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*
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655; and
Department of Cell Biology, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our understanding of how antigenic peptides are generated remains incomplete. The active form of the proteasome, which appears to degrade most cellular proteins, is the 26S proteasome (3). This complex is formed by the association of the 19S regulatory complex with each end of the core 20S proteasome. This 2-MDa structure degrades ubiquitinated and some nonubiquitinated proteins in an ATP-dependent manner (2). Proteins are cleaved within the 20S (700 kDa) core proteasome, which is composed of four stacked rings. The two inner ß-rings contain six proteolytic sites, which differ in substrate specificity; two have chymotrysin-like specificity, two trypsin-like, and two postacidic-cleaving activities (2, 3). These active sites all have a novel proteolytic mechanism, in which the active nucleophile is the hydroxyl group of the amino terminal threonine residue on several of the ß-subunits (2, 3). Proteasome inhibitors have been described that interfere with this catalytic mechanism (3). The most selective inhibitor is the antibiotic lactacystin and its active derivative, ß-lactone, which acylates the amino terminal threonine residues of the ß-subunits and thereby prevent catalytic activity (6, 7).
It is now well established that the ubiquitin-proteasome pathway is
involved in the generation of the majority of class I-presented
peptides (3, 8). For example, proteasome inhibitors, such
as lactacystin or peptide aldehydes, can block the processing and
presentation of specific antigenic peptides and markedly reduce the
overall supply of peptides to class I molecules (8, 9, 10, 11, 12).
In addition, genes encoding the alternative proteasome ß-subunits,
LMP-2 and LMP-7, are encoded in the MHC-gene region (13, 14), and cell lines or mice that lack these subunits have
defects in Ag presentation (15, 16, 17, 18). On the other hand,
IFN-
, which promotes Ag presentation (3),
causes induction of these MHC-encoded subunits and thus alters
proteasomal peptidase activities, apparently to favor generation of
peptides with C termini appropriate for MHC binding (19, 20). Furthermore, in mutant cell lines with a
temperature-sensitive ubiquitin-activating enzyme (E1), the
inactivation of ubiquitin conjugation at the nonpermissive temperature
blocks generation of antigenic peptides from the OVA (21, 22). However, because the presentation of some antigenic
peptides is not reduced by proteasome inhibitors, other proteases might
also produce some class I-presented peptides (23, 24, 25).
In those cases where the proteasome clearly is essential for the generation of class I-presented peptides, it is uncertain whether it makes the final cleavages that yield the presented peptides or if other proteases are needed to trim further the proteasomal products. To bind to class I molecules with high affinity, peptides must be of a precise length, either 8, 9, or 10 residues, depending on the class I molecule (26, 27). The peptides produced by purified 20S and 26S proteasomes during protein degradation range in size from 4 to 24 residues, but two-thirds are too short to serve in Ag presentation (<8 residues) (28, 29). However, most such studies with isolated proteasomes have used highly unphysiological conditions, and therefore it is uncertain whether such results can be extrapolated to the in vivo situation (3).
We have recently shown in intact cells that proteasomes were
responsible for generating the correct C terminus of SIINFEKL from a
longer peptide, but other protease might generate its proper N terminus
(30). Furthermore, in cell extracts, aminopeptidases,
including the IFN--inducible enzyme (31), leucine
aminopeptidase, can trim N-terminally extended versions of SIINFEKL to
generate the correct octomer (32). In an effort to define
the proteolytic activities that make the final carboxyl- and
amino-terminal cleavages that generate natural viral antigenic
peptides, we have extended our studies to the
Db-restricted influenza peptide nucleoprotein
(NP) 366–374 (ASNENMETM) and the Kb-restricted
Sendai NP324–332 (FAPGNYPAL) peptides. By
expressing N- and C-terminally extended versions of these peptides, we
have tested whether proteasomes function directly in these final steps
in the production of antigenic peptides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vaccinia constructs containing the full-length sequences for NP of influenza virus A/Puerto Rico/8/34 (PR8) strain (V-FluNP) (a kind gift from Dr. J. W. Yewdell, National Institute of Allergy and Infectious Diseases, Bethesda, MD) or NP of Sendai virus (V-SVNP) (provided by Dr. D. Kolakofsky, University of Geneva School of Medicine, Switzerland), or T7 RNA polymerase (vTF7-3) (obtained from American Type Culture Collection, Manassas, VA) (VR-2153) were all propagated in thymidine kinase-deficient human 143BTK- osteosarcoma cells (CRL 8303; American Type Culture Collection).
