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Division Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands
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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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A wealth of experimental data has implicated the proteasome in the
generation of antigenic peptides (7). The proteasome is a
multicatalytic particle consisting of structural 
subunits and
catalytic ß subunits arranged as an
7ß7ß77
cylinder (8). The proteasome moves within the cell by diffusion (9) and
is a member of the Ntn-hydrolase family of enzymes, since active ß
subunits posses a catalytic N-terminal threonine residue (10). Up to
five types of catalytic activities have been ascribed to the proteasome
based on studies performed with fluorogenic peptide substrates. These
include a chymotrypsin-like activity and a trypsin-like activity, which
catalyze scission of the peptide bond on the C-terminal side of
hydrophobic and basic amino acids, respectively. An independent
acid-like enzyme activity has been postulated to be involved in
cleavage at the N-terminus of the substrate, with the products of
proteasome digestion being around nine amino acids in length (11). In
the assembled eukaryotic proteasome, only three of the seven types of
ß subunits are catalytically active. The generation of these active
sites takes place during proteasome assembly by cleavage of an
N-terminal pro-sequence that exposes the catalytic threonine (12, 13).
In mammals, these three catalytic subunits, ß1 (Y/
), ß2 (Z), and
ß5 (X/MB1), are co-ordinately replaced upon IFN-
stimulation by
the active ß subunits, LMP2, MECL1, and LMP7, respectively (14, 15).
Examination of the Saccharomyces cerevisae proteasome
crystal structure suggests that the incorporation of LMP2 into the
particle should enhance the generation of peptides with C-terminal
hydrophobic and basic residues at position 9 (11, 16, 17). This makes
them more suitable for binding to MHC class I molecules that
accommodate such residues in their F pockets. Consistent with this,
LMP2- and LMP7-deficient mice and cell lines have been claimed to
express diminished amounts of MHC class I molecules at the cell
surface, although the phenotypes are rather mild (18, 19).
Functional evidence for a role of the proteasome in the class I Ag presentation pathway also comes from studies using broad specificity inhibitors, which block the presentation of endogenous antigens to CTL (20, 21). More recently, a Streptomyces metabolite called lactacystin and its ß-lactone derivative have been shown to bind specifically and covalently to the catalytic subunits of mammalian proteasomes (22, 23, 24). Irreversible inhibition of proteasomes using lactacystin specifically abolishes the presentation of a range of viral proteins to CTL (25), although this may not be the case for all antigenic peptides (26).
In vivo, the proteasome interacts with modulatory protein complexes.
Included among these is the 19S cap, which is involved in the targeting
of ubiquitinated proteins to the proteasome (27), and the
IFN--inducible activator PA28 (28). PA28 exists as a hexamer of
and ß subunits that binds at the ends of the proteasome cylinder
(29). Overexpression of PA28 has been shown to enhance the
generation of some Ag for presentation to CTL (30) and in vitro, PA28
alters the pattern of peptide products generated by purified 20S
proteasome digests (31). However, how PA28 functions in vivo with
respect to the 26S proteasome complex in Ag presentation is
unresolved.
Previously, we used the proteasome-specific inhibitor lactacystin to
demonstrate that the trypsin-like activity of the proteasome was
closely correlated with the peptide loading of MHC class I molecules in
four different cell types (32). Both the trypsin-like and the
chymotrypsin-like activities of the proteasome became related to MHC
class I stability after IFN- stimulation. These results indicated
that in cells optimized for Ag presentation, the proteasome is the
limiting factor in the MHC class I Ag presentation pathway. In this
report we show that while this assertion holds for the majority of MHC
class I alleles, it is not universal. HLA-A3, HLA-A11, and HLA-B35 do
not conform to this model and are resistant to the effects of
proteasome inhibitors at concentrations that inhibit 70 to 80% of the
trypsin-like and chymotrypsin-like activities. This is also observed
across different cell lines. These results imply that while peptide
generation by the proteasome is limiting for most MHC class I alleles,
the peptide loading of other alleles is free from this limitation. The
implications of these findings will be discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proteasome-specific inhibitor lactacystin (22) was obtained
from E. J. Corey, Harvard University (Boston, MA), and was stored
as a powder or as a 10-mM stock solution in sterile water at 4°C. The
proteasome inhibitor PSI
(Cbz-Ile-Glu(O-t-Bu)-Ala-Leu; Calbiochem,
La Jolla, CA) and the proteasome inhibitor z-L3VS (33) (a
gift from Prof. H. Ploegh, Harvard University) were stored as 10-mM
stock solutions at -20°C in DMSO. Human rIFN- was obtained from
Boehringer Ingelheim (Ingelheim, Germany) and was added to HeLa cells
at a concentration of 100 U/ml for 72 h or 200 U/ml for 24 h where
relevant. Other chemicals were purchased from standard suppliers.
