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*
Institute of Biochemistry, Charité, Humboldt University, Berlin, Germany; and
Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible proteasome subunits low molecular mass protein 2
(LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing
and concomitantly that of immunoproteasomes, we established the
tetracycline-regulated mouse cell line MEC217, allowing the titrable
formation of immunoproteasomes. Infection of MEC217 cells with
Adenovirus type 5 (Ad5) and analysis of Ag presentation with
Ad5-specific CTL showed that cells containing immunoproteasomes
processed the viral early 1B protein (E1B)-derived epitope
E1B192–200 with increased efficiency, thus allowing a
faster detection of viral entry in induced cells. Importantly, optimal
CTL activation was already achieved at submaximal immunosubunit
expression. In contrast, digestion of E1B-polypeptide with purified
proteasomes in vitro yielded E1B192–200 at quantities that
were proportional to the relative contents of immunosubunits. Our data
provide evidence that the IFN--inducible proteasome subunits, when
present at relatively low levels as at initial stages of infection,
already increase the efficiency of antigenic peptide generation and
thereby enhance MHC class I Ag processing in infected
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To detect viruses shortly after cellular infection when viral loads are still low, the MHC class I Ag processing pathway should function with high efficiency. Although ER translocation of antigenic peptides, MHC class I binding, and cell surface transport seem not to curtail CTL epitope presentation (2, 3), proteolytic peptide generation has been identified as a rate-limiting step in Ag processing (4, 5).
Proteasomes are multicatalytic enzyme complexes consisting of a
catalytic 20S core that associates with regulatory complexes (19S or
PA700 and 11S or PA28), which tune proteasome function (for review, see
Refs. 6, 7). The induction of cells with IFN-
results in enhanced expression of most components of the MHC class I Ag
processing pathway, including that of the proteasome activator PA28,
which has been demonstrated to enhance MHC class I Ag presentation
(8, 9). IFN- also induces the expression of three
proteasome subunits, low molecular weight protein 2 (LMP2), LMP7, and
multicatalytic endopeptidase complex-like 1 (MECL-1) (7),
which are called the immunosubunits. These subunits replace the
constitutive catalytic subunits in the proteasome complex, resulting in
the formation of so-called immunoproteasomes. The association of the
IFN--inducible subunits alters the hydrolytic activity of
proteasomes against tri- and tetrapeptides and changes cleavage site
usage in polypeptides (10, 11, 12, 13, 14, 15). Both the catalytic
specificity and structural effects imposed on the proteasome by
immunosubunit incorporation may explain the altered cleavage properties
of immunoproteasomes
(16, 17, 18).4
To clarify the contribution of immunoproteasomes to Ag processing, 25- to 40-mer polypeptides harboring CTL epitopes have been digested with purified proteasomes. Indeed, certain antigenic peptides were liberated with greater efficiency in the presence of the LMPs and MECL-1, but the generation of other peptides was not affected (Refs. 14, 19 and our unpublished observations). Although generation of a murine CMV pp89 CTL epitope was enhanced in the presence of LMP2 in vitro, overexpression of this subunit in pp89-transfected fibroblast cells did not enhance recognition by specific CTL (20). Other investigators showed that the absence of the LMPs hampered the processing of a subset of MHC class I-presented CTL epitopes in the human 721.174 and T2 mutant cell lines (21, 22, 23, 24). In one study, the adverse effect of lacking LMP expression was reversed by the introduction of LMP7 (24). Mice lacking either LMP2 or LMP7 expression show only partial deficits in antigenic peptide presentation (25, 26), making a major impact of the LMP subunits on MHC class I Ag processing questionable.
So far, all studies using intact cellular systems only examined the
influence of LMP2 and LMP7 on antigenic peptide generation, neglecting
the potential contribution of the third IFN--induced subunit MECL-1.
Recently, the IFN--inducible subunits were shown to incorporate
interdependently into proteasomes, implicating that under physiological
conditions the assembly of proteasomes containing LMP2, LMP7, and
MECL-1 is favored (27, 28). Thus, because of potential
functional cooperativity, these three subunits should be regarded in
combination to further elucidate their role in MHC class I Ag
processing.
To this aim, transfectant cells were generated expressing LMP2, LMP7,
and MECL-1 under control of an inducible promoter. Uninduced cells
containing constitutive proteasomes and induced cells containing
immunoproteasomes were infected with adenovirus type 5 (Ad5), and MHC
class I presentation of a viral CTL epitope derived from the early 1B
protein (E1B) (29) was studied. Compared with uninduced
cells, induced cells processed the E1B CTL epitope with greater
efficiency, resulting in a better recognition by E1B-specific CTL
already at early time points after infection. Consistent with this, the
presence of the IFN--inducible subunits enhanced the
proteasome-mediated liberation of the antigenic nonamer from E1B
polypeptide in vitro. We discuss the possible mechanisms by which
immunoproteasomes may improve MHC class I Ag processing efficiency.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression vectors pTET-Splice and pTET-tTAk, containing the tetracycline (TET)-regulated tTAk transcription activator, were described by Shockett et al. (30). cDNAs encoding LMP2 (H-2b haplotype), LMP7, and MECL-1 were cloned into pTET-Splice using standard methods.
