and polar aa was observed for both proteasomal
degradation products and MHC class I ligands (12). Intriguingly, similar COOH and NH2 termini as well as a similar
length distribution were found for peptides preferentially
transported by TAP or by certain TAP alleles (13, 14). Together, these results are consistent with the notion that
many of the peptides transported by TAP and many of the
epitopes presented by MHC class I are directly derived by
proteasomal proteolysis.
inducible elements drastically alter the repertoire
of peptide products of proteasome mediated proteolysis (18,
19). Our results suggest that the capacity of proteasomes to
generate potentially immunocompetent peptides, including
the efficient generation of several proven MHC class I ligands,
is highly conserved in eukaryotes and evolved before the
vertebrate immune system. The functional modifications by
the IFN-
-inducible elements suggest an evolutionary adaptation of proteasomes to their novel immune functions.
However, these modifications appear to be mainly quantitative in nature and did not confer fundamentally novel
characteristics to proteasomal proteolysis.
was
from Boehringer Mannheim GmbH (Mannheim, Germany). The
proteasome inhibitor lactacystin was purified as described (20).
The C57BL/6-derived thymoma EL4, the human lymphoblastoid cell line T1, the human erythroblastoid cell line K562, and
Drosophila melanogaster Schneider cells were obtained from American Type Culture Collection (Rockville, MD). Monoclonal antibodies were prepared from the hybridoma Y3 (anti-H2 class I
Kb heterodimers; 21). Rabbit antiserum specific for sequences encoded by exon 8 of the Kb gene and reactive with free or
2m-associated Kb heavy chains was a gift from Dr. S. Nathenson (Albert Einstein College, New York).
exon 8 antiserum were added to the precleared lysates for 2 h. For
the last 90 min of incubation, protein A-Sepharose was added.
Immunoprecipitates were analyzed by SDS-PAGE on 12% gels.
Quantitation of gel bands was performed with the aid of a Fujix
BAS 1,000 phosphorimager.
.
20S proteasomes were purified from EL4 cells cultured with or
without 50 U/ml IFN-
for 6 d, from K562, T1, and D. melanogaster Schneider cells, as well as from Saccharomyces cerevisiae (strain YRG-2) by fractionated precipitation of the cytosol with polyethylene glycol 6,000 followed by anion exchange chromatography on a Mono Q column (HR 5/5; Pharmacia) as previously described for EL4 cells (22). Modifications of the NaCl gradient
(buffer A: 20 mM Tris/HCl [pH 7.2]; buffer B: 20 mM Tris/HCl
[pH 7.2], 1 M NaCl) were as follows: S. cerevisiae: 0-38% B in 50 min, 38-48% B in 25 min, proteasomes eluted at 45% B; D. melanogaster: 0-28% B in 50 min, 28-31% B in 25 min, proteasomes
eluted at 30% B; IFN-
-treated EL4 cells: 0-34% B in 65 min,
34-37% B in 40 min, proteasomes eluted at 36% B; K562 cells:
0-37% B in 55 min, 37-41% B in 40 min, proteasomes eluted at
38% B; T1 cells: 0-35% B in 55 min, 35-38% B in 40 min, proteasomes eluted at 37% B. T1 proteasomes were further purified
on a Phenylsuperose column (Pharmacia). The proteasomes were
recovered in the flow-through fraction. 20S proteasomes from
Rhodococcus sp. and recombinant Thermoplasma acidophilum proteasomes were purified as described (23, 24). The purity of proteasomes was assessed by SDS-PAGE followed by silver staining
as described (22). Purification of recombinant human red blood
cell PA28
is described in references 25 and 26.
was added in a fivefold molar excess over the proteasome. Aliquots of the reaction mixture
were separated by reverse phase HPLC on a SMART system
equipped with a µRPC C2/C18 SC 2.1/10 column (Pharmacia).
Eluent A was 0.1% (vol/vol) TFA/water; eluent B was 80% (vol/
vol) acetonitril/water (0.081% TFA). The identity of peptides in
individual HPLC fractions was established by MALDI-Tof-MS
and aa sequence analysis by Edman degradation.
Fig. 7.
PA28 enhances the rate of accumulation of dual cleavage
products without changing the cleavage site specificity of the proteasome. The substrate OvaY249-269 (for the sequence, see Fig. 5 A) was incubated
with 20S proteasomes from EL4 cells in the absence (A and C) or presence (B and D) of recombinant PA28
. At the time points indicated, the
mixtures were separated by reverse phase HPLC. The peptides in the
peaks marked with numbers are: TEWTS (1), YVSGLEQLE (2), YVSGLEQL (3), ESIINFEKL and the Kb ligand SIINFEKL (4), SIINFEKLTEWTS and ESIINFEKLTEWTS (5), YVSGLE (6), SIINFE (7), and
SIINF (8). The large peak at the right of A and B is undigested substrate.
Peptides were identified by MALDI-Tof-MS and Edman degradation.
[View Larger Version of this Image (23K GIF file)]
Fig. 5.
Proteasomes from vertebrates and from eukaryotic invertebrates show highly conserved cleavage patterns in polypeptides. Synthetic peptides OvaY249-269 (A), Ova239-281 (B), and Ova37-77 (C) were incubated
in the presence of 20S proteasomes isolated from the indicated cell lines
or organisms. After substrate consumption, the mixtures were subjected
to pool sequencing by Edman degradation. Proteasome cleavage sites
were determined and quantitatively estimated by the sequence cycle numbers and the yields of unique amino acids. For example, the strong signal
for asparagine (N) in sequence cycle 4 (panel A) indicates a strong cleavage site four residues towards the NH2 terminus of N between E-S. Although isoleucine (I) is not unique, these signals are necessary for the interpretation of the phenylalanine (F) signal, and are therefore included in panel B. I undergoes racemization to iso- and alloleucine, the latter representing 30-40% and coeluting with F. The phenylalanine signals in cycles
2 and 3 are therefore caused by isoleucine (e.g., EL4 digest). Cleavage
sites are indicated by arrows, with the sizes reflecting estimates of the relative efficiency of cleavage. The CTL epitopes SIINFEKL and KVVRFDKL are underlined.