Cell lines and hybridomas
E36.17.3 (E36/Db) and E36.12.4 (E36/Kb) APC lines used in this study were derived from E36 cells (hamster lung carcinoma cells), which were stably transfected with murine H-2Db or H-2Kb, and ICAM-1 molecules (21) and maintained in RPMI 1640 medium (10% FCS) with G418. Another APC, LB27.4 B, (33) is a lymphoblastoid-presenting cell line, expressing H-2Kb molecules, and was grown in OptiMem (Life Technologies, Grand Island, NY) with 1% normal mouse serum. T cell hybridomas 12.33, specific for Db-restricted NP366–374 ASNENMETM of PR 8, and B3.4D8, specific for Kb-restricted NP324–332 FAPGNYPAL of Sendai virus, were kind gifts from Dr. D. Woodland (St. Jude Children’s Research Hospital, Memphis, TN) (34, 35). All the above cell lines (except LB27.4 B), and the human cervical carcinoma cell line HeLa S3, were maintained in RPMI 1640 medium with 10% FCS (Atlanta Biologicals, Norcross, GA). The T cell Hybridoma RF33.70 specific for OVA257–264 was described previously (36).
Reagents
The proteasome inhibitor lactacystin was obtained from Dr. E.J. Corey (Harvard University, Cambridge, MA), and ß-lactone was a kind gift from Dr. Julian Adams (ProScript, Cambridge, MA). The proteasome inhibitors were dissolved at 10 µM in DMSO and stored at -80°C.
The peptides VQIASNENMETM (influenza
NP363–374), HGEFAPGNYPAL (Sendai
NP321–332), QLESIINFEKL
(OVA254–264), and their N-terminal acetylated
counterparts (Table I
) were synthesized
and purified by HPLC by the Peptide Facility at the University of
Massachusetts Medical School (Worcester, MA).
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Synthetic minigenes encoding the antigenic peptides from
influenza virus NP366–372 (ASNENMETM) or Sendai
NP324–332 (FAPGNYPAL) with or without natural N-
or C-terminal flanking residues were constructed and cloned into
pBluscript SK under the control of the T7 RNA promoter as follows.
Coding oligonucleotides were designed to consist of a Kozak consensus
sequence, a start codon, the relevant DNA sequences encoding the
oligopeptide (see Table I
) followed by two stop codons, and an internal
NcoI site (to facilitate the identification of recombinant
plasmids). In addition, these oligonucleotides were flanked at both
ends by EcoRI and HindIII sites for cloning into
the multiple cloning site of the pBluscript SK plasmid. The
oligonucleotides were phosphorylated with T4 polynucleotide kinase,
annealed, and ligated into pBluscript SK. The recombinant plasmids were
transformed into DH5
(Life Science Biolab, Gaithersburg, MD).
Clonies were picked and plasmids isolated using Wizard plus minipreps
kit (Promega, Madison, WI). The correct plasmid was identified by the
presence of NcoI site and sequenced. All the enzymes used in
these constructions were purchased from New England Biolabs
(Beverly, MA).
Ag presentation assay
When Ags were expressed by recombinant vaccinia virus infection,
the APCs (E36/Db or E36/Kb
cells) were first seeded onto a 6-well plate and incubated for 24
h. They were then infected with vaccinia virus V-FluNP or V-SVNP at a
multiplicity of infection of 10 for 5 h at 37°C, after which
time they were harvested and fixed. Alternatively, when Ags were
expressed from transfected plasmids, E36 APCs were first infected with
vTF7-3 (multiplicity of infection of 10) for 30 min in OptiMem medium.
