From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Knowledge about the sizes of peptides generated
by proteasomes during protein degradation is essential to fully
understand their degradative mechanisms and the subsequent steps in
protein turnover and generation of major histocompatibility complex
class I antigenic peptides. We demonstrate here that 26 S and activated 20 S proteasomes from rabbit muscle degrade denatured, nonubiquitinated proteins in a highly processive fashion but generate different patterns
of peptides (despite their containing identical proteolytic sites).
With both enzymes, products range in length from 3 to 22 residues, and
their abundance decreases with increasing length according to a
log-normal distribution. Less than 15% of the products are the length
of class I presented peptides (8 or 9 residues), and two-thirds are too
short to function in antigen presentation. Surprisingly, these
mammalian proteasomes, which contain two "chymotryptic," two
"tryptic," and two "post-acidic" active sites, generate
peptides with a similar size distribution as do archaeal 20 S
proteasomes, which have 14 identical sites. Furthermore, inactivation
of the "tryptic" sites altered the peptides produced without
significantly affecting their size distribution. Therefore, this
distribution is not determined by the number, specificity, or
arrangement of the active sites (as proposed by the "molecular
ruler" model); instead, we propose that proteolysis continues
until products are small enough to diffuse out of the proteasomes.
The ubiquitin-proteasome pathway is the major proteolytic system
in the cytosol of eukaryotic cells, where it catalyzes the selective
degradation of short lived regulatory proteins and the rapid
elimination of proteins with abnormal conformation (1, 2). In mammalian
cells, this system also seems to be responsible for the breakdown of
the bulk of cell proteins (3, 4). The critical protease in this pathway
is the 26 S proteasome, an ATP-dependent proteolytic
complex, which is formed by the association of the barrel-shaped 20 S
proteasome (700-kDa) and two 19 S (700-kDa) regulatory complexes (5,
6). The 19 S complexes activate peptide hydrolysis within the 20 S
proteasome (7) and are responsible for the recognition of ubiquitinated
proteins (8). It contains six different ATPases, which probably unfold
protein substrates and facilitate their entry into the 20 S particle
(9-11). This cylindrical structure is composed of four stacked rings
(5, 6). Each of the outer two rings contains seven different
As part of the continuous turnover of cell proteins, the great majority
of peptides generated by proteasomes must be rapidly degraded into
amino acids by cytosolic peptidases. In mammalian cells, some of the
proteasomal products escape complete degradation and are presented to
the immune system on the cell surface in complexes with
MHC1 class I molecules (3-5,
19, 20). These antigenic peptides are 8 or 9 residues long (21).
Proteasomes are essential for the formation of the C terminus of most
antigenic peptides but may not be required for the generation of their
N termini (22). Thus, if proteasomes generate N-terminally extended
versions of antigenic peptides, they can be trimmed by cytosolic
peptidases to the presented epitopes (22, 23). Obviously, information on the sizes and nature of the products of protein breakdown by mammalian proteasomes is essential for a full understanding of both MHC
class I antigen presentation and the postproteasomal steps in the
complete degradation of proteins to amino acids.
Homologous 20 S proteasomes are also found in archaea and certain
eubacteria (5, 6, 24, 25) that do not possess the ubiquitin system or
26 S complexes. In the archaeal particle, there is only one type of
These findings on the archaeal proteasome cannot be automatically
applied to their mammalian counterparts for several reasons. Eukaryotic
proteasomes have fewer active sites (6 versus 14) (13, 26).
These sites are asymmetrically distributed in the eukaryotic particle
(13). Eukaryotic proteasomes have three different types of active
sites, and therefore they cleave a much larger range of peptide bonds.
The sensitivities of mammalian and archaeal proteasomes to inhibitors
are different (27). In fact, with oligopeptide substrates (11-44
residues), eukaryotic and archaeal proteasomes generate different
products (31, 32). Therefore, it is important to analyze systematically
the products of protein degradation by 26 S proteasomes from mammalian tissues.
Most prior biochemical studies of proteasome activity have focused on
20 S particles because of the difficulties in purification of the 26 S
complexes, their instability (5, 33), and the inability to obtain
ubiquitinated proteins in amounts necessary for chemical studies.
However, the physiological relevance of findings on the 20 S particles
is uncertain. In fact, when isolated rapidly in the presence of
glycerol, 20 S proteasomes exhibit little or no activity against
protein substrates (5, 33). These latent 20 S particles can be
activated in vitro by a variety of treatments
(e.g. by the addition of detergents, such as 0.02% SDS, or
by removal of glycerol), but it is unclear if, after such treatments,
the 20 S proteasomes function in the same way as when they are
associated with 19 S regulatory complexes as part of the 26 S particle.
