From the Department of Pathology, Harvard Medical
School, Boston, Massachusetts 02115,
Mikrobiologiskt och
Tumôr Biologiskt Centrum, Karolinska Institutet, Stockholm
S-171 77, Sweden, and the §§ Department of
Biochemistry and Biophysics, University of California,
San Francisco, California 94143
Received for publication, November 3, 2002
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ABSTRACT |
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Degradation of cytosolic proteins depends largely
on the proteasome, and a fraction of the cleavage products are
presented as major histocompatibility complex (MHC) class I-bound
ligands at the cell surface of antigen presenting cells.
Proteolytic pathways accessory to the proteasome contribute to protein
turnover, and their up-regulation may complement the proteasome when
proteasomal proteolysis is impaired. Here we show that reduced reliance
on proteasomal proteolysis allowed a reduced efficiency of MHC class I
ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor
adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal Several cytosolic proteases, including the 26 S proteasome,
bleomycin hydrolase, puromycin-sensitive amino peptidase and
leucine-aminopeptidase, contribute to the generation of
MHC1 class I ligands (1-3).
However, the 26 S proteasome, a large multicatalytic proteinase
complex, carries out the bulk of both cytosolic protein degradation and
MHC class I ligand production (1, 2). This protease has a multisubunit
20 S core structure containing two sets of three distinct catalytic
sites, X ( In the case of an immunological challenge, mammalian cells express
IFN- We show that lymphoma cells with reduced reliance on proteasomal
activity no longer efficiently produced MHC class I ligands, although
cytosolic proteolysis continued, and proliferation was not altered
compared with control cells. Assembly of H-2Db molecules
was dramatically reduced, and endogenous tumor antigens were not
presented efficiently under these conditions. This phenotype contributed to escape from tumor rejection in tumor graft experiments in syngeneic C57Bl/6 mice. Using a GFP reporter to measure
proteolysis, we show in live cells that non-proteasomal serine
peptidase activity participated in protein degradation, but inhibition
of these enzymes failed to have a significant effect on the assembly of
H-2Kb molecules. Continued proteolysis in
proteasome-impaired cells afforded the cell the requisite housekeeping
functions while preventing the full display of the usual set of MHC
class I-restricted epitopes.
Cells and Transfections--
EL-4 is a benzopyrene-induced
thymoma cell line of the H-2b haplotype, derived from
C57Bl/6 mice. RMA is a Rauscher's virus-induced T cell lymphoma cell
line and is also derived from C57Bl/6. Adaptation to the proteasomal
inhibitor NLVS was obtained by incubation of these cells in RPMI 1640 medium containing 5% fetal calf serum, 1% penicillin/streptomycin,
1% glutamine, and 10 µM NLVS. Gradually outgrowing cells
were selected and cultured in 50 µM NLVS over a period of
several weeks as described previously (10). EL-4.Ub-R-GFP and
EL-4.Ub-M-GFP cells were obtained by electroporation of EL-4 cells with constructs Ub-R-GFP and Ub-M-GFP (16), respectively, and
stable clones were selected with 0.5 mg/ml G418. Electroporation was
preformed in a Bio-Rad Gene-Pulser at 250 V and 960 microfarads.
Proteasomal Inhibitors--
NLVS (14) covalently modifies all
catalytically active subunits of the proteasome, but with preference
for the Peptide Substrates and Peptidase Assays--
To assay the
activity of the proteasome, we used the fluorogenic substrates
succinyl-LLVY-AMC, benzyloxycarbonyl-GGL-AMC, t-butyloxycarbonyl-LRR-AMC, and benzyloxycarbonyl-YVAD-AMC
(Sigma). To assay tripeptidyl-peptidase II activity, we used AAF-AMC
(Sigma). Cell extracts or proteasome-enriched fractions and substrate
(100 µM) were mixed in 50 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, and
2 mM ATP in a final volume of 100 µl. Peptide hydrolysis
was monitored by fluorescence spectroscopy (PerSeptive Biosystems, Framingham, CT) with excitation at 380 nm and fluorescence reading at
460 nm.
Proteasome Purifications--
Preparation of proteasome-enriched
fractions was performed using 0.5-1 × 109 control or
adapted EL-4 cells and C57Bl/6 livers. Cells were washed with
phosphate-buffered saline and lysed by vortexing with glass beads in 50 mM Tris base (pH 7.5), 250 mM sucrose, 5 mM MgCl2, 1 mM dithiothreitol, and
2 mM ATP. Glass beads and cell debris were removed by
sequential centrifugations at 3000 and 14,000 rpm, respectively.
