(Received for publication, August 22, 1996, and in revised form, November 25, 1996)
From the Division of Lymphocyte Biology, Dana-Farber
Cancer Institute, Boston, Massachusetts 02115, the
§ Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115, the ¶ Department of Chemistry and
Chemical Biology, Harvard University, Cambridge, Massachusetts 02138,
and the
Department of Pathology, Harvard Medical
School, Boston, Massachusetts 02115
The antibiotic lactacystin was reported to
covalently modify -subunit X of the mammalian 20 S proteasome and
inhibit several of its peptidase activities. However, we demonstrate
that [3H]lactacystin treatment modifies all the
proteasome's catalytic
-subunits. Lactacystin and its more potent
derivative
-lactone irreversibly inhibit protein breakdown and the
chymotryptic, tryptic, and peptidylglutamyl activities of purified
20 S and 26 S particles, although at different rates. Exposure to
these agents for 1 to 2 h reduced the degradation of short- and
long-lived proteins in four different mammalian cell lines. Unlike
peptide aldehyde inhibitors, lactacystin and the
-lactone do not
inhibit lysosomal degradation of an endocytosed protein. These agents
block class I antigen presentation of a model protein, ovalbumin
(synthesized endogenously or loaded exogenously), but do not affect
presentation of the peptide epitope SIINFEKL, which does not require
proteolysis for presentation. Generation of most peptides required for
formation of stable class I heterodimers is also inhibited. Because
these agents inhibited protein breakdown and antigen presentation
similarly in interferon-
-treated cells (where proteasomes contain
LMP2 and LMP7 subunits in place of X and Y), all
-subunits must be affected similarly. These findings confirm our prior conclusions that
proteasomes catalyze the bulk of protein breakdown in mammalian cells
and generate the majority of class I-bound epitopes for immune
recognition.
MHC1 class I molecules typically bind
8-9-residue peptides derived from cellular or viral proteins. Most of
these peptides are generated by protein breakdown in the cytosol and
are transported by the transporter associated with antigen presentation
transporter into the endoplasmic reticulum (1). Here the peptide, a MHC class I heavy chain, and a 2-microglobulin molecule
associate, and the complex is then transported through the Golgi
apparatus to the plasma membrane (1). This process allows T lymphocytes to screen for cells that are synthesizing foreign or abnormal proteins.
The mechanisms responsible for the generation of the class I-presented
peptides had been unclear until recently. However, a variety of recent
evidence has suggested that the proteasome plays a primary role in this
process and that during the turnover of cytosolic and nuclear proteins
a fraction of the peptides generated by the proteasome are utilized for
MHC class I presentation (1-3).
Proteasomes are found in the nucleus and cytosol of all cells and are
essential components of the ATP-ubiquitin-dependent pathway
for protein degradation. The 20 S proteasome is a 700-kDa particle
with multiple peptidase activities, including a chymotryptic-, tryptic-, and peptidylglutamyl-like activity (4-6). It is a
cylindrical-shaped structure composed of four rings, the outer two each
contain seven -subunits and the inner two each contain seven
-subunits (7, 8). Proteolysis occurs in the central chamber of this
particle and is catalyzed through a nucleophilic attack on the peptide bond by the N-terminal threonine hydroxyl groups on certain
-subunits, named X (
), Y(
), Z (HCO), and their homologues
LMP2, LMP7, and LMP10 (MECL-1) (9-12). The 20 S particle functions as
the proteolytic core of a larger 26 S (2000 kDa)
ATP-dependent proteasome complex which selectively degrades
proteins that are modified by conjugation to multiple ubiquitin
molecules (6, 13, 14). Although the ubiquitin-proteasome pathway is
clearly essential for the rapid degradation of short-lived or highly
abnormal proteins and polypeptides in yeast (15) and mammalian cells
(16-19), its role in degradation of the bulk of cell proteins, which
are long-lived, is uncertain (16, 20).
Several lines of evidence had suggested that the proteasome was also
responsible for the generation of some class I-presented peptides. Two
of the proteasome's catalytic -subunits, LMP7 and LMP2, are encoded
in the MHC region (21, 22), and the absence of these subunits in mutant
cells or mice decreases the efficiency of presentation of certain
antigens (23-26). Furthermore, inhibiting ubiquitin conjugation in a
TS mutant decreased antigen presentation of a model protein (27). On
the other hand, modifications of a protein that stimulate its
ubiquitinylation and degradation by 26 S proteasomes enhance its rate
of MHC class I presentation (28). Finally, when 20 S proteasomes are
incubated with a protein for extended periods, they can generate some
class I-binding peptides, although such experiments involve highly
nonphysiological conditions (29-31).