Therefore, an important goal of this work was to compare the nature of
the products generated by 26 S proteasomes and activated 20 S
proteasomes during the degradation of full-length proteins. One
potential complication in interpreting results of such studies is the
heterogeneity of 26 and 20 S proteasome subunits in many mammalian
tissues (5). Therefore, rabbit skeletal muscle was chosen as the source
of the proteasomes because of their homogeneous composition in this
tissue (34).2 Unlike other
tissues (e.g. liver), muscles express exclusively X, Y, and
Z catalytic subunits and do not contain the Purification of 20 and 26 S Proteasomes--
20 and 26 S
proteasomes were simultaneously purified to homogeneity from rabbit
psoas muscle. After the muscles were minced to small pieces, they were
homogenized in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.25 M
sucrose, 5 mM MgCl2, 2 mM ATP. The
homogenate was centrifuged for 15 min at 10,000 × g to
remove cell debris and then was centrifuged for 1 h at
100,000 × g. The supernatants were spun for 6 h
at 150,000 × g. The resulting proteasome-containing
pellets were dissolved in buffer A (20 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 10% glycerol, 5 mM MgCl2, 1 mM ATP) and loaded onto
a DEAE AffiBlue (Bio-Rad) column. After washing with 40 mM
NaCl (in buffer A), proteasomes were eluted with buffer A containing
0.15 M NaCl and directly loaded on an Uno Q-12 column
(Bio-Rad). Fractions containing proteasomal activity were identified by
their ability to hydrolyze Suc-LLVY-Amc. Complete separation of the 26 and 20 S proteasomes was achieved by a gradient of 0.15-0.45
M NaCl in 200 ml (Fig. 1).
The peak of the activity of 20 S proteasomes was stimulated by 0.02%
SDS, while the 26 S activity was markedly inhibited by this
concentration of SDS and by removal of ATP. Fractions containing 20 S
proteasomes were dialyzed against 50 mM HEPES, 1 mM DTT, 10% glycerol, pH 7.5, and finally purified by
chromatography on a heparin-Sepharose HiTrap column (Amersham Pharmacia
Biotech). The 20 S proteasome was eluted by a 0-0.3 M
gradient of KCl in 10 column volumes and stored at Peptidase Assay for Proteasome Activity--
Each sample (1-10
µl) was added to 100 µl of 100 µM Suc-LLVY-Amc in 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1%
Me2SO, 5 mM MgCl2, 1 mM
ATP. After a 20-min incubation at 37 °C, the reaction was stopped by
the addition of 900 µl of 1% SDS, and fluorescence of released Amc
was measured (excitation, 380 nm; emission, 460 nm). In order to
distinguish between 20 and 26 S proteasomes, the same incubation was
repeated in the presence of 0.02% SDS instead of ATP. Peptide
substrate incubated without proteasomes served as control.
Alternatively, proteasomal activity was measured in a continuous assay.
The proteasome sample (0.02-1 µg) was added into the cuvette
containing 500 µl of substrate preincubated at 37 °C. Fluorescence
of released Amc was monitored continuously for 10-20 min, and the
reaction velocity was calculated from the slopes of the resulting
reaction progress curves. Consumption of substrate at the end of
incubation never exceeded 1%.
Protein Substrates--
Ovalbumin, bovine Protein Degradation by Proteasomes--
Denatured IGF,
lactalbumin, casein, and ovalbumin were incubated with 20 or 26 S
proteasomes at 37 °C in 50 mM Bis-tris propane, 1 mM DTT, 2-5% glycerol (30). In addition, for experiments
with the 26 S particles, reaction buffer contained 0.5 mM
ATP, 5 mM MgCl2, and, for 20 S proteasomes,
0.02% SDS was added to stimulate the particles unless stated otherwise
(as in Fig. 4). Aliquots were analyzed for new amino groups using
fluorescamine (27), which forms a fluorescent adduct with N termini of
peptides, generated by proteasomal cleavage. A mixture of standard
peptides was used to calibrate the assay (27).
Inactivation of the Trypsin-like Sites in 20 S
Proteasomes--
Pure 20 S proteasomes (~0.8 µM) were
incubated with 0.5 mM AEBSF (Pefablock SC, Boehringer
Mannheim) for 1.5 h at room temperature, and all three peptidase
activities were measured in continuous assay, using as substrates
Suc-LLVY-Amc for the chymotrypsin-like activity,
tert-butyloxycarbonyl-LRR-Amc for the trypsin-like activity, and Ac-YVAD-Amc for the postacidic activity. All substrates were obtained from Bachem (Bubendorf, Switzerland). The covalently modified
enzyme was used in degradation reactions with casein and IGF as substrates.
Degradation of Nonubiquitinated Proteins by 26 S
Proteasomes--
It is well established that 26 S proteasomes catalyze
the degradation of proteins conjugated to ubiquitin (1, 5), but they
also have been reported to degrade a few nonubiquitinated proteins in
an ATP-dependent manner (35-38). Because ubiquitinated substrates cannot be generated in homogeneous form in quantities necessary for analysis of the products, we tested whether 26 S proteasomes from rabbit muscle can degrade other proteins without ubiquitination. Indeed, in the presence of ATP, the highly purified 26 S particles degraded several denatured proteins (IGF, lactalbumin, ovalbumin) and casein at linear rates (Fig. 2A).