Microsomes were removed by centrifugation for 1 h at 100,000 × g, and large cytosolic proteins or protein complexes containing proteasomes and tripeptidyl-peptidase II were then
sedimented at 100,000 × g for 5 h. The resulting
pellet was dissolved in 50 mM Tris base (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 2 mM ATP, and 30% glycerol.
Pulse-Chase Experiments--
Cells were starved in
methionine/cysteine-deficient medium for 45-60 min, pulsed with
[35S]methionine for 15 min, and chased for the indicated
times. Cells were collected by centrifugation and lysed in 0.5%
Nonidet P-40 lysis buffer, and MHC class I molecules were
immunoprecipitated with rabbit anti-p8 serum (H-2Kb
cytoplasmic tail), monoclonal antibody Y3 (H-2Kb
Generation of CTLs and 51Cr Release
Assays--
For the generation of CTLs specific for tumor antigens
expressed by EL-4 cells, we primed B6 mice two to three times with control or adapted EL-4 cells transfected with B7.1. Priming of responses with EL-4 cells not expressing B7.1 led to very low CTL
responses or no response at all. To generate RMA cell-specific CTLs, we
primed C57BL/6 mice with irradiated RMA cells two to three times; and
for the subsequent generation of in vitro effector cells,
25 × 106 splenocytes were restimulated in
vitro with 1-2 × 106 irradiated tumor cells for
5 days. The H-2Db restricted CTL clone ln17 was acquired from
Elisabeth Wolpert and Vanoohi Fredriksson (MTC, Karolinska Institutet).
The conditions for generation and analysis of this CTL have been
described previously (23).
Tumor Growth Experiments--
Control EL-4 and EL-4ad cells
(cultured in RPMI medium 1640 supplemented with 5% fetal calf serum)
were washed with phosphate-buffered saline and resuspended in
200 µl/inoculate. The cells were inoculated into the right
flanks of syngeneic C57Bl/6 mice at 104 or 106
cells/animal. Some of the mice were irradiated with 400 rads prior to
tumor inoculation to inhibit antitumor immune responses. Outgrowth of
the tumors was monitored by palpations weekly.
Tumor Cells with Reduced Reliance on Proteasomal Proteolysis Fail
to Efficiently Produce MHC Class I Ligands--
EL-4 cells can adapt
to proliferate in the presence of high concentrations of NLVS, a
covalent proteasomal inhibitor (denoted EL-4ad cells) (10). MHC class I
molecules show allelic variation in their ability to undergo assembly
and transport during proteasomal inhibition, and H-2Db is
one allele that fails to assemble in cells that are treated with
proteasomal inhibitors for short-term periods (19-21). We therefore
investigated H-2Db ligand production in EL-4ad cells, which
proliferate with low proteasomal activity. In control EL-4 cells,
almost all folded H-2Db molecules were transported
from the endoplasmic reticulum within 120 min after onset of the chase,
as judged from their acquisition of Golgi-specific glycan modifications
(Fig. 1a, left
panel). In EL-4 cells treated with NLVS (50 µM,
3 h), only a minimal fraction of H-2Db heavy chains
were transported even after long chase times (Fig. 1a,
middle panel), as reported previously (21). Stabilization of
H-2Db molecules in cell lysates of NLVS-treated EL-4 cells
by addition of the influenza nucleoprotein-(366-374) peptide
confirmed that most of these H-2Db heavy chains were devoid
of peptide ligand (+ lanes) (22). In EL-4ad cells, a
fraction of H-2Db resumed folding, although at much lower
levels compared with control EL-4 cells. The majority of
H-2Db heavy chains in EL-4ad cells remained unassembled in
the endoplasmic reticulum devoid of peptide, as indicated by the
stabilizing effect of nucleoprotein-(366-374) added to lysates
of these cells (Fig. 1a, right panel). In RMAad
cells, which were similarly adapted to NLVS, maturation of
H-2Db molecules was comparable to that observed in EL-4ad
cell (Fig. 1b). Despite normal proliferation, tumor cells
can therefore avoid production of most H-2Db ligands by
reduced reliance on proteasomal activity.