More definitive evidence for the proteasome's general importance in antigen presentation in vivo requires methods to specifically inhibit proteasome function in intact cells. Recently, certain peptide aldehydes (such as N-acetyl-L-leucinyl-L-leucinal-L-norleucinal, LLnL, and N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal, MG115) have been shown to strongly inhibit multiple peptidase activities of proteasomes and to reduce protein hydrolysis (3). Moreover, these agents can enter cells and block the degradation of most cellular proteins and the generation of the majority of class I-presented peptides (3). Although these peptide aldehydes can also inhibit the cysteine proteases found in lysosomes and calpains (3), several findings argued that the inhibition of protein degradation and antigen presentation was due to effects on the proteasome. For example, the rank order of potencies of different peptide aldehydes in inhibiting the proteasome was the same as for blocking protein degradation and antigen presentation and did not correlate with efficacy against cysteine proteases (3). Also, inhibition of these cysteine proteases did not affect protein breakdown or antigen presentation. Nevertheless, more selective proteasome inhibitors are needed to establish definitively a major role for the proteasome in these processes.
A chemically distinct type of proteasome inhibitor is the antibiotic
lactacystin which was isolated from Streptomyces by Omura and colleagues (32) and synthesized by Corey and colleagues (33).
Fenteany et al. (11) have shown that
[3H]lactacystin bound covalently to a polypeptide
identified as proteasome -subunit X in bovine brain cells and that
lactacystin could irreversibly inhibit the chymotryptic- and
tryptic-like peptidase activity and reversibly inhibit the post-acidic
activity of purified proteasomes. Lactacystin spontaneously hydrolyzes into clasto-lactacystin
-lactone, which appears to be the
active inhibitor that reacts with the N-terminal threonine of subunit X
(34). Because lactacystin and the
-lactone appeared to be highly
specific inhibitors that do not affect cysteine or serine proteases,
they are potentially very useful research tools. We therefore examined
whether they inhibit proteasomes that contain
-type subunits not
expressed in neurons (e.g. LMP7, LMP2, and MECL-1), whether
they reduce protein hydrolysis in vitro and in cells, and
whether they can block MHC class I antigen presentation.
Chicken ovalbumin and keyhole limpet hemocyanin (KLH) were purchased from Sigma. The SIINFEKL peptide was synthesized by the molecular biology core of the Dana-Farber Cancer Institute (Boston, MA).
Monoclonal antibodies were purified from culture supernatants of
hybridomas Y3 (anti-class I Kb heterodimers) (35), B22
(anti-Db heterodimers) (36), and S19.8.503
(anti-2-microglobulin) by affinity chromatography on
protein A-Sepharose. Rabbit anti-Kb exon 8 was provided by
Dr. Ian York.
LLnL was purchased from Sigma. Lactacystin and
clasto-lactacystin -lactone were kindly provided by
ProScript (Cambridge, MA) and Dr. K. Omura (Kitasato Institute, Tokyo,
Japan). [3H]Lactacystin was prepared as described
(11).
Recombinant human IFN- was a kind gift from Biogen Inc. (Cambridge,
MA). FITC-casein was prepared as described previously (37). Synthetic
substrates for peptidase assays were purchased from Bachem
(Switzerland).
LB27.4 B lymphoblasts
(38), E36.12.4 hamster carcinoma cells (27), RF33.70 (OVA 257-264 plus
Kb-specific) T-T hybridomas (39), RMA T lymphoblasts (40),
U937 monocytoid cells, and A3.1A7 murine macrophage cells (41) have been described previously. 143B.TK human osteosarcoma cells were kindly provided by J. Yewdell and J. Bennink. The human lymphoblastoid cell lines 721 and .174 were kindly provided by R. DeMars (42). A
retroviral-transduced B16 melanoma line, which constitutively secretes
murine IFN-
, was a gift from Dr. Glenn Dranoff (Dana-Farber Cancer
Institute, Boston, MA). DAP34.8 cells are mouse L cells stably
transfected with class I Kb and CD54.
For electroporation loading experiments LB27.4 cells were grown in Opti-MEM (Life Technologies, Inc.) supplemented with normal mouse serum (1%). In all other experiments cells were grown in RPMI 1640 or Dulbecco's modified Eagle's medium (Irving Scientific, Santa Ana, CA) supplemented with fetal calf serum (10%) and antibiotics. Transfected cells were passaged with geneticin (Life Technologies, Inc.).