Cell lines and transfections
The MEC-18 cell line was derived from a primary culture of embryonal cells (MEC) of a C57BL/6 p53-/- mouse and cultured in IMEM (Biochrom, Berlin, Germany) with 10% FCS, 2 mM L-glutamine, 20 µM 2-ME, penicillin, and streptomycin. Human WEHI-164 clone 13 cells were maintained as described (31). The CTL clones 100B6 (28) and 100 PKOB4 (31) are specific for the Ad5 CTL epitope E1B192–200 in the context of H-2Db MHC class I molecules.
Transfectant cell lines were established using calcium-phosphate precipitation. MEC-18 cells were transfected with pTET-tTAk and pLXSH, and positive clones were selected with hygromycin B (200 µg/ml) in medium containing 400 ng/ml TET. The presence of tTAk was confirmed by PCR analysis using the oligonucleotides 5'-CCTGGAGACGCCATCCACGC-3' and 5'-CGTTTAAGGGCACCAATAACTGC-3', which are specific for pTET-Splice. TET-inducibility of tTAk expression was verified by Northern analyses. MEC/tTAk clone 29 (MEC-29) was further transfected with pTETsplice-LMP2, -LMP7, and -MECL-1 and pLXSP. Transfectant cells were selected with puromycin (50 µg/ml) in medium with 200 µg/ml hygromycin and 400 ng/ml TET. Inducible expression of the proteasome subunits was examined by immunoblot analysis.
Western blot analysis
Western blot analyses were performed as described (28). Expression of the proteasome immunosubunits was detected with polyclonal rabbit antisera raised against recombinant mouse LMP2 and against the synthetic peptides SDVSDLLYKYGEAAL and TAGGAKLQRALSTPTEPVQ corresponding to residues 265–276 of mouse LMP7 and 218–236 of mouse MECL-1, respectively.
Metabolic labeling and immunoprecipitation
A total of 2 x 106 MEC217 cells were seeded in flasks in Iscove’s medium with 400 ng/ml TET, washed after adherance, and placed in medium without or with TET (400 ng/ml or 10 ng/ml) for 10 h. Cells were labeled overnight with translabel [35S]methionine (50 µCi/ml), washed twice, chased for 4 h in medium with 400 ng/ml TET, and lysed in lysis buffer (1% Nonidet P-40, 20 mM Tris.Cl (pH 7.5), 10 mM EDTA, 100 mM NaCl) with 1 mM PMSF, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aproptinin as described (3). Centrifuged detergent lysates were precleared with 25 µl protein A-Sepharose (50% slurry) and 25 µl rabbit preimmune serum o/n at 4°C. 20S proteasomes were immunoprecipitated with 25 µl protein A-Sepharose and 25 µl polyclonal 20S-proteasome-specific rabbit antiserum for 1 h at 4°C. Sepharose beads were washed with NET buffer (50 mM Tris.Cl (pH 8), 150 mM NaCl, 5 mM EDTA) with 0.5% Nonidet P-40, resuspended in nonequilibrium pH-gradient gel electrophoresis (NEPHGE) sample buffer, and subjected to NEPHGE two-dimensional gel electrophoresis. Gels were exposed for autoradiography. Radioactive signals were quantified using a Molecular Dynamics (Sunnyvale, CA) phosphor imager.
Proteasome isolations
20S proteasomes used in this study were freshly prepared as described (14) and kept on ice. The purity of the proteasome preparates, checked by Coomassie-stained SDS-PAGE, was >95%.
NEPHGE two-dimensional gel electrophoresis
Purified proteasomes were subjected to NEPHGE-PAGE as described (14). Gels were Coomassie-stained according to standard procedures.
Ad5 and infections
Ad5 dl7001 (33) lacking the E3 region was propagated in 911 cells as described (34). Viral stocks were aliquoted and stored at -80°C in 10% glycerol. Adhered MEC217 cells were infected at a multiplicity of infection of 100 in PBS with 0.5% BSA for 35 min at room temperature. This procedure results in infection of 100% of cells. Infected cells were cultured in Iscove’s medium at 37°C until use in a CTL assay.
Eu3+-release assay
MEC217 cells were cultured for 3 days in the absence or presence of TET and then infected with Ad5 dl7001 or left uninfected. After 24 h of infection, the cells were harvested and used as targets in a 16-h Eu3+-release assay with CTL clone 100B6 as described (35).