Interestingly, the absolute rates of peptide bond cleavage (measured by
the appearance of new amino groups) were faster with the smaller
substrates (IGF and lactalbumin) than with casein or ovalbumin. To
confirm that this degradation (Fig. 2A) was indeed due to 26 S proteasomes, we analyzed the reaction mixtures by native
polyacrylamide gel electrophoresis (Fig. 2B). At the outset
and after 3 h of incubation (lane 3), only
26 S proteasomes were detectable. Thus, the hydrolysis of these
nonubiquitinated proteins was not due to contaminating 20 S particles
or to 20 S generated by breakdown of 26 S proteasomes. Clearly,
in vitro, and presumably in vivo, 26 S particles,
like activated 20 S proteasomes, can hydrolyze some denatured proteins without ubiquitination.
20 and 26 S Proteasomes Degrade Proteins Processively--
In
order to test whether mammalian 26 and 20 S proteasomes degrade
proteins processively, we incubated these enzymes with casein and
analyzed at different times the nature of the products of the reaction
by SDS-PAGE. 20 S proteasomes were purified in the latent state in the
presence of glycerol but then were activated by the addition of
0.02% SDS. Despite the disappearance of casein, no Coomassie-stainable
polypeptide fragments were detected in the gel (not shown), indicating
that the substrate was degraded all the way to oligopeptides. To
increase the sensitivity of the detection of individual peptide
products, we initially used casein as a substrate that was covalently
modified with FITC at multiple sites (27). Incubation conditions were
chosen to ensure a linear rate of breakdown of the FITC-casein and the
presence of the substrate in large excess. At different times, the
fluorescent products released by proteasomes were analyzed by
HPLC on a reverse-phase column (Fig. 3).
With both the 26 and 20 S particles, a large number of products were
generated, indicating that the protein was cleaved at multiple sites.
As the reaction proceeded, the areas under the individual product peaks
increased in parallel with each other, but the relative amounts of
these fluorescent products did not change, and no new peaks appeared.
Thus, no peptides were generated that were degraded in subsequent
proteolytic rounds.
These results together demonstrate that activated 20 S proteasomes and
the ATP-dependent 26 S particles degrade proteins in a
highly processive manner into oligopeptides without dissociation of the
substrate. A similar mechanism of protein breakdown was found earlier
for the archaeal 20 S proteasomes (27). By contrast, when chymotrypsin,
a typical nonprocessive protease, was incubated under similar
conditions with casein (24 kDa), polypeptide fragments ranging from 14 to 20 kDa were generated (not shown), and with FITC-casein as the
substrate, the pattern of fluorescent products varied with incubation
time. Thus, unlike the proteasome, chymotrypsin released large products
that were subsequently cleaved further (27).
20 and 26 S Proteasomes Generate Different Patterns of
Peptides--
Because proteolysis within the 26 S complex is catalyzed
by its core 20 S proteasome, it has been widely assumed that the pattern of peptide bond cleavage by the 20 S proteasome reflects proteolysis by the larger 26 S particle. However, careful analysis of
the spectra of peptides produced by the 20 and 26 S proteasomes showed
the unexpected result that they generated different patterns of
products. (See, for example, peptides eluted between 12 and 14 min in
Fig. 3.) These differences in product patterns suggest that 20 and 26 S
proteasomes can cleave proteins at different sites. However, it was
also possible that these differences were an artifact due to 0.02%
SDS, which was used to activate 20 S proteasomes and might also be
altering the substrate. In addition, these differences might possibly
be due to some conformational difference in the substrate due to the
Mg2+ and ATP used to stabilize the 26 S particles.
Alternatively, the covalent modification of casein by very hydrophobic
FITC residue might lead to a differential binding to these different
proteasome particles.
To exclude these possibilities, we compared the pattern of peptides
generated by 20 and 26 S proteasomes from IGF (which had not been
modified with FITC) during incubation under the exact same conditions
(i.e. in the presence of Mg2+ and ATP). As was
found with FITC-casein, the patterns of peptides generated by the 26 S
and latent 20 S proteasomes from IGF were different (Fig.
4, a and b).
Moreover, when the 20 S proteasomes prior to incubation were activated
by dialysis against buffer lacking glycerol, the pattern of the
products differed to an even greater extent than with latent particles
(Fig. 4c). Finally, when the 20 S proteasomes were activated
by the addition of 0.02% SDS (Fig. 4d), they generated a
distinct pattern of the products from those generated by the 26 or 20 S
proteasomes activated in other ways. These distinct patterns were
reproducible with different enzyme preparations. Thus, the observed
differences in the pattern of the products are due to differences in
the behavior of 20 and 26 S particles. Since these two particles have
the same active sites, the associated 19 S complexes in the 26 S
proteasomes as well as the nature of the treatment used to activate 20 S particles must influence how proteins are cut within the 20 S
proteasome.
Product Sizes--
To analyze the lengths of peptides generated by
mammalian proteasomes, we used a size-exclusion chromatography method
developed earlier to study the products of the archaeal proteasomes
(30). Methylated casein, IGF, and ovalbumin were incubated for 2-4 h with SDS-activated 20 S proteasomes or 26 S particles (in the presence of ATP). Under these conditions, degradation rates (i.e. the generation of new amino groups) were linear, and
usually less than 20% of the original substrate amount was consumed.