Cell lines
The human cell lines used in this study and their HLA types are as follows. AF cells (HLA-A1, -A11, -B35, and -B51) were provided by Dr. R. Khanna (Queensland Institute of Medical Research, Brisbane, Australia). OSH cells (HLA-A2, -A11, -B27, and -B35) were donated by Dr. F. Claas (AZL, Leiden, The Netherlands). HRC-5 cells (HLA-A11, -B7, and -B18) were obtained from the ECACC (Porton Down, U.K.). All these B cell lines were cultured in RPMI supplemented with 8% FCS. The cervical carcinoma cell line HeLa (obtained from the American Type Culture Collection, Rockville, MD) was believed to express HLA-A3, -A28, and -B35, but was found to be HLA-B75 positive and HLA-B35 negative and to express the HLA-A68 subtype of HLA-28 by DNA typing of the HeLa cells used in our experiments. This was confirmed by isoelectric focusing. HeLa was maintained in DMEM supplemented with 8% FCS.
Antibodies
The conformation-specific mAb W6/32 recognizes correctly folded
MHC class I complexes (34). The proteasome Abs used were MCP21 (a gift
from K. Hendil, August Krogh Institute, Copenhagen, Denmark), which
detects the HC3 (2) subunit (35) and IB5 (Organon Teknika, Belgium),
which recognizes the iota (1) subunit (36).
Proteasome activity assays
Proteasome activity assays were performed essentially as
described previously (32). Cells cultured in the presence of given
proteasome inhibitor concentrations were lysed in a hypotonic buffer
(10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, and 0.25 M
sucrose, pH 7.4), disrupted using an EMBL cell cracker, and
subsequently kept at 4°C. Equal amounts of cytosolic proteins (
300
µg) were used to immunoprecipitate proteasomes after extensive
preclearance. Specific immunoprecipitations were performed for 1.5
h using either MCP21 (anti-proteasome) or W6/32 as a negative
control. After washing five times, the pellets were split into equal
portions and assayed against 100 µM of the peptide substrates
Suc-Leu-Leu-Val-Tyr-AMC and z-Ala-Ala-Arg-AMC (Novabiochem,
Läutelfingen, Switzerland, and Bachem, Bubendorf, Switzerland) in
a total volume of 200 µl of hypotonic buffer at 37°C. These
substrates give an accurate determination of the chymotrypsin-like and
the trypsin-like activities of the proteasome, respectively. Aliquots
of 10 µl were quenched in duplicate into 1 ml of ethanol after 8
h, and the fluorescence of the free AMC (
excitation
= 370 nm, emission = 460 nm) was measured using a
spectrofluorometer (Perkin-Elmer, Den Bosch, The Netherlands).
Incubation buffer plus peptides, beads, or Ab alone gave negligible
fluorescence. Chymotrypsin and trypsin digestion of peptides was used
as a positive control. Proteasome activity was calculated by
subtracting the mean W6/32 (control) values from the mean MCP21 values
at each concentration and setting the figure obtained at 0 µM
lactacystin to 100% for each substrate. Control values were always
<10% of specific values.