TNF-
production assay
MEC217 cells cultured for 2 days without or with 10 ng/ml or 400
ng/ml TET were infected with Ad5 dl7001 or were left uninfected. After
12 h of infection, the cells were harvested and used as targets in
a TNF- production assay. Target cells were placed in the wells of
96-well plates (1 x 104 cells/well) in a
volume of 50 µl. CTL clone 100 PKOB4 cells (5000 cells/well) were
added in a volume of 50 µl, and the plates were incubated at 37°C
for 2.5, 10, and 22 h. Sixty microliters of supernatant of
independent triplicate wells were transferred into the wells of fresh
96-well plates and were assayed for the presence of TNF- by
measuring the cytotoxic effect on WEHI cells as described
(31). Conversion of MTT substrate by WEHI cells incubated
in CTL supernatant was determined by measuring the OD570s of the
microcultures with a Dynatech (Chantilly, VA) MR5000 ELISA reader. The
OD570s of wells with WEHI cells cultured with mouse recombinant TNF-
(35 pg/ml) were used as a reference for total WEHI cell
death.
Peptides
Z-LLE-ßNA was purchased from Bachem (Heidelberg, Germany). Synthetic AKVALRPDCKYKISKLVNIRNCCYISGNGAEVEIDTEDRV (Ad5 E1B E1B176–215) and VNIRNCCYI (E1B192–200) were kindly provided by Dr. P. Henklein (Institute of Biochemistry).
Peptide digestion assays and quantitation of CTL epitope generation
Digestion assays using Z-LLE-ßNA as a substrate were performed
as described (14). To determine proteasome-mediated
cleavage of Ad5 E1B 40-mer, 15 µg of polypeptide and 1 µg of
purified 20S proteasomes were incubated in 100 µl assay buffer (20 mM
HEPES/KOH (pH 7.8), 2 mM MgAc2, 5 mM DTT) at
37°C for the time periods specified in the figure legends, and then
they were frozen. Samples were analyzed by reversed phase HPLC
(RP-HPLC) (HPLC system HP1100 (Hewlett-Packard, Waldbron, Germany)
system equipped with a µRPC C2/C18 SC 2.1/10 column (Pharmacia,
Freiburg, Germany); eluent A, 0.05% trifluoroacetic acid ; eluent B in
30 min, 63–95% eluent B in 4 min; flow rate, 50 µl/min). Analysis
was performed on line with an ion trap mass spectrometer (ThermoQuest,
Egelsbach, Germany) equipped with an electronspray ion source. Each
scan was acquired over the range m/z = 300-1300
in 3 s. The peptides were identified by their molecular masses
calculated from the m/z peaks of the single or
multiple charged ions and were confirmed by mass spectrometric
sequencing analyses. To measure E1B192–200
generation, the digests were diluted in PBS with 0.5% BSA and 80 µM
2-ME and were titrated on IFN--induced uninfected MEC217 cells in
96-well plates (2 x 104 cells/well). After
30 min at 37°C, cells were washed with PBS used as targets in a
TNF- production assay. A standard curve generated in the same
experiment by diluting synthetic E1B192–200 on
MEC217 cells was used to quantify E1B192–200 in
the digests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the effect of LMP2, LMP7, and MECL-1 on
proteasome-mediated Ag degradation and CTL epitope generation,
transfectant mouse embryonal cell (MEC) lines expressing these subunits
under a TET-regulatable inducible promoter were generated as described
in Materials and Methods. A total of 54% of the established
MEC lines showed inducible expression of the three introduced subunits
in immunoblot analyses, as demonstrated for a representative clone
(MEC217) in Fig. 1
. This clone was
selected for complete processing of the tranfected subunits into the
mature form, lacking the prosequence, which indicates efficient
incorporation into 20S proteasomes. MEC217 cells cultured in the
presence of TET, which inactivates tTAk, did not express the LMPs or
MECL-1 at detectable levels, and thus lack any significant expression
of the inducible proteasome subunits. In the absence of TET, both LMPs
and MECL-1 were clearly detectable (Fig. 1). Kinetic experiments
established that the levels of induced LMP2 increased from the first up
to the third day of TET removal and then stabilized. In contrast,
TET-mediated repression of gene transcription resulted in a substantial
drop of LMP2 levels within 2 days, probably due to cell division (not
shown). The expression of the transfected subunits did not influence
the levels of endogenous PA28 or ß (not shown), implicating the
absence of stabilizing effects of immunoproteasomes on PA28, which
could serve as a mechanism to optimize MHC class I Ag processing. Thus,
in MEC217 cells the levels of immunosubunits are tightly regulated by
TET, and immunoproteasomes are expressed in the absence of elevated
levels of PA28.
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To further study the efficiencies of subunit exchange, MEC217
cells were cultured in the presence of graded concentrations of TET and
then subjected to pulse-chase analysis (Fig. 2). As expected, high concentrations of
TET (400 ng/ml) fully suppressed expression of the transfected
subunits, resulting in a 100% constitutive proteasome population. In
contrast, culture in 10 ng/ml TET effected a roughly 55% replacement
of constitutive for induced subunits among newly assembled proteasomes,
as measured by quantifying the radioactive signals for 
and LMP2
(Fig. 2). In the absence of TET, a nearly complete exchange of for
LMP2 and MB1 for LMP7 was reached (Fig. 2). Thus, LMP2, LMP7, and
MECL-1 efficiently replace their constitutive homologues , MB1, and
MC14 in induced MEC217 cells.