The generated peptides were separated from the undegraded protein on a
reverse-phase column and subsequently fractionated on a gel filtration
column, which resolves peptides according to their molecular masses in
a 500-10,000-Da (4-90-amino acid) range (30). The abundance of
products in the different fractions (measured by the fluorescamine
assay) was plotted against their elution times, which are proportional
to the logarithms of their molecular weights. The resulting curves were
reproducible and characteristic of each substrate and the type of
proteasome (Fig. 5).
The overall size distribution of the peptide products from these
different proteins was quite similar with the 20 and 26 S proteasomes.
In each case, when plotted against the logarithms of the molecular
weights, the curves resembled a normal distribution. In other words,
the size distribution of proteasome products seemed to fit a log-normal
distribution. This fit was stronger with the longest substrate
(ovalbumin), probably because of the greater total number of peptide
products generated. The sizes of the products covered a wide range,
from less than 500 Da (i.e. 4-5 residues, which corresponds
to the lower separation limit of the column) to about 22 residues with
most protein substrates, but when casein was the substrate, few
peptides of up to 3500 Da (30 residues) were generated. Although the
peptides released by the 20 and 26 S particles fell within the same
size range, they did not appear to be exactly the same sizes. With both
IGF and ovalbumin as substrates, 26 S proteasomes consistently
generated more products smaller than 1000 Da (8-9 residues) than did
the 20 S proteasomes, and the mean size of the products of the 26 S
proteasome was on the average 1 or 2 residues shorter (Fig. 5, Table
I).
To analyze better the relative distributions of peptides of different
sizes, we graphed these data in the form of cumulative frequency curves
(Fig. 6). For each time point (Fig. 5),
the fraction of peptides with molecular weights equal to or less than
those of peptides eluting at this specific point was calculated, and then graphed against the molecular weights and apparent lengths on a
linear scale (Fig. 6). On such an integral plot, the slope of the curve
at each point equals the fraction of peptides of this particular size.
The size distribution plots of peptides generated by 20 S proteasomes
(Fig. 6A) from the three different proteins of very
different lengths were indistinguishable. Similarly, the peptides
generated by 26 S proteasomes (Fig. 6B) from ovalbumin and
IGF had a very similar size distribution, but products of casein
degradation were slightly longer, as had also been found with this
substrate and archaeal 20 S proteasomes (30). Casein degradation may
yield larger products because casein is phosphorylated at multiple
sites or because every seventh residue in it is proline, both of which
may retard cleavages.
Although the products cover a wide size range (95% percent were
between 3 and 22 residues) (Fig. 6), the median sizes of peptides were
5 residues for 26 S proteasome products and 6 residues for the 20 S
proteasome. At least two-thirds of the products of both particles were
shorter than 8 residues, and only 15% were 8 or 9 residues long, which
corresponds to the length of MHC class I antigenic peptides. Thus,
mammalian proteasomes do not preferentially generate peptides of an
appropriate size to bind to MHC class I molecules. A small fraction of
the peptides (15-25%) was longer than 10 residues. None of the
peptides generated by 26 S proteasomes from IGF or ovalbumin was longer
than 20 residues, but some of the 20 S products were up to 30 residues
long. In addition, about 2% of the peptides generated from casein
appeared to contain more than 30 residues, perhaps as a consequence of
casein's unusual primary structure, as discussed above.
Product Sizes Are Not Consistent with the "Molecular Ruler"
Model--
Surprisingly, despite the very different number,
arrangement, and specificities of their active sites, the size
distribution of the products of the mammalian proteasomes (Fig. 5) had
a similar shape, mean size (Table I), and size range as the peptides
generated by proteasomes from archaea T. acidophilum (30).
For example, with IGF as a substrate, the mean sizes of peptides
released by rabbit 20 S proteasome were the same as those generated by
its archaeal counterparts (Table I). These similarities in the size distributions of the products of eukaryotic and archaeal particles strongly suggest that this log-normal distribution is a fundamental feature of the 20 S proteasome that was conserved during
evolution. Thus, the number, catalytic properties of the active sites,
and distance between them have little or no influence on the sizes of
the products.
To further test whether the nature of the individual active sites and
the distances between them influence the size distribution of the
products, we selectively inactivated the two trypsin-like active sites
in 20 S proteasomes by reaction with the specific irreversible
inhibitor of these sites, AEBSF, as was described recently (39). 20 S
proteasomes were preincubated with AEBSF at room temperature, resulting
in the inhibition of the trypsin-like activity by almost 90% (Table
II). The two other activities were not
affected by this treatment. This loss of trypsin-like activity did not
affect the rate of cleavage of peptide bonds in the protein substrates
(Table II), in agreement with observations on a yeast mutant affecting
the homologous activity (15, 16). However, this modification did cause
some changes in the pattern of peptides generated (Fig.
7, compare peaks eluting at 40-50 min
and at 80-100 min). Nevertheless, this inactivation of the two
trypsin-like sites did not affect the log normal distribution of the
size of proteasome products. In fact, when these peptides were analyzed by size-exclusion chromatography, little or no difference was detected
between proteasomes with inactivated trypsin-like sites and fully
active proteasomes (Fig. 8). Thus, the
number, specificity of individual active sites, or the distances
between them do not significantly affect the general size distribution
of the peptides generated by proteasomes.