The vTF7-3-containing media was then removed and replaced with various
plasmids that had been incubated with liposomes (lipofectin; Life
Technologies, Grand Island, NY) in OptiMem medium. E36 cells were then
incubated at 37°C for 5 h before fixation. When Ags were
introduced into the cytosol by electroporation, LB27.4 or E36Db cells
were permeablized in electroporation buffer (0.4 M mannitol, 10 mM
HEPES in PBS) as described (8) in the presence of N
terminus-modified or unmodified peptides (0.2–40 µg) (Table I).
Cells were then either fixed immediately after electroporation or after
a further 2 h incubation at 37°C.
For fixation, APCs were incubated with 0.0025% glutaldehyde (Sigma, St. Louis, MO) for at least 10 min at room temperature. Cells were then washed with PBS three times before being plated onto 96-well plate with T cell hybridomas (1 x 105/well). T cell responses were evaluated by their production of IL-2 in the supernatant using the CTLL assay (37).
In experiments using proteasome inhibitors, E36 cells were pre-incubated with specified concentrations of proteasome inhibitors for 30 min before viral infection, and inhibitors were continually present in the culture medium during all subsequent incubation before fixation.
In vitro degradation of oligopeptides
Cytosolic extracts were prepared from HeLa S3 cells as
previously described (32). Briefly, cells were homogenized
with glass beads, and cytosolic extracts were prepared by
centrifugation of the homogenates for 20 min at 10,000 x
g and 1 h at 100,000 x g and for an
additional 6-h centrifugation at 100,000 x g for
removal of proteasomes. The residual proteasome activity in these
extracts was <15%, and this was completely inhibited by the addition
of the proteasome inhibitor MG132 (100 µM) for 15 min at room
temperature. Next, 15 nmol of various oligopeptide substrates (Table I)
were incubated with 2 µg of the proteasome-free cell extracts or with
0.5 µg of leucine aminopeptidase (Sigma) for 30 min at 37°C in 50
mM Tris-HCl buffer, pH 8.5. Reactions were terminated by adding 0.4%
TCA and incubated for 15 min on ice. The peptide-containing supernatant
was then subjected to reverse-phase HPLC on a 4.6 x 250-mm Vydac
C18 column (Vydac, Hesperia, CA) in 0.06% TCA with a flow rate of 1
ml/min. Elution was performed with 30 min linear gradient from 4 to
48% acetonitrile, and the eluting peptides were detected by measuring
absorbance at 214 nm. The relative concentrations for each eluted
peptide were calculated by integration of the peptide peaks on the
chromatograms. To study the effects of bestatin, the extracts were
preincubated with the inhibitor for 30 min at room temperature.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether the generation of the antigenic peptide
ASNENMETM requires the proteasome, vaccinia constructs that encoded
full-length NP of influenza A Puerto Rico/8/34 strain were used to
express the antigenic protein in E36/Db APCs in the presence or absence
of the proteasome inhibitor, lactacystin. Peptide presentation was
assayed by measuring the responses of a T hybridoma specific for
influenza NP366–374 (ASNENMETM) presented on
Db molecules. As shown in Fig. 1A, the presentation of this
influenza NP-derived epitope was completely inhibited by 2 µM
lactacystin. Similarly, the presentation of NP in cells infected with
influenza virus was also inhibited by 2 µM lactacystin (data not
shown). In contrast, the presentation of the antigenic peptide
ASNENMETM expressed from a minigene, which does not require further
proteolytic cleavage, was not significantly affected by this proteasome
inhibitor (Fig. 1B).