LB27.4 and DAP34.8 cells were incubated in the presence or absence of
murine IFN- (20% supernatant from confluent cultures of
IFN-
-transduced B16 cells, containing approximately 10 units/ml IFN-
) for 3 to 4 days at 37 °C. 721 cells and U937 human
macrophages were treated with 3000 units/ml recombinant human IFN-
for 3 or 7 days, respectively.
20 S and 26 S proteasome-rich cytosolic fractions ("proteasome fractions") were isolated from cultured cells as described previously (43, 44), and 20 S and 26 S particles were purified from rabbit skeletal muscle as described in Ref. 6.
Activity against the fluorogenic peptide substrates was determined by continuously monitoring the fluorescence of 7-amino-4-methylcoumarin (excitation wavelength 380 nm, emission wavelength 460 nm) on a McPherson Spectrofluorimeter FL-750 connected to a Waters 740 Data Module. The assays were performed in 50 mM Tris-HCl buffer (pH 7.5) in a 0.75-ml final reaction volume. All peptide stocks were prepared and stored in Me2SO, and the final concentration of the solvent in the assay never exceeded 2%, which did not affect proteasome activity. The rates of substrate hydrolysis were determined at 25 °C from the initial linear portions of curves (2-5 min). The peptide substrate concentrations used were Suc-Leu-Leu-Val-Tyr-AMC, 67 µM, Boc-Leu-Arg-Arg-AMC, 100 µM, and Z-Leu-Leu-Glu-AMC, 100 µM. Enzyme concentration was 0.1-0.5 nM. Duplicate assays were performed for every time point.
The continuous assay of proteasome proteinase activity was performed in 50 mM Tris-HCl (pH 7.5) containing 7.5 µg of FITC-casein in a 0.75-ml volume. The mixture was equilibrated in the cuvette at 25 °C until a steady fluorescence intensity was observed (generally after 5 min). 1-3 µg of enzyme was then added, and the linear increase of fluorescence was recorded for up to 30 min at 490 nm excitation and emission of 521 nm. A blank without proteasome was run in parallel.
The rate of inactivation of proteasome by inhibitors was calculated
under pseudo first-order conditions (i.e. [I] [E]). Apparent rate constants
(kobs) were determined from semi-logarithmic plots of ln Vt/V0 against
incubation time of inhibitor with enzyme (V0 is
enzyme activity in the absence of inhibitor and Vt
is residual activity in the presence of inhibitor). Apparent
second-order rate constant ka is calculated from
ka = kobs/[I]. The
half-life of the free enzyme is given by t1/2 = 0.693/kobs. The irreversibility of inhibitors was tested by measuring the enzyme activity after dilution of reaction
1000 times.
vTF7-3 vaccinia virus, encoding T7 RNA
polymerase, was obtained from ATCC (VR-2153) (45). Viral stocks
(approximately 108 plaque-forming units per ml) were
prepared from infected 143B.TK cells.
The plasmid pBS.OVA was constructed by cloning ovalbumin cDNA into
pBluescript SK (Promega, Madison, WI), using HindIII and XbaI (Life Technologies, Inc.), such that the orientation of
the ovalbumin gene was under the control of the T7 RNA polymerase promoter. The plasmid pBS.miniOVA was constructed from the primers 5-AGCTTCACCATGTCTATAATAAACTTTGAGAAGTTATAGTGACCATGGG-3
and
5
-AGTGGTACAGATATTATTTGAAACTCTTCAATATCACTGGTACCCTTAA-3
. Primers were
phosphorylated with polynucleotide kinase and then annealed and ligated
into pBluescript SK, using HindIII and EcoRI (Life Technologies, Inc.). The gene expressed under control of the T7
RNA polymerase promoter encoded the SIINFEKL peptide with an initiating
N-terminal Met and contained an NcoI site near the 3
end
for selection after ligation and transformation. cDNAs were
sequenced at the Dana-Farber Cancer Institute molecular biology core
facility.
Cells (E36.12.4, LB27.4, RMA, or A3.1A7) were incubated with [3H]tyrosine (5 µCi/ml) for 6 h at 37 °C to assay degradation of long-lived proteins and for 1 h to assay degradation of short-lived proteins. Assays for the measurement of degradation of such proteins have been previously described in Ref. 3.