Mechanism of Protein Breakdown by 20 and 26 S Proteasomes--
The
present study demonstrates that protein substrates within the mammalian
20 or 26 S particles are cut at many sites (Table I) to yield small
oligopeptides, without the release of longer fragments. Such a highly
processive mode of degradation seems to be a fundamental feature of
intracellular proteases, including 20 S proteasomes from yeast (40),
the simpler proteasomes from archaebacteria (27), and the bacterial
ATP-dependent proteases, ClpAP (41), La (42), and HslUV
(43). This processivity must help to ensure that cell proteins targeted
for destruction are rapidly eliminated without the generation of large
fragments that might retain some biological activity and be highly
toxic to the cell. Since the 20 S proteasome by itself exhibits this
highly processive behavior, this particle must have structural features and enzymatic mechanisms to ensure that the substrate once bound is not
released until its degradation is completed. Once a polypeptide has
entered the 20 S particle, its exit is probably only possible through
the narrow openings in the
Within the 26 S particle, the association of the 19 S regulatory
complex with the 20 S proteasome enhances its peptidase activities (7)
and confers the ability to digest ubiquitinated proteins (5). In
addition, even in the absence of ubiquitination, the 26 S complexes can
degrade a number of denatured polypeptides (Fig. 2), as had been
reported for casein (38), ornithine decarboxylase (36), and c-Jun (35).
Degradation of these nonubiquitinated proteins still requires ATP,
which is necessary for the stability and function of the 26 S complex
(5). Since this ubiquitin-independent process can occur in
vitro, presumably it is also occurring to some extent in
vivo, at least for unfolded substrates (44). The maximal rates of
degradation of these different nonubiquitinated proteins varied widely
(Fig. 2A), presumably because they retain some secondary
structure or tend to aggregate, both of which should retard entrance
into the proteolytic chamber. The actual influence of ubiquitination on
the rate of degradation of these unfolded proteins will be interesting
to study. Possibly, the attachment of multiple ubiquitin moieties to
these proteins simply facilitates their binding to the 19 S complex.
Pattern of Peptides Generated by 20 and 26 S Proteasomes
Differ--
In most published studies of proteasome function,
activated 20 S proteasomes have been used as models of proteasome
function in vivo, and it has been widely assumed, especially
in studies of the production of antigenic peptides (45-48), that
activated 20 S proteasomes and 26 S particles generate identical
spectra of peptides. Clearly, this assumption is not valid. The
patterns of peptides released by 20 and 26 S proteasomes are not
identical, and the spectrum of peptides generated by the 20 S particle
depends on its mode of activation. In the present studies, 20 S
proteasomes were isolated and maintained in a latent state by the
presence of 10% glycerol, but their activity could only be studied
after activation with 0.02% SDS, the addition of Mg2+ (5 mM), or dialysis against a buffer lacking glycerol. Since the patterns of peptides generated from IGF by the 20 S particles under
these three different conditions differed from each other in
reproducible ways and also differed from the peptides released by the
26 S particle (Fig. 4), the specific cleavages made in a protein
substrate by the 20 S proteasome vary with the precise incubation
conditions and whether the particle is free or part of the 26 S complex.
As shown in Figs. 3 and 4, the association of the 20 S proteasome with
the 19 S complex alters the pattern of peptides it generates (Figs. 3
and 4). Because skeletal muscle contains only one type of 20 S particle
(i.e. it lacks alternative interferon-inducible subunits)
(34),2 the differences between 20 and 26 S proteasomes are
not due to the presence of different groups of Determinants of the Sizes of Peptide Products--
The molecular
ruler hypothesis has been proposed to explain the proteasome's
mechanism for degrading proteins. Accordingly, peptide products are
generated by the coordinate actions of two adjacent active sites (26,
28, 29), and the distance between these sites must be a major factor
determining the products' sizes. However, a variety of evidence
indicates that the proteasome is unlikely to function by such a
mechanism. 1) The length of most peptides generated by the archaeal
proteasomes does not equal the distance between its adjacent active
sites, which corresponds to a peptide of 7 or 8 residues. In fact, the
sizes of its products actually ranged in length from 3 to 30 residues
and followed a log-normal distribution (30), although the mean size was
approximately 8 residues in length. 2) In place of the 14 chymotrypsin-like active sites in the archaeal proteasomes, the
eukaryotic particles contain two chymotrypsin-like sites, two
trypsin-like sites, and two active sites cleaving after acidic
residues. Within a single
It was most surprising to find that the archaeal and SDS-activated
mammalian particles, despite their different catalytic properties and
functional organizations, generate products with very similar mean
sizes (Table I) and size distributions. It seems quite unlikely that
the similarities in product size and in the log normal distributions of
products found for archaeal and mammalian proteasomes (even after loss
of trypsin-like sites) is coincidental. Instead, this common feature is
likely to reflect some fundamental property of the particle that has
been conserved through the evolution. These findings clearly indicate
that the specificities of the different active sites and the distances between them are not major determinants of product size. Instead, it
seems more likely that cleavage of peptide bonds continues randomly
within the central chamber until peptides are small enough to escape
further proteolysis by diffusing out of the 20 S particle. The rate of
diffusion of a peptide should be inversely proportional to its
hydrodynamic radius, and as the radius is approximately proportional to
the logarithm of its molecular weight, a log-normal distribution of the
products could result. Moreover, the small openings in the
Strong support for this type of mechanism was the finding that loss of
trypsin-like sites did not alter significantly the sizes of peptide
products. To test this model further, it would be interesting to
analyze the effects on product size of inactivation of the postacidic
site, but potent inhibitors of their activity are not known. Although
potent irreversible inhibitors of the chymotrypsin-like site are
available, their effects on product size would be difficult to
interpret for several reasons (e.g. the chymotrypsin-like
site appears to be rate-limiting in protein breakdown (3, 4, 15, 17),
and occupancy of one such site allosterically activates the other
(47, 51).