Biosynthetic labeling and MHC class I immunoprecipitations
MHC class I stability assays. Approximately 2.5 x 106 cells/immunoprecipitation were incubated for 2 h in the presence or the absence of proteasome inhibitor. Cells were then starved for 1 h in cysteine/methionine-deficient RPMI medium and 10% FCS (also in the presence or the absence of inhibitor) and metabolically labeled with 125 µCi [35S]cysteine/methionine (Amersham) per sample for 20 min at 37°C. Aliquots were quenched in 10 ml of cold PBS and lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 150 mM NaCl, and 1% (w/v) Nonidet P-40. Nuclei were removed by centrifugation, and the lysates were precleared overnight on protein A-Sepharose beads. Aliquots of 5 µl were taken from the lysates and TCA precipitated to monitor the incorporation of [35S]cysteine/methionine into proteins. Lysates from equal amounts of TCA-precipitable radioactivity were split into two equal halves. One aliquot was incubated at 37°C for 2 h, and the other aliquot was kept at 4°C. All other steps were performed at 4°C. The lysates were precleared for 1 h with normal mouse serum on protein A-Sepharose beads, and MHC class I molecules were subsequently immunoprecipitated using the conformation-specific mAb W6/32. Pellets were washed four times in lysis buffer before analysis by isoelectric focusing (1D-IEF). Quantitation of gels was performed using a Fuji X-OMAT PhosphorImager (Fuji, Tokyo, Japan) equipped with TINA software. The amount of stable MHC class I molecules retrieved in the absence of proteasome inhibitors was set at 100% so that the relative percent stability of each MHC class I molecule could be compared.
Thermoinstability of HLA-A3 and -A11 during early biosynthesis. AF cells (10 x 106 cells/sample) or HeLa cells (2 x 106 cells/sample) were starved in cysteine/methionine-deficient medium, labeled in a small volume (AF cells in 200 µl/dish, HeLa cells in 1 ml/dish) with 250 µCi of [35S]methionine/cysteine, and chased for various times. Cells were lysed in 1 ml of 1% Nonidet P-40. Nuclei were removed from the lysates by centrifugation, and the lysates were split into two equal portions. One-half was incubated at 37°C for 30 min, whereas the other half was maintained at 4°C. MHC class I molecules were immunoprecipitated from the lysates with W6/32 and analyzed by 1D-IEF.
Pulse-chase analysis of MHC class I molecules. Biosynthetic labeling was performed as described above, except that after the radioactive pulse, cells were chased (in the continuous presence of proteasome inhibitors where relevant) with fresh medium containing 1 mM cysteine and 1 mM methionine. At the given chase points, equal amounts of cells were quenched in 10 ml of ice-cold PBS, pelleted, and lysed in lysis buffer. The lysates were precleared for 2 h with normal mouse serum on protein A-coupled Sepharose beads and then subjected to immunoprecipitation with W6/32 as before. After washing, the immunoprecipitates were analyzed by 1D-IEF.
Western blotting
Material from the cell lysates used in the proteasome activity assays was monitored to check for equal amounts of proteasomes. Ten micrograms of protein was dissolved in reducing loading buffer and subjected to 10% SDS-PAGE. Proteins were transferred onto nitrocellulose filters at 150 mA for 1.25 h. After transient staining with 0.4% (w/v) Ponceaux S to verify equal loading and correct transfer, the filters were blocked with 1% (w/v) milk powder before probing with a 1/500 dilution of the proteasome-specific Ab IB5. After stringent washing in 150 mM NaCl, 0.05% Tween-20, and 10 mM Tris, pH 8.0, secondary rabbit anti-murine peroxidase-coupled Abs were added and detected using an ECL kit (Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous work we analyzed cell lines that had been
stimulated with or without IFN- and treated in the presence or the
absence of the specific proteasome inhibitor lactacystin. We observed
that the stability of all alleles analyzed decreased in a titratable
manner with increasing concentrations of lactacystin and that this
correlated with a decrease in proteasome activity assayed under the
same conditions (32). However, all the MHC class I alleles that were
examined had a preference for a hydrophobic amino acid at position 9 in
the antigenic peptide. We were therefore interested to see whether MHC
class I alleles capable of accommodating a basic amino acid in this
position also followed this pattern, i.e., whether their peptide
loading was related to the trypsin-like activity of the proteasome.