Tumor cells often acquire deficiencies in MHC class I antigen
presentation to escape from host immune detection. To test whether reduced reliance on proteasomal activity has functional consequences, we tested presentation of endogenous tumor antigens to CTLs by EL-4ad
and RMAad cells. Both EL-4 and RMA cells express
H-2Kb-restricted (gagL75-83) as well as
H-2Db-restricted (env189-196) murine
leukemia virus-derived peptides and, in addition, an endogenous
H-2Db-restricted tumor epitope (23). We generated antitumor
CTLs by priming C57Bl/6 mice and subsequent in vitro
restimulation of splenocytes with B7.1-transfected EL-4 cells. We found
that tumor antigen-specific CTLs performed efficient killing of control EL-4 cells, whereas EL-4ad cells were not efficiently recognized, although the latter were killed significantly better than
C4.4-25
The chymotrypsin-like activity of the proteasome is required for the
production of most MHC class I ligands and is normally rate-limiting
for intracellular proteolysis (1, 4, 7). To visualize protein turnover
in EL-4ad cells, we performed pulse-chase experiments with
[35S]methionine and displayed labeled protein by
SDS-PAGE. As expected from the proliferation rates of these cell lines
(10), we observed a similar rate of decay of labeled proteins when
comparing control EL-4 and EL-4ad cells (Fig. 2d). We
conclude that NLVS-adapted cells have a severely inhibited chymotryptic
proteasomal activity, as deduced from experiments employing active
site-directed covalent probes. When complemented by the induction of
other cytosolic proteases (10-12), the remaining proteasomal activity
is adequate for normal protein turnover, but not for production of all
class I ligands.
Up-regulation of Deubiquitinating Enzymes and Non-proteasomal
Peptidases in EL-4ad Cells--
The activity of the ubiquitin-specific
protease USP14 is associated with the 19 S cap proteasome and is
up-regulated when the proteasome is inhibited (24). More generally,
inhibition of proteasomal proteolysis should lead to accumulation of
ubiquitin-conjugated substrates. Adaptation to proteasomal inhibitors
might well include increased activity of deubiquitinating enzymes to
deal with such accumulation. We therefore examined whether this was the
case also in EL-4ad cells using 125I-labeled
ubiquitin-vinyl sulfone (24). Cellular fractions of control EL-4 and
EL-4ad cells (cytosolic as well as proteasome-enriched fractions) were
incubated with ubiquitin-vinyl sulfone, and covalently modified
polypeptides were separated by SDS-PAGE. We found increased labeling of
IsoT1, USP14, and UCH-L1 in EL-4ad cells compared with control
EL-4 cells (Fig. 3a), in line
with what was observed in acutely treated EL-4 cells (24). More active
ubiquitin removal could prepare these substrates for degradation by
other proteases.
Two additional active-site probes with different peptide scaffolds were
used (25), [125I]AAF-VS and
[125I]LLG-VS, to examine whether residual proteasomal
activity is mediated by the Proteolysis Accessory to the Proteasome Supports Protein
Degradation, but Is Relatively Ineffective in MHC Class I Ligand
Production--
We next made stable EL-4 transfectants expressing
Ub-R-GFP to monitor proteasomal degradation of a protein substrate in
live cells (16). GFP was converted into an N-end rule substrate
and was degraded in EL-4 cells due to its N-terminal arginine, whereas Ub-M-GFP was comparatively stable (Fig.
4, a-c) because of the presence of a methionine residue. NLVS treatment of EL-4.Ub-R-GFP cells
led to accumulation of fluorescence, as detected by FACS. In line with
data from yeast mutants (28, 29), efficient inhibition of primarily the
chymotryptic proteasomal activity was sufficient for accumulation of
fluorescence (Fig. 4, c and d). In addition, the
accumulation of R-GFP fluorescence observed in cells exposed to
10 µM NLVS also correlated with the induction of cellular
toxicity and subsequent cell death (data not shown). These data further confirm that NLVS is indeed an efficient inhibitor of proteasomal protein degradation in live cells.
We next tested whether inhibition of accessory pathways has any effect
on cytosolic proteolysis. To do this, we accumulated high levels of the
R-GFP substrate in live EL-4.Ub-R-GFP cells and then blocked protein
synthesis to study changes in the steady state of the substrate (Fig.
5, a-d). This revealed
residual substrate degradation in the continued presence of 10 µM NLVS because a substantial fraction of the substrate
was removed after 8 h. However, this was inhibited by treatment
with AAF-CMK, an efficient inhibitor of tripeptidyl-peptidase II and
other serine oligopeptidases (Fig. 5, c and d).
Although inhibition of oligopeptidases by AAF-CMK had minor effects on
untreated EL-4.Ub-R-GFP cells, we observed a significant effect on
R-GFP accumulation upon treating EL-4.Ub-R-GFP cells with both NLVS and
AAF-CMK in combination (Fig. 5e). These data show that
non-proteasomal oligopeptidase activity indeed contributes to cytosolic
proteolysis, especially during situations of limiting or insufficient
proteasomal activity.