To measure lysosomal degradation of exogenous proteins, keyhole limpet hemocyanin (KLH) was used as a substrate. 200 mg of KLH (Sigma) was reacted with [125I]sodium iodide (3 mCi) to a specific activity of 3 µCi/mg by the IODO-GEN method. Assays for the uptake and catabolism of 125I-KLH were then performed according to the method of Gray et al. (46). 125I-KLH-pulsed A3.1A7 cells were incubated in triplicate (4 × 106 per group) for 4 h at 37 °C in the presence or absence of inhibitors. Culture medium and cell pellets were treated with trichloroacetic acid, and radioactivity in supernatants and pellets was measured. Protein breakdown was evaluated by measuring the conversion of radiolabeled protein to trichloroacetic acid-soluble form appearing in culture medium.
Antigen Presentation AssaysTo assay for the presentation
of endogenously synthesized antigen, E36.12.4 cells or DAP34.8 cells
were seeded overnight in 6-well plates to a density of
106 cells/well. Cells were then preincubated for 30 min
in the presence or absence of inhibitors, in Opti-MEM medium. vTF7-3
virus was then added to wells at 10 plaque-forming units/cell for 30 min. Media containing vTF7-3 was then removed and plasmid (pBS.OVA (5 µg) or pBS.miniOVA (0.7 µg)) in Lipofectin (Life Technologies, Inc.) was added in 1 ml of Opti-MEM, in the presence or absence of
inhibitors. Cells were incubated for 140 min (E36.12.4 cells) or 200 min (DAP34.8 cells) at 37 °C and then fixed in 1% paraformaldehyde for 10 min at room temperature. The presence of peptide-Kb
complexes on the surface of these cells was measured by quantifying the
amount of interleukin-2 produced by the
ovalbumin-Kb-specific T-T hybridoma RF33.70, after
stimulation with antigen-presenting cells in duplicate cultures, as
described previously (39).
To assay for presentation of antigen introduced into the cytosol by electroporation, LB27.4 cells or DAP34.8 cells were permeabilized as described (3) in the presence or absence of inhibitors, with ovalbumin (30 mg/ml) or SIINFEKL peptide (0.5 µg/ml) in electroporation buffer. Cells were either fixed with paraformaldehyde immediately after electroporation or were incubated for 2 h at 37 °C and then fixed. The inhibitors were present where appropriate during the 0.5-h pretreatment, electroporation, and 2-h incubation steps. The presence of SIINFEKL·Kb complexes on the cell surface was determined using RF33.70 T-T hybridomas as described above and in Ref. 47.
Electrophoretic Methods and Western BlottingFractions from
proteasome-rich cells or purified proteasomes were incubated with
[3H]lactacystin (100 µM) for 3 h at
37 °C. Since proteasomes from different cell types, or cells treated
with IFN-, differ in their 20 S subunit composition, control and
IFN-
-treated macrophages or lymphoblasts were used as sources of
proteasomes to ensure that all 20 S subunits would be present in
detectable amounts in the preparations. Two-dimensional PAGE
(O'Farrell system) was used to detect and identify proteasomal
subunits modified by [3H]lactacystin. Proteins from the
two-dimensional gels (12.5%) were transferred to nitrocellulose, and
the filters were incubated with specific antibodies to
-subunits
(kindly provided by K. Tanaka and K. Hendil) and then with
125I-protein A.
-Subunits of the 20 S proteasome
recognized by antibodies were visualized on the filters using
PhosphorImaging (Molecular Dynamics). Antibody-125I-protein
A complexes were then stripped from the filters until no detectable
125I remained. The filters were reprobed with other
anti-
-subunit antibodies and 125I-protein A, and finally
stripped filters were treated with Amplify enhancer (Amersham Corp.),
dried, and exposed to Kodak AR film to obtain fluorograms with
visualized proteins labeled by [3H]lactacystin. The
subunits that [3H]lactacystin covalently modifies were
identified on the fluorograms obtained from two-dimensional gels and
compared with Western blot phosphorimages.
The assembly of MHC class I molecules was measured as described previously in Ref. 3.
The 20 S proteasome contains distinct activities
that cleave model peptides after hydrophobic, basic, and acidic
residues (4-6), and Fenteany et al. (11) demonstrated that
lactacystin blocks these activities in crude extracts. Both lactacystin
and the -lactone rapidly inactivated the chymotryptic activity of 20 S and 26 S proteasomes purified from skeletal muscle (Table I). The
-lactone appeared approximately 20-fold more
effective than lactacystin, in accord with prior findings that
lactacystin must first be converted to the lactone to inhibit
proteasomes (34). Unlike the peptide aldehydes, these agents were more
potent against the 26 S particles than 20 S. The lactone also
inhibits the tryptic and peptidylglutamyl-hydrolyzing activities, but
these inhibitory reactions are much slower than inhibition of the
chymotryptic activity and require higher concentrations of the
-lactone.