Fate of Peptides Generated by Proteasomes--
In mammalian cells,
a small fraction of the peptides generated by the proteasome are
utilized for MHC class I antigen presentation (19, 20). Exactly why
only certain peptides are selected and presented on the surface is
uncertain. Only 8-9-mer peptides with hydrophobic or basic residues at
their C termini can bind tightly to MHC class I molecules (21). It was
therefore widely assumed that peptides of this size were the
predominant products of the proteasome (53). However, only 15% of all
peptide products fall within this size range (Fig. 6). Moreover,
two-thirds of all peptides are shorter than 8 residues and therefore
cannot be used in class I antigen presentation. Another 15% of the
proteasomal products are longer than 8 residues and may be utilized for
antigen presentation after subsequent trimming by cellular
exopeptidases (22). In fact, this N-terminal trimming reaction can also
be stimulated by
The great majority of the proteasome products in vivo are
rapidly digested to amino acids. These peptides released by proteasomes have mean lengths of 6-9 residues (Table I). In other words, the
proteasomes cleave only 10-15% of peptide bonds in proteins. In the
conversion of cell proteins into amino acids, the remaining 85-90% of
the peptide bonds must be quickly hydrolyzed by cellular endo- and
exopeptidases, since free peptides are not found in cell extracts (54).
These enzymes must be highly active in the cytosol, but their identity
remains unclear.
INTRODUCTION
Top
Abstract
Introduction
References
-subunits, which surround a narrow opening through which substrates
appear to enter (12). Each of the inner two rings is composed of seven different
-subunits, which enclose the central chamber where proteolysis occurs. On three of these
-subunits are found the active
sites (13), one of which is "chymotrypsin-like" in specificity, one
of which is "trypsin-like," and one that cleaves after acidic residues (14-17). In vivo, 20 S proteasomes exist not only
as a part of the 26 S complexes, but also as free particles (18); however, it is not clear whether this free form ever functions in
protein degradation in vivo.
-subunit and one type of
-subunit, and thus this 20 S particle
contains 14 identical chymotrypsin-like active sites, which are
positioned at equal distances around the
-rings (26). Unlike
traditional proteases, which release the substrate after each cleavage
event, proteasomes from the archaebacterium Thermoplasma
acidophilum degrade proteins in a highly processive fashion into
small peptides and do not dissociate from the substrate between
cleavage events (27). It has been proposed that this complex digests
proteins according to a "molecular ruler" mechanism, in which the
length of peptides produced would correspond to the distance between
active sites (7 or 8 residues) (26, 28, 29). However, we have recently
found that archaeal proteasomes generate products that range from 3 to
30 residues in length. The abundance of these peptides decreases as
their size increases, and this relationship follows a log-normal
distribution (30).
-interferon-inducible homologs (i.e. the immunoproteasomes). In these studies,
four proteins of different lengths (casein, lactalbumin, insulin-like growth factor 1 (IGF), and ovalbumin) were used as substrates after
denaturation, which appears to be necessary for proteins to traverse
the narrow opening in the
-rings of the 20 S proteasomes (12). The
present studies of 26 S function were made possible by the finding that
in the presence of ATP, such denatured proteins are also degraded by
the 26 S particles.
EXPERIMENTAL PROCEDURES
70 °C.
Fractions from the Uno Q-12 column containing 26 S proteasomes
(i.e. peptidase activity that was inhibited by 0.02% SDS)
were concentrated to 1 ml and loaded on a 38-ml glycerol gradient
(23-37% glycerol in 25 mM HEPES, pH 7.5, 1 mM
DTT, 0.5 mM ATP, 5 mM MgCl2). After
centrifugation for 22 h at 100,000 × g, the
gradient was fractionated, and the active fractions were pooled and
concentrated. The resulting preparations showed one major band on the
native gel and were not cross-contaminated (Fig.
2B).
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Fig. 1.
Separation of 20 and 26 S proteasomes on the
Uno Q ion-exchange column. After chromatography on DEAE AffiBlue
column, the proteasome-rich fractions were loaded onto an Uno Q-12
(Bio-Rad) column, equilibrated with buffer A, containing 0.15 M NaCl. Elution was performed with a linear gradient of
NaCl from 0.15 to 0.45 M in 200 ml at a flow rate of 2 ml/min, beginning at the zero time point. 5-ml fractions were collected
and assayed for proteasomal activity using Suc-LLVY-Amc as the
substrate. Dotted line, UV absorbance (total
protein); open circles and dashed
line, activity in the presence of 0.02% SDS;
closed squares and solid
line, activity in the presence of 1 mM ATP and 5 mM MgCl2. Horizontal bars
indicate fractions, used for final purification of 20 and 26 S
particles.