To further study whether oligopeptidases inhibitable by AAF-CMK are
important in generating MHC class I ligands, we performed a pulse-chase
experiment with [35S]methionine metabolic labeling and
precipitation of H-2Kb molecules. A substantial fraction of
H-2Kb molecules continue to assemble in EL-4ad cells (10).
We examined whether this may be due to ligands produced by
oligopeptidases inhibitable by AAF-CMK. We found that this treatment
had minor effects on the assembly and transport of H-2Kb
molecules in EL-4ad cells and also in control EL-4 cells with active
proteasomes (Fig. 5f). We conclude that pathways accessory to proteasomal proteolysis that are inhibited by AAF-CMK support protein degradation, but reveal poor yields of MHC class I ligands.
Evidence for Continued Cell Survival and Growth without Significant
Proteasomal Activity--
EL-4ad cells continue to depend on
proteasomal Escape from in Vivo Immune Detection by Tumor Cells with Reduced
Reliance on Proteasomal Activity--
In vivo tumors
frequently acquire mutations in genes encoding proteins of the MHC
class I-processing pathway, and these are believed to be the result of
immune selection (33). Therefore, we explored whether an adapted
phenotype similar to that of EL-4ad cells was selected during growth
in vivo. We inoculated control EL-4 and EL-4ad cells into
syngeneic C57Bl/6 mice and observed tumors in most mice inoculated with
EL-4ad cells at both 106 and 104 cells/animal,
whereas control EL-4 cells failed to grow and produce tumors (Fig.
7a). The tumor-forming ability
of EL-4ad cells was dependent, at least in part, on escape from immune
recognition because both EL-4ad and control EL-4 cells formed tumors in
mice with a deficiency of perforin and RAG-1
(PKOB/RAG This study shows that tumor cells may avoid efficient production
of MHC class I ligands and hence immune recognition by modulation of
proteasomal activity. Pathways accessory to proteasomal proteolysis can
reduce the extent to which cells depend on proteasomal activity. In our
case, cells adapted to growth in the presence of proteasomal inhibitors
were unable to maintain normal levels of MHC class I ligand production.
In addition, using the vinyl sulfone inhibitor Ada-Ahx3-Leu3-VS, we showed that inhibition of
all catalytic Although MHC class I processing is a rather inefficient process
overall, in which most (>99%) of the cleaved peptides are never
displayed at the cell surface, it is an adequate method for screening
of the bulk of cellular protein content for the presence of foreign
antigens (26). The steady-state level of MHC class I at the surface of
cells depends on both its transport and removal from the cell surface
(35), and transport of H-2Db is substantially inhibited in
EL-4ad cells. Despite this, the cell-surface H-2Db (and
also H-2Kb) levels detected by FACS are almost normal,
suggesting that the rate of decay at the cell surface may be reduced
when transport is slow (data not shown). Earlier data on MHC class II
transport in cathepsin S Reduced expression of several components of the MHC class I
antigen-processing pathway is often observed in human tumors. This
includes down-regulation of the IFN--subunits (
1,
2, and
5)
was compatible with continued growth in cells that up-regulate
accessory proteolytic pathways, which include cytosolic proteases as
well as deubiquitinating enzymes. However, under these conditions, we
observed poor assembly of H-2Db molecules and inhibited
presentation of endogenous tumor antigens. Thus, the tight link between
protein turnover and production of MHC class I ligands can be broken by
enforcing the substitution of the proteasome with alternative
proteolytic pathways.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5), Y (
1), and Z
(
2), associated with one or two 19 S regulatory accessory complexes (1, 2). The proteasome generates a wide range of
peptide cleavage products (3-24 amino acids in length) that are
ultimately degraded into free amino acids (4-6). In mammalian cells, a
minor subset of peptides is rescued from further degradation and is
translocated from the cytosol into the endoplasmic reticulum for
assembly with MHC class I molecules. The MHC class I pathway is thereby
assured constitutive production of ligands through cytosolic proteolysis.
-inducible proteasomal
-subunits (LMP7/
5i,
LMP2/
1i, and MECL-1/
2i) that replace the
constitutively expressed subunits in newly synthesized proteasomes (1).