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C). In contrast, the presentation of the
antigenic peptide, FAPGNYPAL, expressed from a minigene was not
inhibited under the same conditions (Fig. 1D). The failure of lactacystin to block the presentation of the antigenic peptides from minigenes was anticipated, because the encoded oligopeptides do not require further proteolytic cleavage. Similar findings with other antigenic peptides and other proteasome inhibitors have been reported previously (8, 10, 24). Moreover, this result demonstrates that the proteasome inhibitor does not affect other steps in the Ag presentation pathway. Therefore, the ability of lactacystin to prevent presentation of influenza NP-derived ASNENMETM and Sendai NP-derived FAPGNYPAL from full-length proteins indicates that the proteasome is required to generate these presented peptides. However, this result does not resolve whether the proteasome directly produces the 9-residue presented peptide or whether other proteases may also be participating in this process, perhaps after the proteasome-dependent step.
Proteasomes are required for cleavage at the C termini of antigenic peptides
To examine whether the proteasome was responsible for the
cleavages that yield the C terminus of the influenza-derived peptide,
we tested whether ASNENMETM presentation from a construct with a
C-terminal extension could be blocked by a proteasome inhibitor. A
plasmid containing a minigene-encoding ASNENMETM plus the 5 aa that
normally flank its C terminus (p.ASNENMETM + 5) was constructed under
the control of a T7 promoter (Table I). This construct was expressed by
transfection into APCs that were infected with a vaccinnia
recombinant-encoding T7 polymerase (vTF7-3). The presentation of the
extended oligopeptide was then studied in the presence or absence of a
proteasome inhibitor.
As shown in Fig. 2A, ASNENMETM
could be presented on MHC class I molecules from the C-terminally
extended construct, ASNENMETM + 5. This presentation was inhibited in a
dose-dependent manner by the proteasome inhibitor, ß-lactone,
although much higher concentrations of this agent were required than
for the full-length construct. For example, the presentation was not
inhibited by ß-lactone at 9 µM, but was partially blocked at 18
µM and completely inhibited at 36 µM. This effect of the inhibitor
on the presentation of ASNENMETM + 5 was specific, because ß-lactone
did not block the presentation of the expressed minimal antigenic
peptide, ASNENMETM, at any of the concentrations tested (Fig. 2B). Therefore, the proteasome is required to cleave within
the five carboxyl-terminal flanking residues of the ASNENMETM
construct.
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). Then the relative extent of presentation of ASNENMETM from these
different expressed peptides was examined. As shown in Fig. 3, Ag presentation from ASNENMETM + 3
(Fig. 3C) and ASNENMETM + 4 (Fig. 3D) was
apparent even when only 0.35 µg of the plasmids were transfected, and
the presentation increased with greater amounts of plasmid in a
dose-dependent manner. Surprisingly, no presentation occurred with
ASNENMETM + 1 (Fig. 3A) and ASNENMETM + 2 (Fig. 3B), even when the dose of plasmids used was increased to
8.1 µg or when the length of time after the transfection was
increased to 24 h (data not shown). This result was confirmed with
other recombinant clones made independently and with different
preparations of plasmids. Because presentation was observed with
ASNENMETM and ASNENMETM + 3, but not for ASNENMETM + 1 or ASNENMETM +
2, the removal of the P1' and P2' residues cannot occur readily in the
cytosol by carboxypeptidase trimming. This result is in accord with our
prior failure to find cytosolic carboxypeptidase activity
(32). Instead, these C-terminal cleavages must involve an
endopeptidase, perhaps the proteasome, whose activity required
minimally the presence of the P3' residue. Presumably, the protease
that makes the correct cleavage to generate ASNENMETM can only cleave
off the C-terminal residues if the extended C-terminal sequence is
sufficiently long or contains a specific residue in the P3'
position.
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, Ag
presentation from ASNENMETM + 3 (Fig. 4A) was blocked by
ß-lactone at 30 µM. Thus, the proteasome was responsible for the
removal of C-terminal tripeptide from ASNENMETM and there was lack of
other activities that could efficiently remove these residues.