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0, 40, and 60% for
cultures with 400 ng/ml TET, 10 ng/ml TET, and without TET,
respectively (not shown). Because replacement of reduces the
proteasomal peptidyl glutamyl hydrolyzing acivity (14),
the subunit exchange efficiencies were further established by measuring
the enzymatic activity of the isolated proteasomes against Z-LLE-ßNA.
Whereas constitutive proteasomes cleaved this substrate efficiently,
immunoproteasomes of cells cultured with 10 ng/ml TET or without TET
hydrolyzed Z-LLE-ßNA to relative levels of roughly 50 and 20% only
(Fig. 3), reflecting the gradual
substitution of for LMP2 in the proteasome preparates. Thus, these
experiments (Figs. 2
and 3) reveal an efficient assembly of
immunoproteasomes in induced MEC217 cells that can be leveled by
titration of the TET concentration in the medium.
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To examine the effects of LMP2, LMP7, and MECL-1 on MHC class I Ag
processing, we used Ad5, against which specific CTL clones exist as a
model system. MEC217 cells were cultured in the presence or absence of
TET and infected with Ad5 mutant dl7001. Dl7001 lacks the viral E3
region and thus the E3-19K protein, which retains MHC class I molecules
in the ER. Viral infection by itself did not affect proteasome subunit
composition in MEC217 cells (data not shown). Presentation of
the viral E1B192–200 epitope by
H-2Db MHC class I molecules was tested in an
Eu3+-release assay with CTL clone 100B6 (Fig. 4A). Whereas uninduced
infected MEC 217 cells (+TET) were not lysed above background levels,
induced infected cells (-TET) expressing the proteasome immunosubunits
appeared highly susceptible to lysis by
E1B192–200-specific CTL. Infected MEC-29 cells
containing tTAk only and cultured with and without TET were
not lysed (not shown), indicating that tTAk expression or the presence
of TET did not influence the results. Observed effects were reproduced
in a second independent experiment (not shown). Thus, the presence of
immunoproteasomes enhances the generation of
E1B192–200 in infected cells.
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production by activated CTL. MEC217 cells
cultured for 2 days with different concentrations of TET (400 ng/ml and
10 ng/ml) or without TET were infected with Ad5 dl7001 and then
cocultured for different time periods with E1B-specific CTL clone 100
PKOB4. TNF- secretion was measured (Fig. 4B). Because 100
PKOB4 CTL lack perforin expression and thus do not lyse their target
cells efficiently and because Ad5 infection is not lytic in mouse
cells, decreases in target cell numbers during the assay were only
minimal. Both uninduced and induced infected MEC217 cells were
recognized by E1B192–200-specific CTL (Fig. 4B). However, cells expressing LMP2, LMP7, and MECL-1
induced significantly higher levels of TNF- release than did cells
cultured with 400 ng/ml TET, already at early time points after
infection. Thus, the presence of immunoproteasomes enhanced the
processing efficiency of E1B192–200, resulting
in a faster recognition of infected cells by virus-specific CTL.
Moreover, the kinetics of CTL activation by cells cultured in the
presence of 10 ng/ml TET and in the absence of TET appeared identical
(Fig. 4B), indicating that the lower levels of
immunosubunits sufficed to effect efficient
E1B192–200 presentation. Enhanced antigenic peptide generation by immunoproteasomes
To explain the observed enhancement of MHC class I Ag processing
in induced MEC217 cells, the effects of the inducible proteasome
subunits on E1B degradation and E1B192–200
generation were further analyzed in vitro. A 40-mer E1B polypeptide
(E1B176–215) encompassing the E1B CTL epitope
was synthesized and incubated with purified 20S proteasomes from
induced and uninduced MEC217 cells. Generated digestion products were
analyzed by RP-HPLC and on-line analyzed with a mass spectrometer. The
identity of detected peptide fragments was confirmed by ms/ms analysis.
Remarkably and consistent with earlier findings with murine CMV pp89
25-mer (U. Kuckelhorn and P. M. Kloetzel, manuscript in preparation),
we found that over the course of digestion (as measured at 3 and 6
h), immunoproteasomes degraded the E1B 40-mer faster than constitutive
proteasomes did (not shown). After 3 h of digestion, when turnover
of the E1B 40-mer was still incomplete, the
E1B192–200 flanking fragments
E1B176–191 and E1B201–215
were among the predominant peptide products (data not shown). The E1B
epitope VNIRNCCYI was just discernable from background in the HPLC-mass
spectrometry of digests of constitutive proteasomes (Fig. 5, upper left panel; at 21.63
min). In contrast, immunoproteasomes produced this peptide efficiently,
as is evidenced by a clearly distinguishable peak at 21.74 min. (Fig. 5, upper right panel). The same relative differences in
E1B192–200 abundancies were found in the
6-h-digestion products (Fig. 5, lower panels). Because not
only E1B192–200 but also the N- and C-terminal
flanking fragments (176–191 and 201–215, respectively) were most
prevalent in immunoproteasome digests, we infer that the observed
better liberation the E1B epitope is caused by a more efficient usage
of the Leu191-Val192 and
Ile215-Ser216 cleavage
sites. Peptides with the correct COOH terminus of the epitope but
prolonged at the N terminus were not detected, implying that both types
of proteasomes generated the E1B CTL epitope at exact size.