Such replacement leads to increased proteasomal production of peptides
with hydrophobic C termini, usually preferred for both TAP transport
and MHC class I binding (7). However, the majority of potential MHC
class I ligands, as deduced from their primary structure, are not
efficiently processed, although the correct motifs for TAP transport
and MHC class I binding are contained in the protein sequence (8, 9). Such failure may depend on proteolysis by cytosolic proteases inefficient at generating the requisite cleavage products. Thus, it is
possible that MHC class I processing may be regulated by differential
participation of non-proteasomal peptidases in cytosolic protein
degradation. Impaired proteasomal activity can be functionally compensated, at least in part, by another large cytosolic peptidase, tripeptidyl-peptidase II (10-13). Despite covalent inhibition by NLVS
(14) or lactacystin (15), EL-4 lymphoma cells adapted to growth in the
presence of this inhibitor (denoted EL-4ad) maintain cytosolic
proteolysis and cell viability by a mechanism that includes compensatory up-regulation of tripeptidyl-peptidase II (10-12). However, it is unknown whether the adapted state has functional consequences at the level of MHC class I ligand generation and antigen
presentation to CTLs.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits with chymotryptic specificity. Several derivatives
of NLVS were obtained by variations in the peptide scaffold:
4-hydroxy-5-iodo-3-nitrophenylacetyl-Ala-Ala-Phe-vinyl sulfone (AAF-VS)
and
4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Gly(cis)-vinyl sulfone (LLG(cis)-VS). LLG-VS was obtained in the
cis- and trans-isomers due to the absence of a
side chain on the P1 glycine. Whereas the trans-form of
LLG-VS modifies proteasomal
-subunits, the cis-form
modifies yet uncharacterized targets in the cytosol distinct from the
proteasome.
Adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone (Ada-Ahx3-Leu3-VS) is an N-terminally extended
vinyl sulfone inhibitor that blocks all proteasomal
-subunits in a
covalent manner (17).
1
2), or antibody B22.249.1
(H-2Db
1
2) as described
previously (18). Viral peptide epitopes are known to stabilize the MHC
class I complex at 4 °C. We added Db-binding peptide
ASNENMDAM (influenza NT60, amino acids 366-374) at 10 µM to detect the presence of unfolded heavy chains.
Immune complexes were removed by adsorption to staphylococcus A and
analyzed by SDS-PAGE as previously described (10).
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Impaired assembly of MHC class I molecules in
cells with reduced reliance on proteasomal activity. a,
pulse-chase experiments were performed on control EL-4 cells, EL-4
cells treated with 50 µM NLVS for 16 h, or EL-4ad
cells. H-2Db molecules were immunoprecipitated
(IP) with anti-H-2Db antibody B22.249.1 in the
presence or absence of an H-2Db-binding peptide, followed
by SDS-PAGE analysis and autoradiography. b, RMA cells left
untreated or treated with 50 µM NLVS for 3 h
(3h), adapted (ad) to 50 µM NLVS
(RMAad), or adapted to NLVS and washed (wash) and RMA-S
cells were subjected to pulse-chase experiments. H-2Db
molecules were immunoprecipitated in the presence or absence of an
H-2Db-binding peptide, followed by SDS-PAGE analysis and
autoradiography. Glycosylated heavy chains (GHC) that were
transported from the endoplasmic reticulum, heavy chains
(HC), and 2-microglobulin
(
2m) are indicated with
arrows.
, a
2-microglobulin-deficient
variant of EL-4 (Fig. 2a and
data not shown). We also found that RMAad cells likewise had a reduced ability to present endogenous tumor antigens compared with control RMA
cells. Although RMAad target cells were killed at higher levels than
TAP-deficient RMA-S cells, 5-10 times more CTLs were required to
obtain the same degree of killing as seen on RMA target cells (Fig.
2b). Even more pronounced differences were obtained using CTL clone ln17, specific for tumor antigen-specific peptide NKGENAQAI restricted by H-2Db (20). In line with previous data, we
found that ln17 detected the presence of the tumor-specific epitope on
RMA cells, but failed to recognize RMA-S cells (Fig. 2c).
Furthermore, no recognition of RMAad cells was observed. MHC class
I-restricted presentation of a tumor antigen-specific peptide can
thereby be inhibited when proteasomal proteolysis is inhibited in a
suitable manner. Because we used IFN-
secretion as readout for
antigen detection by ln17, these data also exclude that the differences
in CTL killing were due merely to differences in target cell apoptosis
when comparing control and NLVS-adapted target cells. These data
support the conclusion that EL-4ad cells fail to display the full
repertoire of MHC class I-associated antigens at the cell surface.
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Fig. 2.