Thus, HeLa cells that had been stimulated with IFN- were treated in
culture with increasing concentrations of the proteasome inhibitor
lactacystin.
The cells were metabolically labeled for 20 min, lysed, and split into
two equal portions, one of which was incubated at 37°C and the other
of which was kept on ice. The conformation-specific mAb W6/32 was then
used to solely immunoprecipitate those 37°C-resistant molecules that
had acquired stably bound peptides. Temperature stability as a result
of peptide binding is a well-documented and sensitive assay for
antigenic peptide loading of MHC class I molecules (37). The
peptide-loaded MHC class I molecules were analyzed by 1D-IEF (Fig. 1
A), which resolves
different class I alleles according to their isoelectric point (38).
Although the stabilities of HLA-A68 and HLA-B75 are clearly reduced
with increasing concentrations of lactacystin, HLA-A3 remains stable
even up to 10 µM lactacystin. The stability and peptide loading of
HLA-A3 are also unaffected in HeLa cells that have not been stimulated
with IFN- (Fig. 1B) and in HeLa cells that have
been treated with another proteasome inhibitor, z-L3VS
(Fig. 1C). This compound also covalently modifies the
active sites of proteasome ß subunits and specifically inhibits their
activity (33), producing results similar to those obtained with
lactacystin (A. M. Benham, M. Grommé, and J. Neefjes,
unpublished observations).
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D). Western
blotting of samples of the cell lysates used in this experiment
verifies that equal quantities of proteasomes were analyzed in the
activity assay (Fig. 1D, inset). Proteasomes isolated from
cells treated with the covalent inhibitor lactacystin show irreversible
and titratable inhibition. As we have previously observed, the
chymotrypsin-like activity of the proteasome is more readily inhibited
than the trypsin-like activity. Next, we compared the inhibition of the
proteasome with its effect on MHC class I peptide loading by
quantitating the temperature-dependent destabilization of class I
molecules in the presence of lactacystin (Fig. 1D).
Quantitation of HLA-A3 stability confirms that HLA-A3 is stable even
when 70 to 80% of the total proteasome activity is inhibited. This
contrasts with HLA-A68, expressed in the same cells, for which peptide
loading is directly related to proteasome activity, as noted
previously (32). The HLA-A3 superfamily member HLA-A11 is also peptide loaded during proteasome inhibition
HLA-A3 is one of only five alleles described to date with a peptide binding motif that can accommodate the basic residues arginine or lysine at position 9 of the antigenic peptide. This set of HLA-A3, -A11, -A31, -A33, and -A68 constitutes the A3-like supertype, and they all contain negatively charged acidic residues in the hypervariable F pocket of the binding groove to facilitate the loading of a C-terminal basic peptide (39).
To determine whether any other members of the HLA-A3 family were also
resistant to proteasome inhibition, we analyzed HLA-A11 expressed in
the B cell line AF. AF cells were treated in the presence of increasing
concentrations of the proteasome inhibitor z-L3VS and
metabolically labeled for 20 min. MHC class I molecules were
immunoprecipitated from cell lysates and analyzed by 1D-IEF as before.
Figure 2A shows that HLA-A1
and -B51 become titratably unstable upon proteasome inhibition.
However, under exactly the same conditions and within the same cells,
HLA-A11 is completely unaffected by z-L3VS. The mean
stability of HLA-A11 was compared with those of HLA-A1 and -B35 and was
determined in two separate experiments by analyzing the IEF gels using
a PhosphorImager (Fig. 2B). This analysis confirms
that HLA-A11 remains stable while HLA-A1 does not. Note that HLA-B35 is
also quite stable in the presence of z-L3VS.
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B clearly indicates
that while inhibition of the trypsin-like activity of the proteasome is
mirrored by inhibition of the loading of HLA-A1, no such relationship
exists for HLA-A11. Peptide loading of HLA-B35 is also largely
resistant to z-L3VS. Experiments using lactacystin gave
similar results (not shown).
It is theoretically possible that HLA-A3 and HLA-A11, unlike other MHC
class I alleles, are thermostable per se in the absence of peptide and
therefore do not dissociate under the conditions used in these assays.