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To minimize potential losses during HPLC purification,
E1B192–200 generation was quantified from the
unfractionated peptide digests. Dilutions of digests generated with 20S
proteasomes of MEC217 cultured in the presence of 400 or 10 ng/ml TET
or in the absence of TET were loaded on uninfected MEC217 cells and
cocultured with CTL clone 100 PKOB4, and TNF- release into the
culture supernatant was determined using WEHI cells. Relative antigenic
peptide quantities (Fig. 6) were
calculated from a standard curve generated with synthetic
E1B192–200 in the same experiment. Two
independent measurements showed that proteasomes of cells grown without
TET produced the E1B epitope 3-fold more efficiently than proteasomes
of cells grown with 10 ng/ml TET and 10-fold more efficiently than
proteasomes of uninduced cells. Thus, the incorporation of LMP2, LMP7,
and MECL-1 into the proteasome enhanced
E1B192–200 generation (Fig. 6) in a
dose-responsive fashion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible subunits LMP2, LMP7, and MECL-1 are believed
to convert proteasomes into particles with specialized Ag processing
function. However, the actual effects of these subunits on
proteasome-mediated Ag degradation and CTL epitope generation are still
largely unclear. In this paper, we demonstrate for the first time that
the three immunosubunits, simultaneously expressed, enhance MHC class I
presentation of an Ad5-derived epitope by infected cells, resulting in
improved detection of cellular infection by CTL.
The TET-based inducible gene expression system allowed us to mimick the
conditions of IFN- induction and to examine the influences of the
LMPs and MECL-1 on Ag processing detached from other IFN--mediated
effects. By titrating the TET concentration, the effects of the
immunosubunits could be studied in correlation to their expression
levels. We found that immunoproteasome-containing MEC217 cells
processed and presented the Ad5 E1B192–200
epitope more efficiently than uninduced cells did, resulting in a
higher susceptibility to CTL-mediated lysis (Fig. 4A) and an
increased ability to activate CTL at earlier time points after
infection (Fig. 4B). Thus, the presence of the LMPs and
MECL-1 even at relatively low levels (Fig. 4B) allows the
efficient detection of intracellular viruses already at early stages of
infection, resulting in a rapid elimination of infected cells.
Interestingly, neither the Eu3+-release assay nor
the TNF- production assay showed a clear enhancement of MHC class I
presentation of an E1A-derived CTL epitope by induced cells (not
shown), indicating that immunoproteasomes may not significantly
influence the processing of this epitope. Thus, the immunosubunits
might alter the generation efficiency of different epitopes
differentially.
Our in vitro digestion experiments with E1B polypeptide showed that
immunoproteasomes yielded E1B192–200 more
efficiently than constitutive proteasomes did (Figs. 5 and 6),
indicating that the presence of the LMPs and MECL-1 supported the
production of this antigenic peptide. Because digestion with
immunoproteasomes instead of with constitutive proteasomes led to a
faster degradation of E1B polypeptide (not shown) and a more efficient
generation of the E1B176–191 and
E1B201–215 single cleavage products (Fig. 5), we
infer that the observed enhanced antigenic peptide production in the
presence of immunoproteasomes is likely to follow from more frequent
cleavage at the
Leu191-Val192 and
Ile200-Ser201 peptide
bonds. Although our mass spectrometry analyses revealed the presence of
at least one cleavage site within the E1B192–200
sequence (between Cys197 and
Cys198; not shown), truncated E1B epitopes
containing either the correct N or COOH terminus were not detected (not
shown). Thus, although cleavage at internal sites may influence the
usage of the Leu191-Val192
and Ile200-Ser201 peptide
bonds and thereby the efficiency of antigenic peptide liberation, it is
unlikely that E1B192–200 peptide is degraded
after previous generation.
Although the magnitude of antigenic peptide production in vitro
appeared to be determined by the absolute levels of IFN--inducible
subunits (Fig. 6), we found that in intact cells relatively low levels
of immunoproteasomes were sufficient to reach maximal Ag presentation
(Fig. 4B). Therefore, already the smaller enhancement of
E1B192–200 production in infected cells under
conditions of submaximal immunosubunit expression, reflecting initial
IFN- induction, results in enough MHC class I peptide complexes to
achieve maximal CTL activation. Taken together, our observations
underscore the physiological relevance of in vitro peptide digestions
with 20S proteasomes, which is in agreement with previous studies that
showed a correlation between polypeptide digestion analyses in vitro
and MHC class I presentation of the studied antigenic peptides by
intact cells (8, 9, 37, 38, 39).