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B). Therefore, the proteasome was
required to remove the C-terminal residue to generate the FAPGNYPL
peptide. Proteasome inhibitors do not block the N-terminal trimming of extended peptides
To examine the possible involvement of the proteasome in making
the cleavages that define the amino termini of the presented peptides,
we studied the effect of proteasome inhibitors on the presentation of
constructs with N-terminal flanking residues. When we expressed a
construct encoding the influenza NP epitope ASNENMETM plus a
five-residue extension at its amino terminus (5 + ASNENMETM),
corresponding to the sequence in influenza NP, ASNENMETM was
efficiently presented on Db. In contrast to the
C-terminally extended peptide, Ag presentation from this N-terminally
extended form of ASNENMETM was not blocked by ß-lactone (Fig. 2C) at a concentration that did block presentation from the
NP protein (Fig. 1A) and from the C-terminally extended
constructs (Fig. 2A) in these cells. Moreover, the
presentation of 5 + ASNENMETM was also resistant to the highest
concentrations of ß-lactone tested (72 µM, data not shown). A
similar resistance to the proteasomal inhibitors was observed when a
N-terminally extended version of the Sendai NP epitope, FAPGNYPAL, was
expressed from a minigene. This peptide was efficiently presented from
a 5 + FAPGNYPAL construct on Kb, and its
presentation was not inhibited by concentrations of ß-lactone that
block the presentation of the C-terminally extended construct,
FAPGNYPAL + 1 (Fig. 4C). In fact, the presentation of 5 +
FAPGNYPAL was actually enhanced by ß-lactone (Fig. 4), as has been
described by other research groups (23, 24, 38). These
data indicate that the trimming of N-terminal flanking residues is by a
protease that is resistant to the proteasome inhibitor.
N-terminally extended oligopeptides are trimmed by aminopeptidases in cell extracts
The finding that Ag presentation from N-terminally extended
constructs was not blocked by the proteasome inhibitor (Figs. 2C and 4C) suggested that other cellular
proteases could remove these flanking residues. We previously showed
that aminopeptidases could trim amino-terminal flanking residues from
an extended OVA-derived construct, QLESIINFEKL, and we
identified the major aminopeptidase responsible for this activity in
HeLa cells as leucine aminopeptidase (32). Therefore, we
further examined whether the N-terminally extended influenza and Sendai
NP peptides could also be trimmed by cellular aminopeptidases. For
these experiments, we synthesized oligopeptides corresponding to the
influenza (VQIASNENMETM), Sendai
(HGEFAPGNYPAL), and OVA (QLESIINFEKL) epitopes
with three naturally occurring N-terminal flanking residues. When
incubated with purified leucine aminopeptidase, all three peptides were
rapidly degraded (Table II). Similarly,
when these extended peptides were incubated with HeLa cell extracts
that were depleted of proteasomes, the peptides were degraded. This
process was inhibited to a significant (but varying) extent by the
aminopeptidase inhibitor, bestatin. Thus, cytosolic aminopeptidases can
trim the N-terminal flanking residues on all three antigenic peptides
studied. Although bestatin inhibits almost completely the hydrolysis of
simple amino acid-AMC substrates by leucine aminopeptidase, this agent
inhibits only partially (by about 50%) the trimming of an 11-mer
peptide by this enzyme (32), presumably because the longer
peptide has a much higher affinity for the enzyme. Therefore, the
bestatin-resistant degradation of these oligopeptides in the cytosolic
extracts may represent incomplete inhibition of the aminopeptidases.
Alternatively, these substrates may be degraded by other types of
peptidases which are not sensitive to bestatin.