Our previous experiments showed that early during MHC class I
biosynthesis, heavy chain/ß2m heterodimers form that have
not yet become peptide loaded and thus pass through a
temperature-sensitive stage (40). We made use of these observations to
verify that HLA-A3 and HLA-A11 fold normally and are transiently
thermolabile before peptide binding. Hence, a short pulse-chase
experiment was performed using both AF cells (Fig. 3A) and HeLa cells
(Fig. 3B). Cells were metabolically labeled for 2 min
and then chased from 0 to 10 min after the pulse before detergent
lysis. Thermostability was assayed by incubating one-half of the lysate
at 4°C and the other half at 37°C before immunoprecipitation with
W6/32 and analysis by 1D-IEF.
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reveals that during the early stages of biosynthesis, at 1 min
for AF (Fig. 3A) and 3 min for HeLa (Fig. 3B), a large majority of MHC class I molecules,
including HLA-A3 and HLA-A11, are temperature sensitive and not yet
peptide loaded. Conversely, after 10-min chase, all the MHC class I
alleles become stably peptide loaded and no longer dissociate at
37°C. Thus, like the other MHC class I molecules that were previously
tested (40), HLA-A3 and -A11 are transiently thermolabile early during
biosynthesis.
To show that the allele-specific effects on peptide loading seen to
date were indeed due to the specific effects on proteasome activity, AF
cells were cultured in the presence of three different proteasome
inhibitors. z-L3VS is a covalent peptide analogue, PSI is a
noncovalent peptide analogue (41), and lactacystin is a lactone-based
metabolite. AF cells were cultured in the presence of each of these
inhibitors before immunoprecipitation of peptide-filled class I
molecules with W6/32. Figure 4
demonstrates that 10-µM concentrations of all three inhibitors
severely reduce the peptide loading of HLA-A1 and -B51 and have a
partial effect on HLA-B35, but have little effect on the loading of
HLA-A11. The least specific proteasome inhibitor, PSI, reduces the
amount of peptide-loaded HLA-A11 by approximately 10 to 15%.
Concentrations of lactacystin up to 50 µM also have little effect on
the stability of HLA-A11.
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,
HLA-A11 was fully stable in both cell lines when proteasome activity
was substantially inhibited. Similar results were obtained when the
cells were treated with z-L3VS and in cells where HLA-A11
was the only HLA-A locus product expressed (not shown). This suggests
that the unusual loading of HLA-A11 depends upon the allele itself
rather than its specific cellular environment.
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Having noted that the stability of HLA-A11 was independent of
proteasome activity, we next analyzed whether the subsequent maturation
of HLA-A11 and its egress from the ER remained normal. Maturation
of individual class I alleles can easily be monitored by 1D-IEF, since
these polypeptides acquire sialic acid modifications as they pass
through the trans-Golgi, causing them to migrate at
progressively more acidic (lower) positions in the gel. Thus, AF cells
were treated with either DMSO or 10 µM z-L3VS and
metabolically labeled for 20 min. The cells were chased for 0, 20, 60,
or 120 min, and equal numbers of cells were removed at each time point
for lysis and immunoprecipitation of their MHC class I molecules.
1D-IEF analysis (Fig. 6) revealed that
the maturation of HLA-A1 was compromised by proteasome inhibition, as
evidenced by the slower disappearance of the immature band (Fig. 6, species 0). HLA-A11 matured at a similar rate in the presence and the
absence of z-L3VS (Fig. 6, species 1 and 2). Similar
results were obtained when the experiment was repeated using 10 µM
lactacystin (not shown). Thus, HLA-A11 is efficiently loaded with
peptides and exits the ER when the assembly and maturation of HLA-A1
are severely compromised by the inhibition of proteasome activity.
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), and this was
also seen with other cell lines, including OSH and HRC-5 (not shown).
Along with the observation that the stability of HLA-B35 did not
closely correlate with proteasome activity (see Fig. 2B), this suggests that HLA-B35 is an intermediate
allele that is only partially refractory to proteasome inhibitors. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(32). These alleles all
posses a deep, uncharged F pocket to accommodate bulky amino acids.