Nevertheless, the intriguing question of how to translate the data
obtained with polypeptide digestions to the situation in intact cells
remains. Cytosolic (19S-containing) proteasomes are believed to degrade
full-length proteins, mainly targeted by linkage to ubiquitin moieties
which are recognized by subunits of the 19S component. Indeed, studies
examining MHC class I Ag processing of Ags that were introduced by
electroporation or that were intracellularly secreted by bacteria
demonstrated that the substrate-targeting mechanisms limited the rate
of protein degradation and antigenic peptide generation (4, 40). Alternative theories concerning cytosolic protein
degradation propose that nonproteasomal enzymes mediate the first
cleavages in Ags, after which the generated fragments are further
cleaved by proteasomes, thereby circumventing the need for 19S.
Furthermore, in case of endogenously synthesized Ags, so-called
defective ribosomal translation products may serve as an additional
source of antigenic peptides (41). Compatible with either
theory, the incorporation of the IFN--inducible subunits may change
the degradative mechanism of proteasomes, resulting in altered
preferences (11, 14, 15) and frequencies (this study) of
cleavage site usage and consequently in improved antigenic peptide
generation. Preliminary data (not shown) indicate that PA28, which
increases MHC class I Ag presentation of certain viral Ags (8, 9), does not influence epitope generation from the Ad5 E1A and
E1B proteins.
Immunofluorescence studies using LMP2-specific mAbs reveal an asymmetrical localization of LMP2 proteasomes in induced cells (C. Knuehl and P. M. Kloetzel, unpublished observations). Whereas an antiserum specific for 20S complexes shows an equal division of proteasomes over cytoplasm and nucleus, LMP2-containing proteasomes mainly seem to colocalize with TAP around the ER. This finding would corroborate previous biochemical data demonstrating that 20S proteasomes copurify with microsomal fractions (42) and with ER fractions (43). Thus, immunoproteasomes, unlike constitutive proteasomes, may reside in the vicinity of TAP and may deliver the generated peptides for immediate transportation into the ER, which may contribute to an improved MHC class I Ag presentation.
Thus, although not essential for the generation of many CTL epitopes, immunoproteasomes may tune the Ag processing machinery by multiple ways to improve the efficiency of MHC class I Ag presentation, allowing an optimal immunosurveillance by CTL.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter M. Kloetzel, Institute of Biochemistry, Medical School Charité, Humboldt University, Monbijoustrasse 2, 10117 Berlin, Germany.
3 Abbreviations used in this paper: ER, endoplasmic reticulum; Ad5, Adenovirus type 5; E1B, early 1B protein; LMP, low molecular weight protein; MECL, multicatalytic endopeptidase complex-like 1; TET, tetracycline; NEPHGE, nonequilibrium pH-gradient gel electrophoresis; RP-HPLC, reversed phase HPLC.
Received for publication September 15, 1999. Accepted for publication February 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation. J. Immunol. 159:5896.[Abstract]
in antigen presentation. Nature 381:166.[Medline]
-Interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature 365:264.[Medline]
stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. J. Exp. Med. 179:901. and major histocompatibility complex-encoded subunits on peptidase activities of human multicatalytic proteases. Proc. Natl. Acad. Sci. USA 92:584.-inducible 11S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20S proteasome in vitro. J. Biol. Chem. 270:23808.. Eur. J. Immunol. 25:2605.[Medline]
(IFN)-inducible subunits. J. Exp. Med. 187:97.-induced subunit exchange in the 20S proteasome. Eur. J. Immunol. 26:863.[Medline]
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 D. M. W. Zaiss, N. de Graaf, and A. J. A. M. Sijts The Proteasome Immunosubunit Multicatalytic Endopeptidase Complex-Like 1 Is a T-Cell-Intrinsic Factor Influencing Homeostatic Expansion Infect. Immun., March 1, 2008; 76(3): 1207 - 1213. [Abstract] [Full Text] [PDF]
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 P. Deol, D. M. W. Zaiss, J. J. Monaco, and A. J. A. M. Sijts Rates of Processing Determine the Immunogenicity of Immunoproteasome-Generated Epitopes J. Immunol., June 15, 2007; 178(12): 7557 - 7562. [Abstract] [Full Text] [PDF]