Decreased tumor antigen presentation due to
reduced reliance on proteasomal activity. 51Cr release
assays were performed using antitumor-specific CTLs tested against
51Cr-labeled EL-4 (a) and RMA (b)
target cells. RMA-S and C4.4-25 are MHC class I
mutant cell lines used to control for nonspecific target cell killing.
Tumor antigen (NKGENAQAI30)-specific CTL clone ln17 was
used to test antigen presentation by RMA, RMA-S, and RMAad cells
(c). Antigen recognition was quantified by detection of
IFN-
secretion by effector cells, as described previously (31). Bulk
protein turnover was assessed by metabolic labeling of control EL-4 and
EL-4ad cells with 35S for 15 min, and the fate of
radiolabeled proteins was followed by a chase for the indicated times,
SDS-PAGE separation, and autoradiography (d).
2m
/
,
2-microglobulin-deficient; E/T, effector to
target ratio.
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Fig. 3.
Proteolysis accessory to the proteasome
analyzed by covalent vinyl sulfone probes. a,
isopeptidase activities were analyzed using ubiquitin-vinyl sulfone
(UbVS) in cellular fractions from control EL-4 and EL-4ad
(Ad) cells. 1hr pel and 5hr pel,
protein pellets created by centrifugation at 100,000 × g for 1 and 5 h, respectively. This procedure sediments
high molecular mass proteins or protein complexes. b and
c, different radiolabeled peptide vinyl sulfones
([125I]NLVS, [125I]LLG-VS, and
[125I]AAF-VS) exhibiting different specificities were
incubated with cell lysates prepared from untreated ( ), NLVS-treated
(2 h; 2h), or NLVS-adapted (ad) lymphoma cell
lines. Lysates prepared from EL-4 (b) and RMA (c)
cells were separated by SDS-PAGE and analyzed by autoradiography.
Whereas the first and third compounds efficiently labeled the
5- and
5i-subunits of the proteasome,
[125I]LLG-VS labeled other yet uncharacterized
cellular proteases.
5/
5i-subunits
(X/LMP7). None of these probes labeled
5/
5i-subunits (X/LMP7) in lysates of
EL-4ad cells, whereas strong labeling was detected in lysates of
control EL-4 cells (Fig. 3b). This confirms that virtually
no catalytic activity remains for the
5/
5i-subunits (X/LMP7) in EL-4ad cells,
which is important in view of the fact that small amounts of peptide ligand suffice to load MHC class I molecules with peptide (26). Interestingly, using [125I]LLG(cis)-VS, we
detected a series of modified polypeptides distinct from proteasomal
-subunits. Because vinyl sulfones are mechanism-based probes (14,
27), we conclude that these polypeptides correspond to additional, yet
to be identified, proteases. This activity is not inhibited by NLVS,
further supporting the alteration in proteolytic specificity in EL-4ad
cells (Fig. 3b, middle panel). Labeling of the
5/
5i- subunits (X/LMP7) with
[125I]NLVS was likewise inhibited in RMAad cells when
tested with these peptide vinyl sulfones (Fig. 3c).
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Fig. 4.
Measuring proteasomal degradation by
fluorescent ubiquitinated substrates in live cells. Shown are the
results from FACS analysis of EL-4 cells stably transfected with
Ub-M-GFP (a) or Ub-R-GFP (b and c).
EL-4.Ub-R-GFP cells were left untreated (b) or were treated
with 10 µM NLVS (c). The level of proteasomal
activity in EL-4.Ub-R-GFP cells treated with 10 µM NLVS
was measured by cytosolic high molecular mass protein (100,000 × g, 5 h) cleavage of the fluorogenic peptide substrates
succinyl (succ)-LLVY-AMC,
t-butyloxycarbonyl(boc)-LRR-AMC, and
benzyloxycarbonyl (z)-YVAD-AMC (d). Also
shown are the results from FACS analysis of EL-4.Ub-R-GFP cells treated
with up to 50 µM NLVS (e-h).
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Fig. 5.
Degradation of an N-end rule
substrate is influenced by non-proteasomal oligopeptidases. Shown
are the results from FACS analysis of EL-4.Ub-R-GFP cells
incubated with 10 µM NLVS for 16 h (a)
and further incubated for 8 h in the presence of no addition
(b), cycloheximide (c), or 10 µM
AAF-CMK (d). EL-4.Ub-R-GFP cells were incubated in the
presence of NLVS (0, 2, 5, or 10 µM) in combination with
AAF-CMK (0, 5, or 10 µM) (e). Control EL-4 and
EL-4ad cells were pulsed with [35S]methionine, and
transport of H-2Kb molecules was followed by
immunoprecipitation and SDS-PAGE in the presence (+) or absence ( ) of
50 µM NLVS and 10 µM AAF-CMK
(f). The cells were incubated with the protease inhibitors
for 3 h prior to metabolic labeling. GHC, glycosylated
heavy chains; HC, heavy chains;
2m,
2-microglobulin.