[View Larger Version of this Image (31K GIF file)]
A functional pressure of proteasome-mediated proteolysis on the evolution
of MHC class I could be envisaged if proteasomal digestion
provided the major source of peptides for assembly with MHC class I. We examined the assembly of MHC class
I-peptide complexes in the presence of the proteasome inhibitor lactacystin (27). Except proteasome-related particles
in bacteria (28), no other protease has been reported to be
inhibited by this compound. The murine thymoma cell line
EL4, expressing the class I molecules H-2Kb (Kb) and H-2Db
(Db), was incubated with or without lactacystin and metabolically labeled with 35S-methionine plus cysteine. Immunoprecipitation was performed either with mAb Y3, detecting a conformational determinant of Kb present only on
the assembled trimolecular complex, or with an antiserum
(anti-exon 8 antiserum) that recognizes the cytoplasmic tail
of Kb and Db molecules, independent of whether a peptide
is bound or not. As shown in Fig. 1, addition of lactacystin
caused a marked decrease in the amount of Kb molecules
precipitable with the conformation-specific antibody, but
not of that precipitated by the anti-exon 8 reagent. Plateau inhibition was at ~67% as calculated by phosphorimaging
(see Materials and Methods), similar or slightly better than
that reported for the peptide aldehyde inhibitor LLnL (Fig. 1;
reference 10). These data are in agreement with recent results on the inhibition of antigen presentation by lactacystin
(29) and further support the notion that proteasomes participate in the generation of the majority of peptides presented by MHC class I.
Efficient Generation of Proven MHC Class I Ligands by Proteasomes of Eukaryotic Invertebrates.
At early time points in
the processing of short polypeptide substrates by isolated
mouse 20S proteasomes, single cleavage intermediates can
be detected in addition to dual cleavage oligopeptides. After consumption of the original substrate and of the single cleavage intermediates, the reaction appears to slow down
and the mixture of peptide fragments approaches a relatively stable state (12). We believe that the stable state in
vitro most closely resembles the conditions in vivo. Therefore, although several time points have been analyzed to
compare 20S proteasomes at distinct stages of evolution
(Fig. 2), we present data on the relatively stable product
patterns determined after complete substrate turnover.
We have previously shown that the immunodominant
ovalbumin epitope Ova257-264 (SIINFEKL; reference 30) is
the major stable product generated by mouse 20S proteasomes from the 22-mer OvaY249-269 as well as from the 44-mer Ova239-281 (12). Here we show that this octamer is also
a dominant product of digestion of Ova239-281 by 20S proteasomes isolated from D. melanogaster Schneider cells (Fig.
3 A and Fig. 4 A). As a second example, we studied the
generation of the nonamer TLWVDPYEV, an endogenous
peptide derived from the product of the antiproliferative B
cell translocation gene 1 (BTG1) and eluted as a major self-epitope from the human class I molecule HLA-A2.1 (31).
Fig. 3, B and C show that this nonamer peptide is the major dual cleavage product generated by yeast (S. cerevisiae) proteasomes of the synthetic 24 mer encompassing this
peptide in the sequence of BTG1. Moreover, we studied a
21-mer sequence derived from the tyrosine kinase JAK1
containing the nonamer SYFPEITHI, the most abundant
self-peptide presented by mouse H-2Kd molecules of P815
cells (32, 33), and previously shown to be generated by digestion with mouse 20S proteasomes (34). We detected the
epitope as the predominant dual cleavage product of the 21 mer with Drosophila proteasomes (Fig. 3 D). Thus, proteasomes from invertebrate eukaryotes have a high potency to
generate proteolytic fragments that have been proven to
serve as ligands of MHC molecules in the vertebrate immune system.
The Majority of Dual Cleavage Peptides Produced by Invertebrate Proteasomes Are in Size Range of MHC Class I Ligands.
More than 90% of the peptides so far eluted from MHC class I molecules are 8-10 aa in length (3). We have recently shown that more than half of the dual cleavage proteolytic fragments generated by digestion of Ova239-281 with mouse 20S proteasomes are 8-11 aa in length (12). In Fig. 4, A and B, we show that the peptides most efficiently produced from this substrate by Drosophila 20S proteasomes are in the size range of MHC class I-binding peptides (Fig. 4 B, shaded area). In addition, we studied a longer and immunologically undefined substrate, the 123-aa small subunit (SSU) of ribulose 1,5 bisphosphate carboxylase from the garden pea (Pisum sativum L.) (Fig. 4, C-E). Digests were prepared with proteasomes from mouse EL4 cells and from yeast. Most of the abundant masses in the digests represented peptide sizes of 5 to 11 aa for EL4 proteasomes or 5 to 13 aa for yeast proteasomes. In both cases, the majority of the peptides were 8-10 mer (Fig. 4, D and E, shaded areas). The masses of a number of abundant products are identical or nearly identical in the digests in Fig. 4, D and E, indicating that many of the peptides produced from SSU by mouse and yeast proteasomes may be identical.