This relationship also holds, albeit less well, for HLA-A2, which is
partly TAP independent in that it can bind peptides generated in the ER
by signal peptidase (42, 43, 44, 45). When we extended our investigations to
encompass two MHC class I molecules that bind peptides with basic
residues at position 9, and thus have an acidic F pocket, no
relationship between proteasome activity and peptide loading was found.
The phylogenetically related MHC class I molecules, HLA-A3 and HLA-A11,
remain stable in the presence of different proteasome inhibitors, even
at very high drug concentrations (Figs. 1, 2, and 4). These conditions
inhibit overall proteasome activity by over 70 to 80%, as judged by
assays using fluorogenic peptides. HLA-B35, although not entirely
resistant to proteasome inhibitors, also exhibits a poor relationship
between peptide loading and proteasome activity. All other MHC class I
alleles, even when expressed by the same HLA-A3- or HLA-A11-positive
cell lines, have a class I loading pattern that closely correlates with
proteasome activity, as we have previously observed (32).
The resistance of peptide loading to proteasome inhibitors is IFN-
independent and therefore does not rely upon the level of MHC class I
expression, alterations in proteasome subunit composition, or changes
in the peptide profile generated by "immunoproteasomes." The
loading behavior that we observed also cannot be explained by
differential affinities for the TAP transporter between either 1) the
C-terminus of the peptide substrate (6) or 2) the MHC class I molecules
themselves. Although HLA-A3 and HLA-A11 interact strongly with TAP, the
proteasome-dependent alleles HLA-A2, -B51, and -B8 (among others) do so
as well (46).
We anticipated that the behavior of HLA-A3 and HLA-A11 might be a
common feature of the HLA-A3 superfamily, comprising HLA-A0301, -A1101,
-A3101, -A3301, and -A6801. However, analysis of cell lines expressing
HLA-A68 (e.g., HeLa cells), -A31, and -A33 alleles has not shown them
to withstand proteasome inhibition (Fig. 1 and data not shown). HLA-A3
and HLA-A11 share overlapping peptide specificities and are capable of
binding the same peptides with high affinity (47). As well as the
characteristic positively charged amino acid at position 9 of the
antigenic peptide, A3 superfamily members also prefer a
hydroxyl-containing or hydrophobic residue at position 2. It is
unlikely that a specific A3 superfamily motif or mutation segregates
with proteasome unrestricted loading, since only HLA-A11 and HLA-A3
appear to behave anomalously. However, there are some subtle
differences between family members. HLA-A3 and -A11 both strongly
prefer a Lys at peptide position 9, unlike the preference for Arg of
HLA-A31 and -A33 (48). HLA-A3 and -A11 also share hypervariable Q62,
R114, D116, K144, and R145 amino acids within the peptide binding
groove, a combination of residues that is not shared with HLA-A68,
-A31, or -A33 (49). HLA-A3 and HLA-A11 are also located separately from
HLA-A31, -A33, and -A68 on a relatively recent branch of the MHC class
I phylogenetic tree (50). Although it remains speculative, these
differences could reflect a more recently evolved capacity to bind
peptides engendered by a putative protease or peptidase that generates
a class I binding product with a C-terminal lysine.
The participation of an as yet unidentified protease or pathway in the
loading of some MHC class I molecules has been postulated (26, 51, 52).
Such an enzyme could either provide peptides in parallel to the
proteasome or could take over its role in the absence of proteasome
function. If such a molecule were to be a trypsin-like protease that
generated products terminating in lysine residues, then the loading of
HLA-A3 and HLA-A11 could be favored above that of the hydrophobic P9
alleles. Consistent with the idea of another protease, we often see a
small cohort of lactacystin- or z-L3VS-resistant MHC
class I molecules (e.g., HLA-A68; Fig. 2) at high inhibitor
concentrations, when the relationship between proteasome activity and
MHC class I loading breaks down. However, attempts to block the loading
of HLA-A11 and HLA-B35 in AF cells with a variety of other protease
inhibitors have proved unsuccessful to date (data not shown).