|
In vivo, the primary function of the proteasome is the
hydrolysis of proteins into oligopeptides (6). Since the effect of
these inhibitors on this process had not been examined, we tested their
ability to inhibit the hydrolysis of FITC-conjugated casein (Table
II). This protein substrate is degraded in an
ATP-dependent reaction by the 26 S complex without
ubiquitinylation and in an ATP-independent fashion by the 20 S
particles. The -lactone, and to a lesser extent lactacystin,
inhibited casein breakdown by both the 20 S proteasomes and 26 S
complexes. However, the concentrations necessary to inhibit protein
breakdown were consistently higher than to reduce peptide
hydrolysis.
|
Because
lactacystin and its -lactone derivative inhibit protein breakdown by
proteasomes but do not affect other classes of proteases, they appeared
very useful to test further the importance of the proteasome in protein
turnover. We examined the effects of these agents on nonlysosomal
degradation of long-lived proteins in four different cell types as
follows: hamster lung carcinoma cells (E36.12.4) (Fig.
1A), a murine macrophage cell line (A3.1A7) (Fig. 1B) and murine B (LB27.4) (Fig. 1C), and T
(RMA) lymphoblastoid cells (Fig. 1D). Cells treated with the
inhibitors remained fully viable for the duration of the experiment (8 h) (i.e. they excluded vital dyes and showed no reduction in
protein synthesis). In all three cell types lactacystin and the
-lactone inhibited protein degradation in a
concentration-dependent fashion. The
-lactone was
consistently two to four times as effective as lactacystin, in accord
with findings on purified proteasomes (Tables I and II) (11).
Generally, after a 2-h exposure 50% inhibition was obtained with
inhibitors at 1-10 µM. These agents were slightly more
potent in the LB27.4 cell line (Ki of 1-4
µM) (Fig. 1C.) than in A3.1A7, E36.12.4, and
RMA cells (Ki of 3-10 µM) (Fig. 1,
A, B, and D). Maximal inhibition was
generally reached in these assays at concentrations above 20 µM. Under these conditions, lactacystin and the
-lactone generally had a similar potency to the peptide aldehyde
LLnL in the murine cell lines (Fig. 1, B, C, and
D) but were more active than LLnL in the hamster line (Fig.
1A), possibly due to differences in the entry or metabolism of the agents or in their activity against the proteasomes in the
different cell types.
These agents were also found to be effective in inhibiting the
degradation of short-lived proteins in LB27.4 cells, and again the
-lactone appeared two to four times more potent than lactacystin (Fig. 1E). Thus, the nonlysosomal degradation of the bulk of
cellular proteins can be reduced by these highly specific inhibitors of the proteasome.
To verify that these agents did not inhibit other processes in
vivo, we assayed their effects on the intralysosomal protein degradation of an endocytosed protein, 125I-labeled keyhole
limpet hemocyanin (KLH), in murine A3.1A7 cells, a macrophage cell line
active in lysosomal process. At concentrations of lactacystin and the
-lactone (50 µM) that maximally inhibit protein
degradation in A3.1A7 cells (Fig. 1B), there was no
inhibition of the degradation of 125I-KLH over 4 h
(Fig. 2). In contrast, the peptide aldehyde inhibitor of
the proteasome, LLnL, at 50 µM also inhibits lysosomal
degradation of 125I-KLH by approximately 50%, presumably
due to its inhibition of lysosomal cysteine proteases. As expected, the
weak base chloroquine, which raises intralysosomal pH and thereby
inhibits lysosomal protein degradation, also reduced degradation of
125I-KLH. These results clearly demonstrate that
lactacystin and the
-lactone are more specific inhibitors of
proteasomes in vivo than the peptide aldehydes.
Inhibition of MHC Class I Antigen Presentation
Since
lactacystin and the -lactone selectively inhibit proteasome function
in vivo, we used these agents to investigate the importance
of the proteasome in the generation of peptides presented on MHC class
I molecules. Initial experiments examined their ability to inhibit
presentation of ovalbumin expressed transiently (Fig. 3). Antigen-presenting cells were first treated with or
without the inhibitors and then infected with a recombinant vaccinia
virus (vTF7-3) that expresses high levels of T7 RNA polymerase, and subsequently transfected with a plasmid containing ovalbumin cDNA under the control of a T7 promoter. After allowing time for antigen presentation, cells were fixed and tested for the presence of surface
Kb MHC class I molecules containing the ovalbumin-derived
peptide SIINFEKL, using the antigen-specific T-T hybridoma RF33.70.