Conserved General Cleavage Site Specificity of Proteasomes from Vertebrates and from Eukaryotic Invertebrates.We studied the cleavage site (...P3P2P1 P1
P2
P3
...) preferences in
polypeptides of 20S proteasomes isolated from a variety of
organisms including archaebacteria, eubacteria, and nonvertebrate and vertebrate eukaryotes (see Fig. 2). Fig. 5 A shows
the results obtained by pool sequencing of digests of the
22-mer OvaY249-269 containing the immunodominant
Ova257-264 (SIINFEKL) epitope. The cleavage patterns of all
proteasomes of eukaryotic origin, including the murine cell
line EL4, the human cell lines T1 and K562, as well as of insects and of yeast are remarkably similar; the predominant
cleavage sites reside after the same hydrophobic (L264-T265)
and acidic (E256-S257) aa. These cleavage sites precisely coincide with the NH2- and COOH-terminal epitope boundaries. In contrast, the cleavage pattern of archaebacterial proteasomes is clearly different; they prefer to cleave after aromatic (F261-E262) and aliphatic aa (L264-T265, L255-E256), no cleavage after acidic aa is seen, and the major cleavage
site destroys the epitope. Analyses of the degradation of
longer substrates is shown in Fig. 5, B and C. The 44-mer
Ova239-281 (Fig. 5 B) represents a longer fragment containing the immunodominant SIINFEKL; the 41-mer Ova37-77
(Fig. 5 C) contains the poorly immunogenic epitope Ova55-62
(KVVRFDKL), also presented by Kb (22, 35). For bacterial
proteasomes, the data in Fig. 5 C highlight the preference
for aromatic and aliphatic aa in P1, whereas cleavage after
charged aa is rare but not impossible (Fig. 5 B). Cleavage
patterns of all eukaryotic examples, although not fully identical, reflect the same broad but characteristic P1 specificity
spectrum. About 60-65% of the peptide bonds hydrolyzed (e.g., 11/17 in Ova239-281 by mouse EL4-proteasomes; 11/18
by yeast proteasomes) have an aromatic or a hydrophobic
aliphatic aa in the P1 position. Most of the remaining peptide bonds have either a positively (R) or negatively
charged (E, D) aa in the P1 position. In addition, together
with results in Fig. 3 (see above), these data extend to invertebrate eukaryotes our previous finding that proteasomes
have a preference for small or polar aa in the P1
position of
the scissile bond (12). Major cleavage sites are: E256-S257
and L264-T265 in OvaY249-269 (Fig. 5 A), L102-T103 and V111-S112 in BTG197-120 (see Fig. 3, B and C), and F354-S355 in
JAK1348-368 (see Fig. 3 D).
The existence of IFN--inducible proteasomal
elements in vertebrates indicates that the proteasomes themselves have evolved by adapting to their novel immunological role. If the specificity of proteasomal proteolysis was
drastically and qualitatively altered by the IFN-
-inducible elements, a significant restricting role of proteasomes in the
evolution of MHC would be less likely. Most previous
studies on the functional effects of the IFN-
-inducible
subunits LMP2, LMP7, and MECL1 used short (3-4 aa)
fluorogenic substrates, and inconsistent changes in peptidase activities have been reported by different investigators.
By digesting polypeptide sequences more likely to resemble
physiological proteasome substrates, we compared proteasomes from untreated with that of IFN-
-treated EL4
cells. Enhanced expression of LMP2 and LMP7 in induced
compared to uninduced cells was monitored by Western
blot analyses (not shown). Fig. 6, A and B show the HPLC
patterns obtained upon digestion of OvaY249-269, Fig. 6, C
and D that of the BTG1-derived 24 mer. In both cases, we
observe that proteasomes from IFN-
-treated cells generate
increased amounts of fragments with hydrophobic COOH
termini (YVSGLEQL is peak 4 in Fig. 6, A and B, and TLWVDPYEV is peak 4 in Fig. 6, C and D) and decreased
amounts of fragments with acidic COOH termini (YVSGLE is peak 2, and YVSGLEQLE is peak 3 in Fig. 6, A
and B; TLWVDPYE is peak 2 in Fig. 6, C and D). This is
in line with general preferences of MHC class I molecules, although it does not necessarily result in improved production of each individual epitope; production of BTG1103-111
(TLWVYPDEV, peak 4 in Fig. 6, C and D) is improved,
whereas production of Ova257-264 (SIINFEKL, contained in
peak 5, theoretical mass: 963.14, in Fig. 6, A and B) is impaired. In spite of these quantitative changes, however, the
same set of major proteolytic fragments is produced by proteasomes isolated from uninduced and from IFN-
-induced cells.
The IFN--inducible PA28 enhancer, existing in two
homologous forms,
and
(25, 36), binds as a ring-like
hexa/heptameric structure to the
endplates of the 20S proteasome (37). PA28-capped 20S proteasomes exhibit enhanced activity towards short fluorogenic model substrates
(26, 38). Here we analyze the effects of recombinant
PA28
on the digestion of the 22-mer OvaY249-269 by 20S
proteasomes (Fig. 7). Addition of PA28
leads to a slightly increased turnover of the substrate and reverses the ratio
between HPLC peak 4 (SIINFEKL and ESIINFEKL) and
HPLC peak 5 (SIINFEKLTEWTS and ESIINFEKLTEWTS). The peptides in peak 4 represent dual cleavage fragments; those in peak 5 are produced by a single cleavage
only. Thus, in the presence of PA28
, the rate of accumulation of dual cleavage peptides is relatively increased, in
line with results recently reported for native PA28 presumably consisting of both
and
isoforms (34). However,
the dual cleavage peptides that are more efficiently produced in the presence of the activator are generated also
in its absence. Together, these results support the notion
that the IFN-
-inducible proteasomal elements, including PA28 as well as the inducible
-type subunits, modify the
20S-proteasome in such a way that the generation of immunocompetent peptides is quantitatively improved, without drastic alterations of the specificity of proteasomal proteolysis.