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Fig. 2.
26 S proteasomes degrade nonubiquitinated,
denatured proteins. A, denatured IGF (510 µM), lactalbumin (320 µM), casein (90 µM), and ovalbumin (14 µM) were incubated
with 26 S proteasomes at 37 °C in 50 mM Bis-tris
propane, 0.5 mM ATP, 5 mM MgCl2,
and 2.5% glycerol. In order to ensure that 26 S proteasome functions
at Vmax in these assays, substrate
concentrations exceeded by severalfold the Km values
for each protein (A. F. Kisselev, unpublished observations).
Aliquots were analyzed for new amino groups using fluorescamine (27),
which forms a fluorescent adduct with N termini of peptides, generated
by proteasomal cleavage. A mixture of standard peptides was used to
calibrate the assay (27). B, a native 5% PAGE was run using
the system of Ornstein and Davis (52, 55). Lane
1, purified 20 S proteasomes; lane 2,
purified 26 S proteasomes; lane 3, a mixture of
ovalbumin and 26 S proteasome (from Fig. 2A) after a 3-h
incubation. Ovalbumin (43 kDa) migrated out of the gel and was not
detected.
-lactalbumin, and
bovine
-casein were from Sigma, and recombinant human IGF was a kind
gift of Dr. W. Prouty (Lilly). Fluorescein isothiocyanate (FITC)-casein
was prepared as described (27); IGF and lactalbumin were denatured by
reduction of disulfide bonds and carboxymethylation of the cysteins;
and ovalbumin was treated with performic acid (27). Casein, which has
little tertiary structure, does not require denaturation in order to
become a substrate of the proteasome. Finally, in order to reduce the
background in the reaction with fluorescamine, lysine residues and
N-terminal amino groups on all protein substrates were blocked by
reductive methylation (27).
RESULTS
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Fig. 3.
Both 20 and 26 S proteasomes degrade
FITC-casein processively but generate different patterns of
peptides. FITC-casein (8 µM) was incubated with 26 S
proteasomes, as described under "Experimental Procedures," except
that the buffer contained 50 mM Tris-HCl, pH 7.5, instead
of Bis-tris propane. At indicated times, aliquots were analyzed by HPLC
on a C18 column (27). Fluorescent peptides were
detected at an excitation wavelength of 492 nm and emission of 521 nm.
The relative amounts of the peptides generated did not change with
time, although their absolute amounts increased. Less than 20% of the
FITC-casein was consumed during the incubation, as calculated by
integration of its peak, which was eluted at 25 min (not shown).
Proteasomes used in these experiments were prepared as described in
Ref. 4.
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Fig. 4.
20 and 26 S proteasomes generate different
patterns of products from IGF. IGF (360 µM) was
incubated at 37 °C in 50 mM Bis-tris propane, pH 7.5, 1 mM DTT, 5% glycerol, 5 mM MgCl2,
and 0.5 mM ATP with 26 S proteasomes (50 nM)
for 2 h (a), latent 20 S proteasomes (250 nM) for 4 h (b), activated 20 S proteasomes
(10 nM) for 2 h (c), or latent 20 S
proteasomes (250 nM) in the presence of 0.02% SDS for
3 h (d). 20 S proteasomes in c were
activated prior to incubation by an overnight dialysis against 50 mM Bis-tris propane, pH 7.5, 1 mM DTT at
+4 °C. Digests were loaded on a C8 Vydac column
(0.2 × 25 cm) equilibrated with 0.06% trifluoroacetic acid.
Peptides were eluted by a gradient of acetonitrile from 0 to 8% in 20 min, to 28% within the next 100 min, to 36% within the subsequent 20 min, and to 44% in the last 10 min at a flow rate of 0.15 ml/min. The
large peak of undegraded excess IGF, which was eluted after 150 min, is
not evident.
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Fig. 5.
Size-exclusion chromatography of peptides
generated by SDS-activated 20 and 26 S proteasomes (dashed
lines) from different substrates (indicated in
each panel). After a 2- or 4-h incubation as
described under "Experimental Procedures," the products were
separated from the undegraded substrates on a C18 reverse
phase column (30). At the end of the incubation, usually less than 20%
of the protein was degraded. The pooled products were run on a
polyhydroxyethyl aspartamide column as described (30), and the molar
amounts of peptides in each fraction were determined by the
fluorescamine assay. Each curve is an average of two experiments.
Mean sizes of peptides generated and number of cuts made in a single
polypeptide chain by proteasomes
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Fig. 6.
Cumulative frequency curves for 20 and 26 S
proteasome products. The curves were obtained by transformation of
the size-exclusion chromatography data from Fig. 5. For each point, the
fraction of peptides with this and lower molecular mass was calculated.
The scale for peptide length was obtained by dividing the molecular
mass scale by 115 Da, the average molecular mass of an amino acid
residue in these substrates.