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-amino group. Therefore, to test whether the extended antigenic
peptides were being degraded by such enzymes, N-terminally extended
oliogopeptides were synthesized with an acetylated N-terminal residue
(Table I). As shown in Table II, the acetylated peptides were
completely resistant to digestion by purified leucine aminopeptidase
(Table II) and were not hydrolyzed by the bestatin-sensitive component
in cytosolic extracts. These results indicate that additional cellular
aminopeptidases were able to trim these N-terminally extended peptides
in vitro, and the acetyl-group modification at the N terminus blocked
this process. Ag presentation of oligopeptides with blocked N termini is impaired
Because acetylation of the -amino group of the N-terminally
extended ligopeptides blocked their digestion by aminopeptidases in
vitro, we investigated whether this modification would affect their
presentation in vivo. The blocked and unmodified N-terminally extended
oligopeptides (Table I) were introduced into the cytosol of APCs by
electroporation, and their presentation was evaluated using the T cell
hybridoma assay. Immediately after electroporation, none of the
constructs were presented, indicating that peptides were not binding
directly to class I molecules on the cell surface (Fig. 5, backgrounds). However, after a 2-h
incubation, peptides were presented from all three unmodified
constructs, VQIASNENMETM, HGEFAPGNYPAL, and
QLESIINFEKL (Fig. 5). In contrast, presentation of
acetyl-VQIASNENMETM was not detectable when 3 µg of
peptide was used (Fig. 5A) and remained undetectable even
when a larger amount of the peptide was used (Fig. 5B).
Similarly, acetylation of the N termini of HGEFAPGNYPAL and
QLESIINFEKL markedly inhibited their presentation (Fig. 5, C–F). The results strongly suggest that amino-peptidases
play a major role in the trimming of N termini flanking sequences of
these antigenic peptides in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More specifically, the proteasome was shown to be necessary for the generation of the C termini of ASNENMETM and FAPGNYPAL. Because proteasome inhibitors block completely the presentation of these peptides from the C-terminal extended versions, it appears that mammalian cells lack other proteases, such as carboxypeptidases, which can make the correct C-terminal cleavage necessary to generate presented peptides. Accordingly, in HeLa extracts, we failed to demonstrate any carboxypeptidase activity capable of attacking the OVA-derived peptide, SIINFEKL, or model fluorogenic peptides (32). Presumably, it is advantageous to mammalian cells because appreciable carboxypeptidase activity against such oligopeptides might remove the hydrophobic or basic C-terminal residues that are essential for peptide binding to MHC class I molecules and for transport into the ER by TAP.
In sharp contrast to these findings with the C-terminal extended
peptides, the trimming of N-terminal flanking residues was completely
resistant to proteasome inhibitors. Mammalian cells do contain multiple
aminopeptidases (41), such as leucine aminopeptidase
(42), which can sequentially remove the N-terminal
residues from N-extended precursors (32). Therefore,
leucine aminopeptidase or some other aminopeptidase(s) is likely to
catalyze the trimming of these extended precursor peptides to generate
the presented epitopes in vivo. This conclusion is also supported by
the finding that IFN-, which promotes Ag presentation, induces
leucine aminopeptidase (32), as well as those novel
proteasomal subunits (LMP-2, LMP-7, etc.) that alter the particle’s
peptidase activities so as to potentially favor the production of
peptides with C termini appropriate for MHC class I binding (19, 20). Thus, two distinct proteolytic processes can generate the C
and N termini of the antigenic peptides of ASNENMETM and FAPGNYPAL, as
was found previously for SIINFEKL (30) and for the
vesicular stomatitis virus NP-derived peptide RGYVYQGL
(NP52–59) (40). Because similar
results were obtained with four unrelated antigenic peptides, it seems
very likely that the C termini of presented peptides are generally
determined by proteasomal cleavages, while the N terminus can be
generated by leucine aminopeptidase or some other cytosolic
aminopeptidase.
Several studies have reported that the presentation of certain Ags is not blocked or is only partially reduced by proteasome inhibitors (23, 24, 25), including influenza NP55–57 and NP147–155 peptides (43), Kd-restricted epitopes of NP, hemagglutinin, or PB1 proteins of influenza PR8 virus (24), and a human class I-associated peptide of influenza M1 protein (38). While these findings may indicate that other cellular proteases can generate class I-presented peptides, very high concentrations of peptide aldehydes (8) or lactacystin-ß-lactone (10) are needed to completely inhibit intracellular protein breakdown. In this report, we have found examples where high concentrations of the ß-lactone are needed to block the presentation of some extended peptides. Therefore, some caution is needed in interpreting experiments, in which a proteasome inhibitor failed to block Ag presentation, especially when only low or moderate doses of the inhibitors were used, and where only certain of the proteasome’s active sites may be inhibited (see below). In such studies, it is important that the actual extent of inhibition of protein breakdown or of proteasomal activity be measured in the cells.