The work presented in this paper was stimulated by our
observation that each of three proven MHC class I ligands
was found as a major proteolytic fragment upon digestion
of precursor polypeptides with yeast and/or insect proteasomes. One possible way to rationalize this observation was
that the preexisting proteolytic fragments of proteasomes
provided an important evolutionary force in shaping the
peptide binding groove of MHC class I molecules. Such a
mechanism would predict that proteasomal digestion would
be the major source of MHC class I ligands in immunologically competent vertebrates. The evidence in favor of this
notion is in part circumstantial and is a matter of continuing debate. We show that lactacystin, specific proteasome
inhibitor until otherwise demonstrated, inhibits the assembly of about two-thirds of newly synthesized MHC class I
heavy chains with 2m. Inhibition of recognition of a panel of cytotoxic T lymphocyte epitopes by lactacystin has recently been reported (29). Although protease inhibitors
cannot provide definitive proof, the combined data on this
and other (10) proteasome inhibitors suggest that proteasomes are critically involved in the generation of peptides
for assembly with MHC class I. In addition, data from this
and other laboratories (12, 34, 41) suggest that proteasomes
often are involved in the final proteolytic steps of epitope
generation.
A given MHC class I molecule can accommodate large
arrays of different peptides. Typically, peptide specificity is
constrained by two binding pockets, whereas the rest of the
peptide sequence may vary. This is reminiscent of many endoproteases whose active sites can accommodate many different peptide sequences. In many of these enzymes, the primary determinant of substrate specificity is the S1 subsite,
accommodating the P1 residue of the scissile bond P1-P1.
The F pocket of MHC class I molecules accommodates the
side chain of the aa at the COOH terminus of the peptide
with a strong preference for aliphatic, aromatic, and charged
aa. Except for negatively charged residues, the P1 preference of proteasomes closely resembles the preferences of
the F pocket. The F pocket may therefore represent the
structural center of the relationship of MHC class I molecules to the proteasome. Nevertheless, no class I allele has
been found so far whose F pocket prefers peptides with
acidic COOH termini, although these are also efficiently produced by proteasomes of vertebrates and invertebrates.
This may be taken as an indication that MHC class I molecules evolved independently of proteasomes and that their
preference for aliphatic, aromatic, or positively charged
COOH termini has other reasons. However, it is also possible that there was selection against acidic amino acid side
chains in the COOH-terminal ligand position. For example, it is conceivable that an acidic COOH-terminal aa side
chain interferes with the formation of the H-bond system between conserved polar residues of class I and the free
backbone carboxylate of the ligand. During the approach
of a peptide with an acidic COOH-terminal aa side chain,
the side-chain carboxylate, instead of the backbone carboxylate, may become engaged in H-bond formation, preventing proper anchoring of the ligand in the pockets of the
peptide binding groove. Furthermore, peptides with acidic COOH termini may not be correctly handled by other components of the processing and presentation machinery, such
as heat shock proteins (hsp), which have been proposed to
shuttle peptides in the class I pathway (42), or TAP. However, at least human TAP has been shown to translocate
peptides with acidic COOH termini (43).
Our pool-sequencing data strongly support the high degree of conservation among eukaryotes of the cleavage site
usage in polypeptides by 20S proteasomes. Although cleavage efficiencies of individual peptide bonds are not always
identical, the P1 specificities of both vertebrate and invertebrate eukaryotic proteasomes are confined by the same
broad, but well-defined, limits. The same applies to the P1
position where we frequently see small or polar side chains,
particularly in major cleavage sites. For example, in the
BTG1-derived sequence, yeast 20S proteasomes most efficiently hydrolyzed bonds that contain a small and/or polar
P1
residue (L-T, V-S), in addition to a suitable P1 residue.
Similar preferences were seen in OvaY249-269 (E-S, L-T) and
JAK1348-368 (F-S). Statistical analysis of so far eluted MHC
class I ligands revealed an enrichment of small and polar
residues in the NH2-terminal position (most significantly S,
but also G and A) (12). In addition, a recent analysis of
Db-binding ligands has shown a significant enrichment for
S, T, and C in the NH2-terminal position of high affinity
peptides (44). Moreover, small polar peptide NH2 termini
favor hydrogen bond formation in HLA-B27 (5). Also,
TAP is known to have enhanced binding/translocation efficiency for peptides with small or polar residues (45; P. van
Endert, personal communication). We favor the hypothesis
that these structural features of the peptide-binding groove
of MHC class I, and perhaps also of the peptide-binding site of TAP, may represent evolutionary adaptations to
conserved features of proteasomal proteolysis.
A striking property of proteasomes is the defined length of their proteolytic fragments. For archaebacterial proteasomes, peptide length centers around 7 and 8 mers (46). Upon digestion of 22- and 44-mer OVA-derived polypeptides by mouse 20S proteasomes, we have recently observed that the majority of the dual cleavage peptide products were 8-11 mer. Peptides of this length dominated among the dual cleavage products at all time points tested, including early in the time course. Many of the peptides of this length are relatively stable, even upon prolonged digestion by proteasomes (12). Most MHC class I molecules bind 8-10/11-mer peptides (3). Nonamers seem to be preferred by most alleles. In this report, we show that a major proportion of the stable peptides generated by yeast and D. melanogaster proteasomes fall into the size range preferred by MHC class I. Thus, the putative evolutionary relationships between proteasomes and MHC class I may include the length of the peptide-binding groove. In addition, the highly conserved clusters of polar aa at both ends of the peptide binding groove may have evolved to facilitate efficient H-bond formation with the ends of the short peptides produced by proteasomes.