Effect of AEBSF treatment on three peptidase activities of 20 S
proteasomes and protein degradation by this particle
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Fig. 7.
Pattern of peptides generated from IGF by
control and by 20 S proteasomes (top) with inactivated
trypsin-like site (bottom). AEBSF-treated and control
20 S proteasomes were incubated with IGF in the presence of SDS as
described under "Experimental Procedures." Digests were run on HPLC
as described in Fig. 4.
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Fig. 8.
Inactivation of the trypsin-like sites in 20 S proteasomes does not change significantly the sizes of the
products. AEBSF-treated and control proteasomes were incubated
with IGF and casein for 2 h as described under "Experimental
Procedures," and the peptides generated were analyzed by
size-exclusion chromatography, as in Fig. 5. Closed
circles and solid lines, mock-treated
proteasomes; closed diamonds and
dotted lines, AEBSF-treated proteasomes (this
treatment selectively inhibited trypsin-like activity, Table II).
DISCUSSION
-rings, which are located at a
significant distance from the central proteolytic chamber (12, 26). In
addition, interactions of the substrate with the large inner surface of
the particle may promote retention of longer polypeptides. Another
possible mechanism that might reduce substrate dissociation could be
that the polypeptide chain, while covalently attached to one active
site in a transition state complex, is attacked in turn by other active
sites until degradation to small products is completed (26).
-subunits. Therefore,
the 20 S proteasome, depending on the state of the particle, is capable of cleaving proteins in multiple ways, and the structural basis for
these different modes of proteolysis will be important to understand.
Possibly, the 19 S complex injects polypeptides into the 20 S particle
in a highly specific manner and thus may influence the cleavages made.
Alternatively, because the 19 S complex stimulates the peptidase
activities of the 20 S proteasome, proteolysis may occur in a distinct
manner and proceed further before the products are released. It is
noteworthy that PA28, the interferon-induced activator of peptide
hydrolysis by the 20 S proteasome, also changed the pattern of bonds
cut in a 25-residue oligopeptide (49). In fact, with IGF and ovalbumin
as substrates (Fig. 6 and Table I), the products of the 26 S proteasome
appeared to be slightly shorter than those of the 20 S proteasome
alone. Perhaps the 19 S particles at the ends of the 20 S proteasome
retard the release of the peptide products, leading to additional cleavages.
-ring, the trypsin-like and postacidic
sites are located on adjacent subunits at similar distances apart as
active sites of archaeal proteasome (30 Å), while the
chymotrypsin-like site is on the opposite side of the ring (13).
Despite having far fewer active sites and greater distances between
them, the mammalian proteasomes do not generate longer products than
the archaeal particles. A similar finding was noted recently by
Niedermann et al. (31), who studied the products generated
in the breakdown of short polypeptides (22-44 residues) by activated
20 S proteasomes (although this group analyzed the products after
prolonged incubations when many peptides released by the proteasome
were digested further in later catalytic rounds). 3) When the two
trypsin-like sites of the muscle 20 S proteasome were irreversibly
inhibited, the particles generated a pattern of peptide products
distinct from those produced by control proteasomes (Fig. 7). However,
their size distribution was not significantly different (Fig. 8). The
products of both types of particles followed a log-normal frequency
distribution, and the mean length of the peptides generated by the
particle with four active sites was at most 1 residue longer than
proteasome with six functional sites. In accord with these results,
mutations that inactivate each of the active sites in the yeast 20 S
proteasomes or even two of them did not cause production of peptides of
increased lengths (40). 4) About two-thirds of the products are less
than 8 residues long, which is the shortest distance between
neighboring active sites. Such small products, after initially being
cleaved from the substrate, must have undergone additional cleavages, as was suggested by Dick et al. (50).
- and
-rings and possibly the spaces between the subunits may act as a
filter or sieve preventing or retarding the diffusion of larger
fragments out of the particle.
-interferon, which induces leucine aminopeptidase
when it promotes antigen presentation (23).
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ACKNOWLEDGEMENTS |
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We are grateful to C. Dax, E. Tarcsa, P. Cascio, and T. Saric for help with proteasome preparations and to C. Dax, P. Zwickl, O. Kandror, E. Tarcsa, and A. Scott for critical reading of the manuscript.
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FOOTNOTES |
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* These studies were supported by grants from NIGMS, National Institutes of Health; the Human Frontier Science Program; and the Muscle Dystrophy Association.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.
Present address: Wyeth-Ayerst Research, 145 King of Prussia Rd.,
Radnor, PA 19087.
§ Present address: College of Medicine, Dept. of Biochemistry, Soonchunhyang University, Republic of Korea 330-090.
¶ To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1855; Fax: 617-232-0173; E-mail: agoldber{at}bcmp.med.harvard.edu.
The abbreviations used are: MHC, major histocompatibility complex; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; Amc, 7-amino-4-methylcoumarin; Bis-tris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane; FITC, fluorescein isothiocyanate; IGF, insulin-like growth factor 1; Suc, succinyl; DTT, dithiothreitol; HPLC, high pressure liquid chromatography.
2 K. M. Woo, unpublished observations.
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REFERENCES |
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