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A. R. Ciccaglione, E. Stellacci, C. Marcantonio, V. Muto, M. Equestre, G. Marsili, M. Rapicetta, and A. Battistini Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus Core Protein Results in Inhibition of Antiviral and Immunomodulatory Genes J. Virol., January 1, 2007; 81(1): 202 - 214. [Abstract] [Full Text] [PDF]
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M. K. Callahan, E. A. Wohlfert, A. Menoret, and P. K. Srivastava Heat Shock Up-Regulates lmp2 and lmp7 and Enhances Presentation of Immunoproteasome-Dependent Epitopes J. Immunol., December 15, 2006; 177(12): 8393 - 8399. [Abstract] [Full Text] [PDF]
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B. Strehl, T. Joeris, M. Rieger, A. Visekruna, K. Textoris-Taube, S. H. E. Kaufmann, P.-M. Kloetzel, U. Kuckelkorn, and U. Steinhoff Immunoproteasomes Are Essential for Clearance of Listeria monocytogenes in Nonlymphoid Tissues but Not for Induction of Bacteria-Specific CD8+ T Cells J. Immunol., November 1, 2006; 177(9): 6238 - 6244. [Abstract] [Full Text] [PDF]
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 G. Szalay, S. Meiners, A. Voigt, J. Lauber, C. Spieth, N. Speer, M. Sauter, U. Kuckelkorn, A. Zell, K. Klingel, et al. Ongoing Coxsackievirus Myocarditis Is Associated with Increased Formation and Activity of Myocardial Immunoproteasomes Am. J. Pathol., May 1, 2006; 168(5): 1542 - 1552. [Abstract] [Full Text] [PDF]
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J. Chapiro, S. Claverol, F. Piette, W. Ma, V. Stroobant, B. Guillaume, J.-E. Gairin, S. Morel, O. Burlet-Schiltz, B. Monsarrat, et al. Destructive Cleavage of Antigenic Peptides Either by the Immunoproteasome or by the Standard Proteasome Results in Differential Antigen Presentation J. Immunol., January 15, 2006; 176(2): 1053 - 1061. [Abstract] [Full Text] [PDF]
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 D. Bruder, A. K. Nussbaum, D. M. Gakamsky, M. Schirle, S. Stevanovic, H. Singh-Jasuja, A. Darji, T. Chakraborty, H. Schild, I. Pecht, et al. Multiple synergizing factors contribute to the strength of the CD8+ T cell response against listeriolysin O Int. Immunol., January 1, 2006; 18(1): 89 - 100. [Abstract] [Full Text] [PDF]
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C. F. Towne, I. A. York, J. Neijssen, M. L. Karow, A. J. Murphy, D. M. Valenzuela, G. D. Yancopoulos, J. J. Neefjes, and K. L. Rock Leucine Aminopeptidase Is Not Essential for Trimming Peptides in the Cytosol or Generating Epitopes for MHC Class I Antigen Presentation J. Immunol., November 15, 2005; 175(10): 6605 - 6614. [Abstract] [Full Text] [PDF]
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A. K. Nussbaum, M. P. Rodriguez-Carreno, N. Benning, J. Botten, and J. L. Whitton Immunoproteasome-Deficient Mice Mount Largely Normal CD8+ T Cell Responses to Lymphocytic Choriomeningitis Virus Infection and DNA Vaccination J. Immunol., July 15, 2005; 175(2): 1153 - 1160. [Abstract] [Full Text] [PDF]
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M. Basler, N. Youhnovski, M. van den Broek, M. Przybylski, and M. Groettrup Immunoproteasomes Down-Regulate Presentation of a Subdominant T Cell Epitope from Lymphocytic Choriomeningitis Virus J. Immunol., September 15, 2004; 173(6): 3925 - 3934. [Abstract] [Full Text] [PDF]
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S. Tenzer, L. Stoltze, B. Schonfisch, J. Dengjel, M. Muller, S. Stevanovic, H.-G. Rammensee, and H. Schild Quantitative Analysis of Prion-Protein Degradation by Constitutive and Immuno-20S Proteasomes Indicates Differences Correlated with Disease Susceptibility J. Immunol., January 15, 2004; 172(2): 1083 - 1091. [Abstract] [Full Text] [PDF]
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M. Altfeld, M. M. Addo, R. Shankarappa, P. K. Lee, T. M. Allen, X. G. Yu, A. Rathod, J. Harlow, K. O'Sullivan, M. N. Johnston, et al. Enhanced Detection of Human Immunodeficiency Virus Type 1-Specific T-Cell Responses to Highly Variable Regions by Using Peptides Based on Autologous Virus Sequences J. Virol., July 1, 2003; 77(13): 7330 - 7340. [Abstract] [Full Text] [PDF]
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G. Lautscham, T. Haigh, S. Mayrhofer, G. Taylor, D. Croom-Carter, A. Leese, S. Gadola, V. Cerundolo, A. Rickinson, and N. Blake Identification of a TAP-Independent, Immunoproteasome-Dependent CD8+ T-Cell Epitope in Epstein-Barr Virus Latent Membrane Protein 2 J. Virol., February 15, 2003; 77(4): 2757 - 2761. [Abstract] [Full Text] [PDF]