The concentrations of the proteasome inhibitors needed to block Ag
presentation differed widely with these different antigenic constructs.
A much lower concentration of the ß-lactone was needed to inhibit the
presentation from full-length NPs (Fig. 1) than from the corresponding
C-terminally extended oligopeptide constructs (Fig. 2). For example,
5 µM of lactacystin (Fig. 1) or clasto-lactacystin-ß-lactone
(data not shown) was sufficient to inhibit markedly the presentation of
ASNENMETM and FAPGNYPAL from full-length proteins, whereas 
20 µM of
inhibitor was needed for the short constructs. These differences are
not surprising, because the degradation of proteins is highly
processive (44), involving many peptide-bond cleavages,
and partial inhibition of these individual proteolytic cleavage steps
should have additive effects in reducing the breakdown of the protein.
In contrast, to generate the MHC-presented peptide from these short
constructs, the proteasome needs to make only one or at most a couple
of cleavages. It is also noteworthy that the concentration of
clasto-lactacystin-ß-lactone needed to block the C-terminal cleavage
of ASNENMETM- or FAPGNYPAL-extended peptides was much higher (Figs. 2 and 4) than for SIINFEKL-extended peptides (2–5 µM)
(30). Most likely, these differences may be due to these
constructs being cleaved by different active sites in the proteasome,
which differ appreciably in sensitivity to these inhibitors. For
example, the "chymotryptic site" is particularly sensitive to
lactacystin and ß-lactone, while the "BrAAP" activity and the
peptidylglutamyl peptide hydrolyzing activity are quite resistant to
these inhibitors (6, 45). Thus, a relative resistance to
lactacystin or the ß-lactone may well indicate the involvement of
different active sites of the proteasome. The earlier findings that
high concentrations of lactacystin (>20 µM) are required to
maximally inhibit protein degradation in intact cells are consistent
with this interpretation (10).
In those examples where the proteasome is required for Ag presentation, an important issue is whether the proteasome generates peptides of the correct size or extended precursors that must be trimmed by other enzymes. Mammalian proteasomes degrade polypeptides to oligopeptides that range in length from 4 to 24 residues, although about 70% of these peptides are shorter than 8 residues and cannot function in Ag presentation (29). Approximately 10% of these peptides contain 8–10 residues, the precise length required to bind with high affinity to class I molecules. Although purified proteasomes can make the precise cuts in the full-length OVA or ß-galactosidase proteins (39, 46) or in extended synthetic oligopeptides (47) to generate the correct presented peptides, these studies have used highly unphysiological conditions (e.g., incubation for 24 h and activated 20S proteasome), and therefore it is uncertain whether these results can be extrapolated to in vivo situations. By contrast, our approach, using minigene constructs and proteasome inhibitors, has allowed us to characterize where proteasomes cleave substrates in intact cells and to establish that in vivo proteasomes are essential to generate the correct C termini of many (and presumably most) antigenic peptides, but not their N termini.
The specificity of cleavage by proteasomes has been assumed to be
determined by sequence preceding the scissile bond (2).
One unexpected finding with these extended constructs was the marked
influence of certain P' residues on Ag presentation. Although SIINFEKL
+ 1 (30) and FAPGNYPAL + 1 (Fig. 4) constructs were
presented readily, ASNENMETM with one or two additional C-terminal
residues failed to be presented, while ASNENMETM with longer C-terminal
extensions could be presented. A similar observation has been reported
for another influenza NP epitope (NP147–155),
whose presentation from extended minigene constructs also required
three residues of carboxyl-terminal flanking residues (surprisingly,
the presentation of that peptide was not found to be inhibited by
proteasome inhibitors, although it is possible that the inhibitor was
not used at high-enough concentrations to block proteasome function).