-subunit activity, at least to some extent (30). NLVS
fails to block
2- and
2i-subunits
(Z/MECL-1) in vivo, a pattern of inhibition that is shared
between NLVS and other covalent proteasomal inhibitors such as
lactacystin (15) and epoxomicin (31). To examine if residual
proteasomal activity influences the viability of EL-4ad cells, we used
Ada-Ahx3-Leu3-VS, a cell-permeable tripeptide
vinyl sulfone that covalently modifies all proteasomal
-subunits
with comparable efficiency (17). We found that proteasome-enriched fractions from control EL-4 or EL-4ad cells treated with either NLVS or
Ada-Ahx3-Leu3-VS had almost completely blocked
chymotryptic and trypsin-like proteasomal activities, whereas the
caspase-like activity was 70% inhibited (Fig.
6a). Initially, at early time points, we observed an induction of the trypsin- and
caspase-like specificities during inhibitor treatment, possibly due to
allosteric effects on the proteasome upon binding of the inhibitor to
the X/LMP7 site (32). Consistent with the enzyme assays using
fluorogenic peptide substrates, labeling of
-subunits with
Ada-[125I-Tyr]Ahx3-Leu3-VS in
cell lysates followed by separation of the
-subunits by SDS-PAGE
confirmed that all proteasomal active sites were covalently modified
during treatment of live cells with the Ada-Ahx3-Leu3-VS inhibitor (Fig. 6a,
lower panel). Furthermore, EL-4ad cells proliferated
regardless of the presence of Ada-Ahx3-Leu3-VS, whereas control EL-4 cells died within 48 h (Fig. 6b).
To confirm that proteasomes of proliferating EL-4ad cells were indeed
modified, we prepared proteasome-enriched fractions from cells treated
for several days with Ada-Ahx3-Leu3-VS. This
analysis revealed results similar to those observed in acutely treated
cells. Essentially no residual tryptic and chymotryptic activities and
inhibited caspase-like activity were detected (data not shown). Thus,
tumor cells may adapt and proliferate normally even when proteasomal
-subunit activity is almost absent.
View larger version (25K):
[in a new window]
Fig. 6.
EL-4ad cell proliferation despite inhibition
of all catalytic sites of the proteasome by
Ada-Ahx3-Leu3-VS. a, control
EL-4 or EL-4ad cells were incubated with either 50 µM
NLVS or 50 µM Ada-Ahx3-Leu3-VS
for the indicated times, and cell lysates were submitted to
differential centrifugation for partial purification of proteasomes.
The samples were either tested for cleavage of the peptide reporter
substrates succinyl (Suc)-LLVY-AMC,
benzyloxy-carbonyl-GGL-AMC, t-butyloxycarbonyl
(Boc)-LRR-AMC, and benzyloxycarbonyl
(Z)-YVAD-AMC) (upper three panels) or labeled
with Ada-[125I-Tyr]Ahx3-Leu3-VS,
followed by SDS-PAGE and autoradiography (lower panel).
b, EL-4ad cell viability was mostly independent of
proteasomal proteolysis. EL-4 (left panels) or EL-4ad
(right panels) cells were left untreated (upper
panels) or were incubated with 50 µM NLVS
(middle left panel) or 10 µM (middle
right panel) or 50 µM (lower panels)
Ada-Ahx3-Leu3-VS for the indicated times. Live
( ) and dead (
) cells were counted by trypan blue exclusion.
/
) (Fig. 7a). Furthermore, after
low dose irradiation (400 rads) of the C57Bl/6 mice, commonly used to
reduce in vivo transplantation barriers (34), both control
EL-4 and EL-4ad cells were able to grow after inoculation of
106 cells/animal, whereas only EL-4ad cells grew at
104 cells/animal (Fig. 7b). In conclusion, we
have shown that the ability of NLVS-adapted cells to proliferate
independently of proteasomal
-subunit activities leads to reduced
immune recognition in vivo.
View larger version (16K):
[in a new window]
Fig. 7.