IFN- is a pivotal cytokine in the function of the immune system and the incorporation of IFN-
-inducible elements into the structure of proteasomes is highly suggestive of an evolutionary adaptation to the requirements of
the immune system. One of the IFN-
-inducible
subunits, LMP7, is first detected in the nurseshark (47), i.e., at
the same phylogenetic step as most other elements of the
vertebrate immune system (48). It was therefore possible that MHC molecules evolved independently of proteasomes followed by a unidirectional adaptation of proteasomes to the requirements of MHC class I. On the other
hand, as shown in the present paper, the ability of proteasomes to generate potentially immunocompetent peptides
preceded the evolution of the MHC, suggesting the reverse order of adaptation. We were therefore interested in understanding the extent of functional modification inflicted
upon proteasomes by IFN-
-inducible elements.
Based on experiments with fluorogenic tri- and tetrapeptides, IFN--dependent alterations in the substrate specificity of proteasomes were reported. In the first reports on
this subject, the authors observed that proteasomes isolated
from IFN-
-treated cells gained about twofold higher chymotrypsin-like (Suc-LLVY-MCA-hydrolyzing) and trypsin-like (Boc-LLR-MCA-hydrolyzing) activities (49, 50), but
lost about half of the postglutamyl (Clz-LLE-MNA-hydrolyzing) activity (49). The authors suggested that these functional alterations should favor the degradation of proteins to
peptides that terminate in hydrophobic and basic residues
that are usually found bound to MHC class I. These results
were confirmed by some (51), but not by others (18, 52,
53). Studies using polypeptide substrates have yielded results inconsistent with each other and with that obtained
with fluorogenic substrates (18, 41). Taken together, the functional consequences of the incorporation of IFN-
-inducible
subunits remain incompletely understood.
Using as substrates 22- and 24-mer polypeptide sequences corresponding to natural proteins, we show that
the characteristic P1 specificity spectrum of proteasomes remains qualitatively unchanged in proteasomes isolated from
IFN--treated cells. However, as shown for the first time
with polypeptide substrates, we see enhanced hydrolysis of
individual peptide bonds with hydrophobic P1 residues by
proteasomes of IFN-
-treated cells compared to that of
untreated cells. In addition, we see reduced cleavage of
neighboring peptide bonds with acidic P1 residues. Thus,
the data presented here for polypeptide substrates agree
with that first reported by Gaczynska et al. with short fluorogenic substrates (49). However, together with our results,
the rather mild defects of mice genetically deficient in
LMP2 or LMP7 (54, 55), as well as the restoration of antigen presentation in LMP2/LMP7/TAP triple-deficient T2
cells by transfection with TAP alone (56), argue against
drastic qualitative alterations in the cleavage preferences of
proteasomes by IFN-
-inducible
subunits.
Another IFN--inducible element is the enhancer PA28.
Recently, Dick et al. reported for the degradation of 19-25-mer substrates by 20S proteasomes, a substantially enhanced
rate of accumulation of dual cleavage products by addition
of PA28 (34). Here we show that recombinant PA28
is
sufficient to induce this effect. This is in line with the
recent finding that transfection of PA28
is sufficient to
improve the recognition of virus-infected cells by CTLs
(60). However, with and without PA28, the same cleavage sites are used and the same products are generated. Thus,
the highly conserved general cleavage specificity of the 20S
proteasome remains unchanged in the presence of PA28.
Nevertheless, PA28 might have been evolved to optimize
the capacity of the 20S proteasome for oligopeptide generation, in particular from short substrates.
Evolutionary relationships have been invoked between
MHC class I and the hsp70 family of chaperones (61, 62).
However, recent structural studies (63, 64) indicate that the
homologies between the peptide-binding regions of hsp70
and MHC molecules are less than originally anticipated. Observations suggesting evolutionary links between proteasomes, MHC, TAP, and perhaps hsp70 were recently reported by Kasahara et al. (65) and Katsanis et al. (66). Both
the human and the mouse genomes contain three regions
with striking homology to the MHC complex, as they comprise genes coding for proteasome -type subunits, ABC
transporters, hsp70, NOTCH, and complement components. One of these regions, in addition, harbors the gene
for CD1. They speculate that the MHC complex and these
homologous regions might have been generated by duplication of an ancestral syntenic group in jawless fish, i.e., before emergence of T cell recognition. This implies that MHC class I-like molecules, and perhaps also TAP, may
have existed before the adaptive immune system.
Due to their role in the degradation of unfolded polypeptides, proteasomes are adapted to cleave hydrophobic sequences from the inside of proteins. Most of the abundant self-peptides eluted from MHC class I are derived from highly conserved hydrophobic regions of a restricted set of evolutionary conserved ubiquitous intracellular proteins (67). In view of the possible existence of MHC class I before T cell recognition, it is conceivable that the functional cooperation between proteasomes and the precursor of MHC class I was originally designed to present self-peptides and to serve a purpose other than self-nonself discrimination, for example, inhibition of NK killing. MHC polymorphism and T cell recognition may have evolved subsequently, thus accommodating the greater variety of foreign peptides.
Address correspondence to Dr. K. Eichmann, Max-Planck-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany. Phone: 49-761-51-08-541; FAX: 49-761-51-08-545: E-mail: eichmann{at}immunbio.mpg.de
Received for publication 31 March 1997 and in revised form 19 May 1997.