Alternatively, a small amount of proteasome function, particularly of the trypsin-like activity, may be sufficient to provide enough peptides to fully load HLA-A3 and HLA-A11 and to partially load HLA-B35. In our experiments, we can never totally inhibit the trypsin-like activity of the proteasome, and it may be that 20% or less of this activity is sufficient to load some MHC class I molecules that require a basic residue at the C-terminal position. Residual trypsin-like activity would then not be enough to load the majority of alleles that require a hydrophobic C terminal residue. It is possible that peptides generated by the trypsin-like activity of the proteasome predominantly bind to HLA-A3 family alleles and that peptides generated by the chymotrypsin-like activity bind to the other alleles. The N-termini of these peptides are presumably created by a lactacystin/z-L3VS independent and acidic enzymatic component of the proteasome (11). Specific inhibitors of either the chymotrypsin-like or trypsin-like activity would be required to test this hypothesis directly.
In this light, it is intriguing to note that MHC class I molecules appear to have evolved to fit the products of proteasomal digestion, with no known MHC class I molecule able to present peptides with an acidic residue in peptide position 9 (53). Thus, the relatively high abundance of alleles requiring a hydrophobic P9 vs a basic P9 could be an evolutionary consequence of the relative abundance of these proteasomal products in vivo.
The finding that proteasome inhibition results in a differential effect on MHC class I loading is novel and intriguing. Inhibiting proteasome function by 70 to 80% has little effect on HLA-A3, -A11, and -B35, while within the same cells, the loading and maturation of all other alleles are severely compromised. These observations highlight another level of microdiversity in the MHC class I Ag-processing pathway.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jacques Neefjes, Division Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; PSI, Cbz-Ile-Glu(O-t-Bu)-Ala-Leu; 1D-IEF, one-dimensional isoelectric focusing.
Received for publication December 2, 1997. Accepted for publication February 27, 1998.
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B and stabilises a newly phosphorylated form of IB- that is still bound to NF-B. EMBO J. 13:5433.[Medline]
, G. Jung, H.-G. Rammensee. 1994. Peptide motifs of HLA-A1, -A11, -A31 and -A33 molecules. Immunogenetics 40:238.[Medline]
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 S. Guil, M. Rodriguez-Castro, F. Aguilar, E. M. Villasevil, L. C. Anton, and M. Del Val Need for Tripeptidyl-peptidase II in Major Histocompatibility Complex Class I Viral Antigen Processing when Proteasomes are Detrimental J. Biol. Chem., December 29, 2006; 281(52): 39925 - 39934. [Abstract] [Full Text] [PDF]
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L. Li and M. Bouvier Structures of HLA-A*1101 Complexed with Immunodominant Nonamer and Decamer HIV-1 Epitopes Clearly Reveal the Presence of a Middle, Secondary Anchor Residue J. Immunol., May 15, 2004; 172(10): 6175 - 6184. [Abstract] [Full Text] [PDF]
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F. A. Bland, M. K. Lemberg, A. J. McMichael, B. Martoglio, and V. M. Braud Requirement of the Proteasome for the Trimming of Signal Peptide-derived Epitopes Presented by the Nonclassical Major Histocompatibility Complex Class I Molecule HLA-E J. Biol. Chem., September 5, 2003; 278(36): 33747 - 33752. [Abstract] [Full Text] [PDF]
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B. Kessler, X. Hong, J. Petrovic, A. Borodovsky, N. P. Dantuma, M. Bogyo, H. S. Overkleeft, H. Ploegh, and R. Glas Pathways Accessory to Proteasomal Proteolysis Are Less Efficient in Major Histocompatibility Complex Class I Antigen Production J. Biol. Chem., March 14, 2003; 278(12): 10013 - 10021. [Abstract] [Full Text] [PDF]
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M. K. Lemberg, F. A. Bland, A. Weihofen, V. M. Braud, and B. Martoglio Intramembrane Proteolysis of Signal Peptides: An Essential Step in the Generation of HLA-E Epitopes J. Immunol., December 1, 2001; 167(11): 6441 - 6446. [Abstract] [Full Text] [PDF]