Increased in vivo
tumorigenicity of cells with reduced reliance on proteasomal
proteolysis. Control EL-4 (open bars) and EL-4ad
(closed bars) cells were grafted at 104 to
106 cells into the right flanks of syngeneic C57Bl/6 mice
(a) or perforin/RAG-1 /
mice
(PKOB/RAG
/
) (b). Frequency of tumor growth
is displayed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit activities of the proteasome (more
efficiently than achieved with NLVS) was compatible with continued cell
growth of EL-4ad cells. These results indicate that it is possible for
mammalian cells to partly escape from production of MHC class I ligands
by aversion to pathways of protein degradation involving proteases
other than the proteasome.
/
mice have revealed a similar
feature; and also in this case, an absence of T cell detection is
observed for certain antigens (36, 37). In the course of an immune
response, the proteolytic specificity in antigen processing has
profound influence on the generation of MHC class I ligands, as
illustrated by the IFN-
-dependent substitution of
proteasomal
-subunits (7). The fact that pathways accessory to
proteasomal proteolysis, such as tripeptidyl-peptidase II, can
contribute to maintaining proteolysis when the proteasome is inhibited
allows for mammalian cells to alter the spectrum of cleavage fragments
in the cytosol more dramatically (10-13). This notion is supported by
the up-regulation of several deubiquitinating enzymes in EL-4ad cells,
observed otherwise in cells suffering from acute proteasomal inhibition
(24). USP14 is associated with the 19 S regulatory complex, and its
precise role in proteasomal proteolysis remains to be established.
Other deubiquitinating enzymes are also up-regulated in EL-4ad cells,
such as IsoT1 and UCH-L1, which participate in the disassembly of free
polyubiquitin chains (38, 39). When the proteasome is blocked, the
removal of ubiquitin from ubiquitin-conjugated substrates may be a
crucial step to engage alternative proteolytic pathways.
-inducible proteasomal
-subunits (
1i,
2i, and
5i) (40), important for production of MHC class I
ligands, as well as down-regulation of other gene products involved in
antigen processing (41-43). Tumors may fail to produce certain
immunodominant ligands due to altered proteasomal specificity (40, 44).
Our data show that reduced reliance on proteasomal proteolysis biases
cytosolic proteolysis to produce peptides that are less fit for MHC
class I assembly, thereby down-regulating the pool of potential MHC
class I-restricted epitopes. Such down-regulation is stably retained in
rapidly proliferating cells and can be induced at fairly high frequency
(10). Furthermore, EL-4ad cells appear to use this phenotype to avoid
immunological rejection during the formation of tumors in
vivo. An EL-4ad-like phenotype may be preferentially selected in
tumors that are poorly antigenic, a trait observed in many types of
tumors. Interestingly, in Burkitt's lymphomas, the oncogene
c-myc is known to induce down-regulation of a number of
components of the MHC class I-processing pathway, including
down-regulation of proteasomal chymotryptic activity and up-regulation
of tripeptidyl-peptidase II, thus linking the deficiency in antigen
processing directly to oncogene expression (45, 46). This study reveals
a new strategy for regulation of MHC class I processing: reduced
reliance on proteasomal activity to down-regulate generation of MHC
class I ligands
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ACKNOWLEDGEMENTS |
---|
We thank members of the Ploegh laboratory for support and discussions and Elisabeth Wolpert for CTL clone ln17. We also thank Maria Masucci and Hans-Gustaf Ljunggren for discussions and suggestions on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (to H. P.) and from Cancerfonden and the Swedish Research Council (to R. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by a Human Frontier Science Program long-term fellowship.
** Supported by a National Science Foundation graduate student fellowship.
Supported by a Swedish Research Council fellowship.
¶¶ Present address: Leiden Inst. of Chemistry, Gorlaeus Laboratory, 2300 RA Leiden, The Netherlands.
To whom correspondence should be addressed: MTC,
Karolinska Institutet, Theorells Väg 3, Stockholm S-171 77, Sweden. Tel.: 46-8-58589687; Fax: 46-8-7467637; E-mail:
rickard.glas@mtc.ki.se.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M211221200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MHC, major
histocompatibility complex;
IFN-, interferon-
;
NLVS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinyl sulfone;
CTL, cytotoxic T lymphocyte;
GFP, green fluorescent protein;
Ub, ubiquitin;
AAF-VS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-Ala-Ala-Phe-vinyl sulfone;
LLG(cis)-VS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Gly(cis)-vinyl
sulfone;
Ada-Ahx3-Leu3-VS, adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone;
AMC, 7-amino-4-methylcoumarin;
FACS, fluorescence-activated cell
sorter;
CMK, chloromethyl ketone;
APC, antigen presenting cell.
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