Since murine lymphoid cells are resistant to vaccinia infection, we used the hamster lung carcinoma cell line E36.12.4 and the murine L
cell line DAP34.8 (Fig. 3, left and right panels,
respectively) as the antigen-presenting cells; both are stably
transfected with murine Kb and ICAM-1 and support vaccinia
infection.
Both lactacystin and the -lactone inhibited the presentation of the
ovalbumin-derived peptide in a concentration-dependent fashion in both hamster (E36.12.4) and murine (DAP34.8) cell lines (Fig. 3, A and B). These agents completely
blocked this process at 1-2 µM, which is 10 to 20 times
lower than the concentration of LLnL necessary for complete inhibition.
The
-lactone (Fig. 3B) was consistently twice as
effective as lactacystin (Fig. 3A) in achieving a similar
inhibition of ovalbumin presentation.
To determine whether these inhibitors blocked the proteolytic
generation of SIINFEKL from ovalbumin or some other step in antigen
presentation, we examined their effect on the presentation of SIINFEKL
expressed in the cell from a minigene (Fig. 3C). A cDNA
encoding SIINFEKL, with an initiating Met, was placed under the control
of a T7 promoter in a plasmid vector and transfected into
antigen-presenting cells expressing T7 RNA polymerase. Presentation of
this epitope was not affected by concentrations of lactacystin or the
-lactone that completely inhibited the presentation of ovalbumin.
Since the cDNAs for ovalbumin and SIINFEKL were both expressed from
the same plasmids, the differential inhibition was not due to
interference with expression of the transfected gene (also see below).
Therefore, these agents inhibit only the generation of peptide epitopes
from a protein and do not affect other steps in the class I
pathway.
To further test these conclusions, we studied whether lactacystin and
the -lactone could inhibit antigen presentation when ovalbumin or
the SIINFEKL peptide was delivered directly into the cytosol of murine
B lymphoblastoid cells (LB27.4) by electroporation (Fig.
4). LB27.4 cells were grown in serum-free medium to
minimize the binding of peptides to cell surface class I MHC due to
xenogeneic
2-microglobulin in serum. Cells fixed
immediately after electroporation of either ovalbumin or SIINFEKL
(p316) did not present antigen, as expected (Fig. 4, open
squares). After a 2-h incubation both ovalbumin-loaded and
SIINFEKL-loaded cells presented antigen (Fig. 4, closed
squares). Lactacystin and the
-lactone inhibited presentation from ovalbumin (Fig. 4A) but did not affect presentation
after SIINFEKL loading (Fig. 4B). These results provide
further evidence that the inhibitors block the proteolytic step
required for generation of peptide epitopes from a protein antigen. For
reasons that are unclear, 20-40-fold higher concentrations of these
compounds were required to cause a maximal inhibition of the
presentation of electroporated ovalbumin than were required to inhibit
presentation from transfected plasmids. This difference in sensitivity
to the inhibitors was observed even in comparisons of transfection
versus electroporation in the same cell line (DAP34.8 cells)
in the same experiment (data not shown).
To determine whether most peptides presented on MHC class I are
generated by the proteasome, as suggested previously (3), we determined
the effects of lactacystin and the -lactone on the assembly of
stable MHC class I heterodimers in the endoplasmic reticulum. The
formation of stable class I·
2-microglobulin complexes requires peptide binding (48, 49); therefore, agents that reduce the
generation of antigenic peptides should inhibit this process. Murine T
lymphoblastoid cells (RMA) were labeled with [35S]methionine in the presence or absence of inhibitors,
and class I heterodimers were then immunoprecipitated with a
conformation-sensitive antibody that reacts only with assembled
heterodimers (Fig. 5). Treatment of labeled cells with
either inhibitor (Fig. 5, A and B) markedly
reduced the amount of class I heterodimers (Fig. 5, left
panels) but did not reduce the amount of free class I heavy chain
or
2-microglobulin synthesized (Fig. 5,
middle and right panels). Therefore, these agents
caused a concentration-dependent inhibition of the assembly
of these complexes. Maximal effects were obtained at concentrations
similar to those required to maximally inhibit protein degradation and
the presentation of electroporated ovalbumin. Class I assembly was
inhibited almost completely, as was also found with LLnL. Together,
these findings prove that the great majority of peptides presented are
generated by the proteasome.