This surprising influence of the P3' position on presentation may
indicate that one of the proteasome’s active sites must bind at least
three downstream residues for activity. Alternatively, it is possible
the constructs with additional residues at only the P2' and/or P1'
positions may be much more susceptible to destruction by other
cytosolic peptidases.
In all cases studied, Ag presentation from the N-terminally extended
constructs was not affected by the proteasome inhibitors. These results
with N-terminally extended ASNENMETM or FAPGNYPAL were almost identical
with earlier ones with N-extended SIINFEKL (30) and
RGYVYQGL (40). In all cases, a proteolytic activity that
is resistant to ß-lactone seems to trim the N termini of extended
peptides. It is unlikely that this trimming process is mediated by the
proteasome’s peptidylglutamyl peptide hydrolyzing activity site, whose
inhibition requires high concentrations of ß-lactone
(6). This latter site tends to prefer acidic residues in
the P1 position of model peptides, which is not present in the
N-extended ASNENMETM and RGYVYQGL (40). Moreover, our
biochemical studies have identified bestatin-sensitive cytosolic
aminopeptidases in cells that can remove these N-terminal flanking
residues (32). However, the most definitive evidence was
the finding that acetylation of the N-terminal residues of three
different extended peptides (Fig. 5), which blocks trimming by
aminopeptidases, also inhibits Ag presentation.
The N-terminal trimming process by aminopeptidases probably can occur
both in the cytosol and the ER. The first evidence for trimming in the
cytoplasm came from the analysis of antigenic constructs that appear
almost too long (25 + SIINFEKL, 33 residues) to be transported into the
ER without removal of the N-terminal flanking region. Yet the
presentation of this constructs was not blocked by proteasome
inhibitors (30), suggesting a nonproteasomal activity in
the cytosol. More direct evidence comes from the demonstration that the
cytosolic extracts contain aminopeptidases that can remove these
N-terminal flanking residues. However, this trimming process can also
occur in the ER, because N-terminally extended constructs that are
targeted into the ER via a signal sequence can still be trimmed and
presented (30, 48, 49). Because TAP is unable to
efficiently transport peptides with blocked N termini (5, 50), it is possible that the impaired presentation of our
acetylated constructs (Fig. 5) was due in part to their failure to be
transported into the ER. However, even if some trimming occurred in the
ER, it would not alter our conclusion that the N-terminally extended
sequence is removed by a nonproteasomal activity.
Our data certainly do not exclude the possibility that the proteasome also sometimes makes the cleavages that generate the proper N termini of presented peptides. When this happens, proteasome inhibitors might not block presentation because trimming is not rate-limiting (i.e., aminopeptidases are sufficient to catalyze this process) or the proteasome generates relatively few of the correct peptides. In vitro experiments with pure 20S proteasomes (47), although highly artificial, suggest that the proteasome can make the correct cleavages, and a "2-cut" model has been proposed to account for such behavior (51, 52). In addition, the isolated proteasomes also generate peptides that were too long for presentation and would require trimming for presentation (28). Presumably, the ability of the proteasome to generate either directly the presented peptide or N-extended versions depends on the nature of the N-terminal flanking sequence and their susceptibility to active sites of the proteasomes or the "immunoproteasome" variants. Therefore, it remains to be established with different proteins to what extent 26S proteasomes under in vivo conditions actually do generate N-extended precursors, which are then trimmed by cellular aminopeptidases to the presented epitopes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenneth Rock, Department of Pathology, Room S2-109, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; NP, nucleoprotein.
Received for publication June 9, 1999. Accepted for publication September 14, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation. J. Immunol. 159:5896.[Abstract]
: identification by protein microsequencing after purification by preparative two-dimensional gel electrophoresis. J. Biol. Chem. 267:6865. can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J. Biol. Chem. 273:18734. in antigen presentation. Nature 381:166.[Medline]
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