C. Realini and M.C. Rechsteiner were supported by grants from the American Cancer Society and from The National Institutes of Health.We are grateful to Uli Birsner for help with the peptide synthesis and Erika Hug for technical assistance. We also thank Dr. Michael Fischer and Dr. Dieter Wolf for providing yeast proteasomes for initial experiments and Dr. Klaus Früh for providing antibodies.
1. | York, I.A., and K.L. Rock. 1996. Antigen processing and presentation by the class I major histocompatibility complex. Annu. Rev. Immunol. 14: 369-396 [Medline]. |
2. | Wolf, P.R., and H.L. Ploegh. 1995. How MHC class II molecules acquire peptide cargo: biosynthesis and trafficking through the endocytic pathway. Annu. Rev. Cell Dev. Biol. 11: 267-306 . [Medline] |
3. | Rammensee, H.G., T. Friede, and S. Stefanovic. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics. 41: 178-228 [Medline]. |
4. | Madden, D.R.. 1995. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13: 587-622 [Medline]. |
5. | Madden, D.R., J.C. Gorga, J.L. Strominger, and D.C. Wiley. 1991. The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation. Nature (Lond.). 353: 321-325 [Medline]. |
6. | Coux, O., K. Tanaka, and A.L. Goldberg. 1996. Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65: 801-847 [Medline]. |
7. | Löwe, J., D. Stock, B. Jap, P. Zwickl, W. Baumeister, and R. Huber. 1995. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science (Wash. DC). 268: 533-539 [Medline]. |
8. | Seemüller, E., A. Lupas, D. Stock, J. Löwe, R. Huber, and W. Baumeister. 1995. Proteasome from Thermosplasma acidophilum: a threonine protease. Science (Wash. DC). 268: 579-582 [Medline]. |
9. | Groettrup, M., A. Soza, U. Kuckelhorn, and P.-M. Kloetzel. 1996. Peptide antigen production by the proteasome: complexity provides efficiency. Immunol. Today. 17: 429-435 [Medline]. |
10. | Rock, K.L., C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang, and A.L. Goldberg. 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented by MHC class I molecules. Cell. 78: 761-771 [Medline]. |
11. | Monaco, J.J., and H.O. McDevitt. 1986. The LMP antigens: a stable MHC-controlled multisubunit protein complex. Hum. Immunol. 15: 416-426 [Medline]. |
12. |
Niedermann, G.,
G. King,
S. Butz,
U. Birsner,
R. Grimm,
J. Shabanowitz,
D.F. Hunt, and
K. Eichmann.
1996.
The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I
binding peptides.
Proc. Natl. Acad. Sci. USA.
93:
8572-8577
|
13. | Androlewicz, M.J., and P. Cresswell. 1996. How selective is the transporter associated with antigen processing? Immunity. 5: 1-5 [Medline]. |
14. | Van Endert, P.M.. 1996. Peptide selection for presentation by HLA class I: a role for the human transporter associated with antigen processing. Immunol. Res. 15: 265-279 [Medline]. |
15. | Kaufman, J., J. Salomonsen, and M. Flajnik. 1994. Evolutionary conservation of MHC class I and class II molecules-different yet the same. Semin. Immunol. 6: 411-424 [Medline]. |
16. | Takahashi, T., T. Tokumoto, K. Ihshikawa, and K. Takahashi. 1993. Cleavage specificity and inhibition profile of proteasome isolated from the cytosol of Xenopus oocyte. J. Biochem. (Tokyo). 113: 225-228 [Abstract]. |
17. |
Leibovitz, D.,
Y. Koch,
M. Fridkin,
F. Pitzer,
P. Zwickl,
A. Dantes,
W. Baumeister, and
A. Amsterdam.
1995.
Archaebacterial and eukaryotic proteasomes prefer different sites in
cleaving gonadotropin-releasing hormone.
J. Biol. Chem.
270:
11029-11032
|
18. |
Kuckelhorn, U.,
S. Frentzel,
R. Kraft,
S. Kostka,
M. Groettrup, and
P.M. Kloetzel.
1995.
Incorporation of major histocompatibility complex-encoded subunits LMP2 and LMP7
changes the quality of the 20S proteasome polypeptides processing products independent of interferon-![]() |
19. |
Groettrup, M.,
T. Ruppert,
L. Kuehn,
M. Seeger,
S. Standera,
U. Koszinowski, and
P.M. Kloetzel.
1995.
The interferon-![]() |
20. | Omura, S., T. Fujimoto, K. Otoguro, K. Matsuzaki, R. Moriguchi, H. Tanaka, and Y. Sasaki. 1991. Lactacystin, a novel microbial metabolite, induces neuritogenesis in neuroblastoma cells. J. Antibiot. (Tokyo). 44: 113-116 [Medline]. |
21. | Jones, B., and C.A. Janeway Jr.. 1981. Cooperative interaction of B lymphocytes with antigen-specific helper T lymphocytes is MHC restricted. Nature (Lond.). 292: 547-549 [Medline]. |
22. | Niedermann, G., S. Butz, H.-G. Ihlenfeldt, R. Grimm, M. Lucchiari, H. Hoschützky, G. Jung, B. Maier, and K. Eichmann. 1995. Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. Immunity. 2: 289-299 [Medline]. |
23. | Tamura, T., I. Nagy, A. Lupas, F. Lottspeich, Z. Cejka, G. Schoofs, K. Tanaka, R. De Mot, and W. Baumeister. 1995. The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus. Curr. Biol. 5: 766-774 [Medline]. |
24. | Zwickl, P., F. Lottspeich, and W. Baumeister. 1992. Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli. FEBS Lett. 312: 157-160 [Medline]. |
25. |
Realini, C.,
W. Dubiel,
G. Pratt,
K. Ferrell, and
M. Rechsteiner.
1994.