The Activities of LMP-containing Proteasomes Are Inhibited by Lactacystin
Lactacystin was previously reported to modify mainly
the N-terminal threonine on subunit X, and also a residue on the
Z-subunit, in proteasomes from bovine brain (11). However, the ability of this compound to inhibit three major peptidase activities of proteasomes (Table I) suggested that it might react with additional active sites. We therefore tested whether lactacystin inhibits the
peptidase activities of proteasomes from U937 monocytes cultured in
IFN- for 7 days, which have very low levels of subunits X, Y, and Z,
and high levels of LMPs and MECL-1. Lactacystin was found to be a
potent inhibitor of proteasomes whether or not they contained the
X-subunit (Fig. 6). For proteasomes isolated from IFN-
-treated U937 cells, the ka value appeared
30% lower than for control preparations (31 ± 3 and 45 ± 3 M
1 s
1; n = 4, p < 0.05). Similarly, a mutant lymphoblast that lacks LMPs was also sensitive to inhibition of its chymotryptic activity by
lactacystin (data not shown). These findings indicate that lactacystin
does not only react with X (and Z)-subunits.
We therefore investigated the ability of lactacystin and the
-lactone to affect the function of LMP-containing proteasomes in
cells treated with IFN-
. In IFN-
-treated LB27.4 cells (Fig. 7B) protein degradation was found to be
equally, or slightly more, susceptible to these agents than in
nontreated cells (Fig. 7A). Similar findings were obtained
for RMA cells treated with IFN-
(data not shown). Both inhibitors
were also slightly more potent in blocking antigen presentation of
electroporated ovalbumin after IFN-
treatment (Fig.
8). These results demonstrate that lactacystin and the
-lactone inhibit protein degradation and class I epitope generation
mediated by LMP2-, 7-, and 10 (MECL-1) -rich proteasomes and that
subunit X is not the sole target for their effects in vivo.
Lactacystin Covalently Modifies Multiple Proteasome
To confirm that lactacystin can inactivate multiple
-subunits (and not just X), we examined whether radiolabeled
lactacystin could covalently modify subunits of proteasomes isolated
from cells treated with or without IFN-
. Crude cell extracts,
proteasome fractions, or purified 20 S and 26 S particles were
incubated with [3H]lactacystin. SDS-PAGE and fluorography
revealed multiple radioactive bands in the 20-30-kDa region of the
gel, where the subunits of the 20 S proteasome migrate (not shown). To
identify the lactacystin-modified proteins, proteasome preparations
were incubated with [3H]lactacystin, and then subunits
were resolved on two-dimensional PAGE and identified by Western
blotting with specific anti-
subunit antibodies and fluorography.
Six different subunits of the 20 S proteasome were definitively
identified as labeled by [3H]lactacystin: LMP2, LMP7, Y
(
), X (
), Z, and MECL-1 (Fig. 9). All these
subunits, which were visualized and identified on at least two
fluorogram/Western blot pairs, are of
-type. Thus, lactacystin
reacts with all of the
-subunits that are believed to be
catalytically active and is highly specific for only these subunits. No
other proteins were found on the two-dimensional fluorograms to be
reproducibly labeled by [3H]lactacystin.
The present
studies have used the highly specific proteasome inhibitors lactacystin
and its derivative -lactone to examine the role of the proteasome in
intracellular protein degradation. As reported earlier for 20 S
proteasomes (11), we found that these agents inhibit the multiple
peptidase activities of 20 S and 26 S proteasomes, and also the
LMP-containing "immuno-proteasome," and demonstrate for the first
time that these inhibitors also reduce protein hydrolysis by the 20 S
and 26 S particle. Interestingly, for both 20 S and 26 S particles,
the concentrations and incubation times required for inactivation
differed for chymotryptic, tryptic, and peptidylglutamyl activities.
These activities must therefore be mediated by distinct sites that
differ in their affinities and/or rate of reaction with these
compounds. Substantially higher concentrations of the inhibitors and
longer incubation times were required to inhibit casein degradation
than to block the chymotryptic-like activity, as was previously
observed using peptide aldehyde inhibitors (3). Presumably, multiple
peptidase activities, including some with lower affinity for the
lactone than the chymotryptic-like site, participate in protein
degradation and must be inactivated to block this process.