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S. F. Wieland, R. G. Vega, R. Muller, C. F. Evans, B. Hilbush, L. G. Guidotti, J. G. Sutcliffe, P. G. Schultz, and F. V. Chisari Searching for Interferon-Induced Genes That Inhibit Hepatitis B Virus Replication in Transgenic Mouse Hepatocytes J. Virol., December 20, 2002; 77(2): 1227 - 1236. [Abstract] [Full Text] [PDF]
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 D. M. W. Zaiss, S. Standera, P.-M. Kloetzel, and A. J. A. M. Sijts PI31 is a modulator of proteasome formation and antigen processing PNAS, October 29, 2002; 99(22): 14344 - 14349. [Abstract] [Full Text] [PDF]
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K. Yusim, C. Kesmir, B. Gaschen, M. M. Addo, M. Altfeld, S. Brunak, A. Chigaev, V. Detours, and B. T. Korber Clustering Patterns of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1 (HIV-1) Proteins Reveal Imprints of Immune Evasion on HIV-1 Global Variation J. Virol., July 29, 2002; 76(17): 8757 - 8768. [Abstract] [Full Text] [PDF]
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 Y. Sun, A. J. A. M. Sijts, M. Song, K. Janek, A. K. Nussbaum, S. Kral, M. Schirle, S. Stevanovic, A. Paschen, H. Schild, et al. Expression of the Proteasome Activator PA28 Rescues the Presentation of a Cytotoxic T Lymphocyte Epitope on Melanoma Cells Cancer Res., May 1, 2002; 62(10): 2875 - 2882. [Abstract] [Full Text] [PDF]
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M. Skoberne and G. Geginat Efficient In Vivo Presentation of Listeria monocytogenes- Derived CD4 and CD8 T Cell Epitopes in the Absence of IFN-{gamma} J. Immunol., February 15, 2002; 168(4): 1854 - 1860. [Abstract] [Full Text] [PDF]
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 E. S. Schultz, J. Chapiro, C. Lurquin, S. Claverol, O. Burlet-Schiltz, G. Warnier, V. Russo, S. Morel, F. Levy, T. Boon, et al. The Production of a New MAGE-3 Peptide Presented to Cytolytic T Lymphocytes by HLA-B40 Requires the Immunoproteasome J. Exp. Med., February 11, 2002; 195(4): 391 - 399. [Abstract] [Full Text] [PDF]
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S. Khan, M. van den Broek, K. Schwarz, R. de Giuli, P.-A. Diener, and M. Groettrup Immunoproteasomes Largely Replace Constitutive Proteasomes During an Antiviral and Antibacterial Immune Response in the Liver J. Immunol., December 15, 2001; 167(12): 6859 - 6868. [Abstract] [Full Text] [PDF]
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R.E.M. Toes, A.K. Nussbaum, S. Degermann, M. Schirle, N.P.N. Emmerich, M. Kraft, C. Laplace, A. Zwinderman, T.P. Dick, J. Muller, et al. Discrete Cleavage Motifs of Constitutive and Immunoproteasomes Revealed by Quantitative Analysis of Cleavage Products J. Exp. Med., June 25, 2001; 194(1): 1 - 12. [Abstract] [Full Text] [PDF]
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W. Chen, C. C. Norbury, Y. Cho, J. W. Yewdell, and J. R. Bennink Immunoproteasomes Shape Immunodominance Hierarchies of Antiviral CD8+ T Cells at the Levels of T Cell Repertoire and Presentation of Viral Antigens J. Exp. Med., June 4, 2001; 193(11): 1319 - 1326. [Abstract] [Full Text] [PDF]
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 L S YOUNG and V MAUTNER The promise and potential hazards of adenovirus gene therapy Gut, May 1, 2001; 48(5): 733 - 736. [Full Text] [PDF]
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J. H. Kessler, N. J. Beekman, S. A. Bres-Vloemans, P. Verdijk, P. A. van Veelen, A. M. Kloosterman-Joosten, D. C.J. Vissers, G. J.A. ten Bosch, M. G.D. Kester, A. Sijts, et al. Efficient Identification of Novel HLA-A*0201-presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen PRAME by Proteasome-mediated Digestion Analysis J. Exp. Med., January 2, 2001; 193(1): 73 - 88. [Abstract] [Full Text] [PDF]
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T. van Hall, A. Sijts, M. Camps, R. Offringa, C. Melief, P.-M. Kloetzel, and F. Ossendorp Differential Influence on Cytotoxic T Lymphocyte Epitope Presentation by Controlled Expression of Either Proteasome Immunosubunits or PA28 J. Exp. Med., August 14, 2000; 192(4): 483 - 494. [Abstract] [Full Text] [PDF]
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S. Raasi, G. Schmidtke, and M. Groettrup The Ubiquitin-like Protein FAT10 Forms Covalent Conjugates and Induces Apoptosis J. Biol. Chem., September 14, 2001; 276(38): 35334 - 35343. [Abstract] [Full Text] [PDF]
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