Molecular cloning and expression of a ![]() |
26. | Ustrell, V., C. Realini, G. Pratt, and M. Rechsteiner. 1995. Human lymphoblast and erythrocyte multicatalytic proteases: differential peptidase activities and responses to the 11S regulator. FEBS Lett. 376: 155-158 [Medline]. |
27. | Fenteany, G., R.F. Standaert, W.S. Lane, S. Choi, E.J. Corey, and S.L. Schreiber. 1995. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science (Wash. DC). 268: 726-731 [Medline]. |
28. |
Yoo, S.J.,
J.H. Seol,
D.H. Shin,
M. Rohrwild,
M.S. Kang,
K. Tanaka,
A.L. Goldberg, and
C.H. Chung.
1996.
Purification
and characterization of the heat shock proteins HslV and
HslU that form a new ATP-dependent protease in Escherichia
coli.
J. Biol. Chem.
271:
14035-14040
|
29. | Cerundolo, V., A. Benham, V. Braud, S. Mukherjee, K. Gould, B. Macino, J. Neefjes, and A. Townsend. 1997. The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells. Eur. J. Immunol. 27: 336-341 [Medline]. |
30. | Rötzschke, O., K. Falk, S. Stevanovic, G. Jung, P. Walden, and H.G. Rammensee. 1991. Exact prediction of a natural T cell epitope. Eur. J. Immunol. 21: 2891-2894 [Medline]. |
31. | Hunt, D.F., R.A. Henderson, J. Shabanowitz, K. Sakaguchi, H. Michel, N. Sevilir, A.L. Cox, E. Appella, and V.H. Engelhard. 1992. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science (Wash. DC). 255: 1261-1263 [Medline]. |
32. | Falk, K., O. Rötzschke, S. Stevanovic, G. Jung, and H.G. Rammensee. 1991. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature (Lond.). 351: 290-296 [Medline]. |
33. | Harpur, A.G., A. Ziemiecki, A.F. Wilks, K. Falk, O. Rötzschke, and H.-G. Rammensee. 1993. A prominent natural H-2Kd ligand is derived from protein-tyrosine kinase JAK1. Immunol. Lett. 35: 235-238 [Medline]. |
34. | Dick, T.P., T. Ruppert, M. Groettrup, P.M. Kloetzel, L. Kuehn, U.H. Koszinowski, S. Stefanovic, H. Schild, and H.-G. Rammensee. 1996. Coordinated dual cleavages induced by the proteasome regulator PA28 lead to dominant MHC ligands. Cell. 86: 253-262 [Medline]. |
35. | Chen, W., S. Khilko, J. Fecondo, D.H. Margulies, and J. McCluskey. 1994. Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues. J. Exp. Med. 180: 1471-1483 [Abstract]. |
36. |
Ahn, J.Y.,
N. Tanahashi,
K. Akiyama,
H. Hisamatsu,
C. Noda,
K. Tanaka,
C.H. Chung,
N. Shibmara,
P.J. Willy,
J.D. Mott, et al
.
1995.
Primary structures of two homologous
subunits of PA28, a ![]() |
37. | Gray, C.W., C.A. Slaughter, and G.N. DeMartino. 1994. PA28 activator protein forms regulatory caps on proteasome stacked rings. J. Mol. Biol. 236: 7-15 [Medline]. |
38. | Yukawa, M., M. Sakon, J. Kambyashi, E. Shiba, T. Kawasaki, H. Ariyoshi, and T. Mori. 1991. Proteasome and its novel endogeneous activator in human platelets. Biochem. Biophys. Res. Commun. 178: 256-260 [Medline]. |
39. |
Dubiel, W.,
G. Pratt,
K. Ferrell, and
M. Rechsteiner.
1992.
Purification of an 11S regulator of the multicatalytic protease.
J. Biol. Chem.
267:
22369-22377
|
40. |
Chu-Ping, M.,
C.A. Slaughter, and
G.N. DeMartino.
1992.
Identification, purification, and characterization of a protein
activator (PA28) of the 20S proteasome (macropain).
J. Biol.
Chem.
267:
10515-10523
|
41. | Ehring, B., T.H. Meyer, C. Eckerskorn, F. Lottspeich, and R. Tampe. 1996. Effects of major histocompatibility complex encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides. Eur. J. Biochem. 235: 404-415 [Abstract]. |
42. | Srivastava, P.K., H. Udono, N.E. Blachere, and Z. Li. 1994. Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics. 39: 93-98 [Medline]. |
43. | Androlewicz, M.J., and P. Cresswell. 1994. Human transporters associated with antigen processing possess a promiscuous peptide-binding site. Immunity. 1: 7-14 [Medline]. |
44. |
Hudrisier, D.,
H. Mazarguil,
F. Laval,
M.B.A. Oldstone, and
J.E. Gairin.
1996.
Binding of viral antigens to major histocompatibility complex class I H-2Db molecules is controlled
by dominant negative elements at peptide non-anchor residues.