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C. J. Luckey, J. A. Marto, M. Partridge, E. Hall, F. M. White, J. D. Lippolis, J. Shabanowitz, D. F. Hunt, and V. H. Engelhard Differences in the Expression of Human Class I MHC Alleles and Their Associated Peptides in the Presence of Proteasome Inhibitors J. Immunol., August 1, 2001; 167(3): 1212 - 1221. [Abstract] [Full Text] [PDF]
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 J. Yewdell, U Schubert, and J. Bennink At the crossroads of cell biology and immunology: DRiPs and other sources of peptide ligands for MHC class I molecules J. Cell Sci., January 3, 2001; 114(5): 845 - 851. [Abstract] [PDF]
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A. Bai, C. J. Aldrich, and J. Forman Factors Controlling the Trafficking and Processing of a Leader-Derived Peptide Presented by Qa-1 J. Immunol., December 15, 2000; 165(12): 7025 - 7034. [Abstract] [Full Text] [PDF]
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K. Schwarz, R. de Giuli, G. Schmidtke, S. Kostka, M. van den Broek, K. B. Kim, C. M. Crews, R. Kraft, and M. Groettrup The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used to Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses J. Immunol., June 15, 2000; 164(12): 6147 - 6157. [Abstract] [Full Text] [PDF]
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S. D. Wainwright, P. A. Biro, and C. H. Holmes HLA-F Is a Predominantly Empty, Intracellular, TAP-Associated MHC Class Ib Protein with a Restricted Expression Pattern J. Immunol., January 1, 2000; 164(1): 319 - 328. [Abstract] [Full Text] [PDF]
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X. Y. Mo, P. Cascio, K. Lemerise, A. L. Goldberg, and K. Rock Distinct Proteolytic Processes Generate the C and N Termini of MHC Class I-Binding Peptides J. Immunol., December 1, 1999; 163(11): 5851 - 5859. [Abstract] [Full Text] [PDF]
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G. Lauvau, B. Gubler, H. Cohen, S. Daniel, S. Caillat-Zucman, and P. M. van Endert Tapasin Enhances Assembly of Transporters Associated with Antigen Processing-dependent and -independent Peptides with HLA-A2 and HLA-B27 Expressed in Insect Cells J. Biol. Chem., October 29, 1999; 274(44): 31349 - 31358. [Abstract] [Full Text] [PDF]
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T. Serwold and N. Shastri Specific Proteolytic Cleavages Limit the Diversity of the Pool of Peptides Available to MHC Class I Molecules in Living Cells J. Immunol., April 15, 1999; 162(8): 4712 - 4719. [Abstract] [Full Text] [PDF]
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A. Komlosh, F. Momburg, T. Weinschenk, N. Emmerich, H. Schild, E. Nadav, I. Shaked, and Y. Reiss A Role for a Novel Luminal Endoplasmic Reticulum Aminopeptidase in Final Trimming of 26 S Proteasome-generated Major Histocompatability Complex Class I Antigenic Peptides J. Biol. Chem., August 3, 2001; 276(32): 30050 - 30056. [Abstract] [Full Text] [PDF]
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D. R. Johnson and B. Mook-Kanamori Dependence of Elevated Human Leukocyte Antigen Class I Molecule Expression on Increased Heavy Chain, Light Chain (beta 2-Microglobulin), Transporter Associated with Antigen Processing, Tapasin, and Peptide J. Biol. Chem., May 26, 2000; 275(22): 16643 - 16649. [Abstract] [Full Text] [PDF]
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) and HLA-A68 (
)
were quantitated by exposing the gel depicted in A to a
PhosphorImager. HLA-B75 could not be accurately quantitated.
Proteasomes were isolated from IFN-
). Proteasomes were isolated from AF
cells that had been cultured at the same concentrations of
z-L3VS and assayed against 100 µM of either
z-Ala-Ala-Arg-AMC (•) or Suc-Leu-Leu-Val-Tyr-AMC (+). The
fluorescence values obtained for each substrate were calculated as
described, and bars represent 95% confidence intervals. As with HeLa,
the chymotrypsin-like activity of the proteasome is more readily
inhibited than the trypsin-like activity.