In a variety of mammalian cells, these inhibitors blocked degradation
of both long-lived and short-lived proteins similarly. Inhibition of
75-95% of protein degradation was achieved within 1-h exposure to
20 µM of the inhibitors. Therefore, these agents inhibit the nonlysosomal pathway for protein degradation in cells, which, as shown here and previously, is the major degradative pathway
in cells. Nevertheless, these reagents did not reduce cell viability or
protein synthesis for at least several hours (data not shown). These
results provide strong further evidence that the proteasome is
responsible for degrading most proteins in mammalian cells. Previous
experiments with different peptide aldehydes had suggested that the
proteasome was responsible for most protein degradation (3). However,
since peptide aldehydes can inhibit thiol proteases (e.g.
lysosomal proteases) and, as shown here, can reduce breakdown of
endocytosed protein, the prior findings could not exclude the
possibility that some unknown cellular proteases also contributed to
this process. However, very similar results were found here with
lactacystin and the
-lactone which react with proteasome
-subunits, do not inhibit any other known proteases aside from the
proteasome (11), and do not interfere with lysosomal protein
degradation. Both classes of inhibitor appear very useful for studying
the role of the proteasome in physiological processes, although studies
with peptide aldehydes require controls with other inhibitors to rule
out the involvement of lysosomal or calcium-dependent
proteases (3, 6).
Our results with various cell types indicate a greater contribution of the proteasome to overall protein breakdown than previously estimated from studies with temperature-sensitive ubiquitin conjugation mutants (16-18, 27). Such studies had failed to demonstrate a significant effect of ubiquitin on degradation of long-lived cellular proteins. It is possible that ubiquitin conjugation was not completely inactivated in the ubiquitin mutant studies. Alternatively, a significant component of intracellular degradation may involve proteasomes but not ubiquitin conjugation.
Role of the Proteasome in MHC Class I Antigen PresentationAs
had earlier been found with peptide aldehydes, lactacystin and the
-lactone both strongly inhibited presentation of the dominant class
I epitope from ovalbumin. The proteasome thus is responsible for the
endoproteolytic step that liberates the appropriate peptide from
ovalbumin. These agents did not inhibit the class I presentation of the
peptide epitope alone (whether synthesized endogenously or loaded).
Therefore lactacystin and the
-lactone inhibit protein hydrolysis of
the whole protein into peptides, without affecting any other steps in
antigen presentation or cell viability during the experiments. By
reducing the supply of presentable peptides required for assembly of
the complex, the inhibitors also prevented the stable assembly and
movement to the cell surface of MHC class I molecules. Proteasomes must
be the source of the great many peptides presented on MHC class I. The
assembly of MHC class I molecules was not fully inhibited by these
agents, either because the proteasome was not completely inhibited or a
small fraction of antigenic peptides was being generated by other
proteinases in cells (as is well established for the peptides generated
from signal sequences released from secreted proteins in the
endoplasmic reticulum (50)).
Interestingly, the concentration of lactacystin required to inhibit the
presentation of ovalbumin loaded into the cytosol was 10-20-fold
higher and the concentration of the peptide aldehyde was up to 4-fold
higher than was required when ovalbumin was expressed in the vaccinia
transfection system. Typically, near maximal inhibition of antigen
presentation from exogenous ovalbumin required 20 µM lactacystin or the -lactone, which was similar to the concentrations required to inhibit protein degradation and
peptide-dependent assembly of MHC complexes. The reason why
the presentation of endogenously expressed ovalbumin is more sensitive
to the inhibitors remains unclear. We have previously found that the
presentation of native ovalbumin loaded into the cytosol is inhibited
by temperature-sensitive mutations that block ubiquitin conjugation,
whereas the presentation of transfected ovalbumin is not (51). This may
reflect differences in the folding of the two substrates or their
subcellular localization. Possibly, ubiquitinated molecules are more
resistant to inhibition. These differences cannot be explained by
involvement of different species of proteasome, since lactacystin is
more effective against 26 S and the aldehyde against the 20 S
particle.
Lactacystin was previously shown to modify subunits
X and Z in brain extracts (11). However, we found here that six
-subunits (LMP2, LMP7, MECL-1, X, Y, and Z) can react with
lactacystin, all of which have N-terminal threonines, whose hydroxyl
groups catalyze the nucleophilic attack on the substrate (10, 52, 53).
Thus, all the
-subunits that are capable of forming the threonine-based active sites are shown here to be covalently modified by lactacystin. Accordingly, lactacystin was found to inhibit protein
degradation as well as the presentation of ovalbumin by both control
cells and IFN-
- stimulated cells, in which X-, Y-, and Z-subunits
are largely replaced by LMP2, LMP7, and MECL-1.
The difference between these findings and the specificity for the
subunit X noted previously in studies of brain extracts (11) may be due
to the variation in LMP expression in different tissues. Unlike the
proteasomes studied here, brain proteasomes normally contain very low
levels of LMP2 and LMP7 -subunits compared with liver or spleen
(25), presumably because brain also has much lower levels of MHC
molecules and is less active in antigen presentation (54).
Consequently, any reaction of lactacystin with the three
IFN-
-induced subunits would have been missed in the earlier
studies.