J. Biol. Chem.
271:
17829-17836
|
45. | Momburg, F., J. Roelse, J.C. Howard, G.W. Butcher, G.J. Hämmerling, and J.J. Neefjes. 1994. Selectivity of MHC-encoded peptide transporters from human, mouse and rat. Nature (Lond.). 367: 648-651 [Medline]. |
46. | Wenzel, T., C. Eckerskorn, F. Lottspeich, and W. Baumeister. 1994. Existence of a molecular ruler in proteasomes suggested by analysis of degradation products. FEBS Lett. 349: 205-209 [Medline]. |
47. | Kandil, I., C. Namikawa, M. Nonaka, A.S. Greenberg, M.F. Flajnik, T. Ishibashi, and M. Kasahara. 1996. Isolation of low molecular mass polypeptide complementary DNA clones from primitive vertebrates. Implications for the origin of MHC class I-restricted antigen presentation. J. Immunol. 156: 4245-4253 [Abstract]. |
48. | Thompson, C.B.. 1995. New insights into V(D)J recombination and its role in the evolution of the immune system. Immunity. 3: 537-539 . |
49. | Gaczynska, M., K.L. Rock, and A.L. Goldberg. 1993. Gamma-interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature (Lond.). 365: 264-267 [Medline]. |
50. | Driscoll, J., M.G. Brown, D. Finley, and J.J. Monaco. 1993. MHC-linked LMP gene products specifically alter peptidase activities of the proteasome. Nature (Lond.). 365: 262-264 [Medline]. |
51. |
Aki, M.,
N. Shimbara,
M. Takashina,
K. Akiyama,
S. Kagawa,
T. Tamura,
N. Tanahashi,
T. Yoshimura,
K. Tanaka, and
A. Ichihara.
1994.
Interferon-![]() |
52. |
Boes, B.,
H. Hengel,
T. Ruppert,
G. Multhaup,
U.H. Koszinowski, and
P.M. Kloetzel.
1994.
Interferon ![]() |
53. | Ustrell, V., G. Pratt, and M. Rechsteiner. 1995. Effects of interferon gamma and major histocompatibility complex-encoded subunits on peptidase activities of human multicatalytic proteases. Proc. Natl. Acad. Sci. USA. 92: 584-588 [Abstract]. |
54. | Van Kaer, L., P.G. Ashton-Rickardt, M. Eichelberger, M. Gaczynska, K. Nagashima, K.L. Rock, A.L. Goldberg, P.C. Doherty, and S. Tonegawa. 1994. Altered peptidase and viral-specific T cell response in LMP2 mutant mice. Immunity. 1: 533-541 [Medline]. |
55. | Fehling, H.J., W. Swat, C. Laplace, R. Kühn, K. Rajewsky, U. Müller, and H. von Boehmer. 1994. MHC class I expression in mice lacking the proteasome subunit LMP 7. Science (Wash. DC). 265: 1234-1237 [Medline]. |
56. | Arnold, D., J. Driscoll, M. Androlewicz, E. Hughes, P. Cresswell, and T. Spies. 1992. Proteasome subunits encoded in the MHC are not generally required for the processing of peptides bound by MHC class I molecules. Nature (Lond.). 360: 171-174 [Medline]. |
57. | Momburg, F., V. Ortiz-Navarrete, J. Neefjes, E. Goulmy, Y. van de Wal, H. Spits, S.J. Powis, G.W. Butcher, J.C. Howard, P. Walden, and G.J. Hämmerling. 1992. Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation. Nature (Lond.). 360: 174-177 [Medline]. |
58. |
Yewdell, J.,
C. Lapham,
I. Bacik,
T. Spies, and
J. Bennink.
1994.
MHC encoded proteasome subunits LMP2 and LMP7
are not required for efficient antigen presentation.
J. Immunol.
152:
1163-1170
|
59. | Zhou, X., F. Momburg, T. Liu, U.M. Abdel, Motal, M. Jondal, G.J. Hämmerling, and H.G. Ljunggren. 1994. Presentation of viral antigens restricted by H-2Kb, Db, or Kd in proteasome subunit LMP2- and LMP7-deficient cells. Eur. J. Immunol. 24: 1863-1868 [Medline]. |
60. |
Groettrup, M.,
A. Soza,
M. Eggers,
L. Kuehn,
T.P. Dick,
H. Schild,
H.-G. Rammensee,
U.H. Koszinowski, and
P.-M. Kloetzel.
1996.
A role for the proteasome regulator PA28![]() |
61. | Flajnik, M.F., C. Canel, J. Kramer, and M. Kasahara. 1991. Which came first, MHC class I or class II? Immunogenetics. 33: 295-300 . |
62. | Rippmann, F., W.R. Taylor, J.B. Rothbard, and N.M. Green. 1991. A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA. EMBO (Eur. Mol. Biol. Organ.) J. 10: 1053-1059 [Abstract]. |
63. | Morshauser, R.C., H. Wang, G.C. Flynn, and E.R.P. Zuiderweg. 1995. The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology. Biochemistry. 34: 6261-6266 [Medline]. |
64. | Zhu, X., X. Zhao, W.F. Burkholder, A. Gragerov, C.M. Ogata, M.E. Gottesmann, and W.A. Hendrickson. 1996. Structural analysis of substrate binding by the molecular chaperone DnaK. Science (Wash. DC). 272: 1606-1614 [Abstract]. |
65. | Kasahara, M., J. Nakaya, Y. Satta, and N. Takahata. 1997. Chromosomal duplication and the emergence of the adaptive immune system. TIG (Trends Genet.). 13: 90-92 . |
66. | Katsanis, N., J. Fitzgibbon, and E.M.C. Fisher. 1996. Paralogy mapping: identification of a region in the human MHC triplicated onto human chromosomes 1 and 9 allows the prediction and isolation of novel PBX and NOTCH loci. Genomics. 35: 101-108 [Medline]. |
67. | Hughes, A.L., and M.K. Hughes. 1995. Self peptides bound by HLA class I molecules are derived from highly conserved regions of a set of evolutionary conserved proteins. Immunogenetics. 41: 257-262 [Medline]. |