From the Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115
The 20 S proteasome processively degrades cell
proteins to peptides. Information on the sizes and nature of these
products is essential for understanding the proteasome's degradative
mechanism, the subsequent steps in protein turnover, and major
histocompatibility complex class I antigen presentation. Using
proteasomes from Thermoplasma acidophilum and four unfolded
polypeptides as substrates (insulin-like growth factor, lactalbumin,
casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide
and the time needed to degrade it increase with length. The average
size of peptides generated from these four polypeptides was 8 ± 1 residues, but ranged from 6 to 10 residues, depending on the protein,
as determined by two new independent methods.
However, the individual peptide products ranged in length from
approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit
a log-normal distribution. No length was predominant, and more than
half were shorter than 10 residues. Peptide abundance decreased with
increasing length, and less than 10% exceeded 20 residues. These
findings indicate that: 1) the proteasome does not generate peptides
according to the "molecular ruler" hypothesis, and 2) other
peptidases must function after the proteasome to complete the turnover
of cell proteins to amino acids.
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INTRODUCTION |
The 20 S proteasome is a 700-kDa proteolytic complex that is
present in all eukaryotic cells, archaea, and certain bacteria (1, 2).
In eukaryotes, the proteasome is an essential component of the
ATP-ubiquitin-dependent pathway for protein degradation (3,
4). The 20 S particle functions as the proteolytic core of the 26 S
proteasome complex, which catalyzes the rapid degradation of many short
lived regulatory proteins and of proteins with abnormal conformation
(1, 4-6). In mammals, the proteasome is also responsible for the
breakdown of most long lived cell proteins and for the generation of
most peptides presented to the immune system on
MHC1 class I molecules (5,
7).
The 20 S particle is a barrel-shaped structure composed of four stacked
rings. Each outer ring contains seven related
-subunits, and each of
the inner rings seven related
-subunits, which catalyze peptide bond
cleavage. The active sites are located within the central chamber of
the 20 S particle, into which protein substrates must enter through a
narrow openings in the
- and
-rings. Proteasomes cleave peptide
bonds by a novel catalytic mechanism, in which the hydroxyl group of
the threonine on the N terminus of the
-subunit serves as the active
site nucleophile (8-12).
Recently, we have shown that the 20 S proteasome degrades proteins by a
highly processive mechanism to oligopeptides (10). A typical protease
makes a single cleavage in a polypeptide substrate and then releases
the fragments, which may be cleaved to smaller products in subsequent
enzymatic rounds. In contrast, the proteasome appears to make many
cleavages in the polypeptide and to digest it to small products without
dissociation of the partially degraded substrates. This novel mode of
degradation appears highly important for an intracellular proteolytic
system, since the release of large protein fragments could interfere
with cell function and regulation. However, definitive proof of this
mode of degradation requires knowledge of the number of cleavages made
in a protein substrate and the sizes of peptides generated. If the
proteasome makes repeated cuts processively along the length of the
polypeptide, one would predict that the enzyme should make a greater
number of cleavages and take more time to digest longer polypeptides than shorter substrates. One goal of the present study was to test
these predictions.
Knowledge about the size distribution of peptides produced by the
proteasome is important for understanding the subsequent steps in the
protein degradative pathway and the process of MHC class I antigen
presentation. In vivo, most of the peptides generated by the
proteasome must be rapidly hydrolyzed to amino acids, which are
utilized in synthesis of new proteins or in intermediary metabolism. In
mammalian cells, some of these peptides are utilized in antigen presentation, possibly after further proteolytic processing to the
final 8-9-residue peptides presented on the cell surface (13). These
latter steps are poorly understood, in part because of a lack of
precise information on the sizes of the peptides released by the
proteasome during protein breakdown.
It is widely believed that the proteasome degrades polypeptides
according to a "molecular ruler" to yield products of rather uniform size (8, 12, 14, 15), as first proposed by Wenzel et
al. (16). This group reported that a large fraction of the peptides generated during the breakdown of hemoglobin or insulin
-chains by archaeal proteasomes were between 7 and 9 residues long
(16). Since the distance between adjacent active sites corresponded to
an octapeptide in an extended conformation (8, 16, 17), it was proposed
that peptides of 7-9 residues were routinely generated as a result of
coordinated cleavages by neighboring active sites. However, evidence
for such a molecular ruler is quite limited. In the study by Wenzel
et al. (16) or in other studies of peptides generated by the
proteasome (18-23), the relative amounts of peptides of different
sizes were not quantified. In addition, Wenzel et al. (16)
analyzed peptide products after prolonged incubations, during which the
products were likely to undergo repetitive cleavage by the proteasome
(20, 22, 23). The present studies were undertaken to determine the mean
size of peptides generated from full-length proteins, to measure the relative amounts of peptides of different sizes, and thus to critically test the molecular ruler model.
Most prior studies have focused on peptides ranging from 4 to 44 residues in length, which may be degraded differently from proteins. In
contrast, in this study, we have investigated the digestion of proteins
of different sizes ranging in length from 70 to 471 residues. We have
introduced several new approaches to evaluate the mean number of cuts
made in each protein substrate, the mean sizes of the peptides
generated by proteasome, and the relative amounts of products of
different lengths. Since only unfolded molecules can enter the 20 S
particle and be degraded (24), the present studies utilized denatured
polypeptides. Conditions were chosen where the protein substrate was
present in large excess, such that the released peptides were not
digested further. We have employed 20 S proteasomes from the archaea
Thermoplasma acidophilum, because this particle is
structurally simpler than the eukaryotic 20 S proteasome. It contains
seven identical
- and
-subunits, and thus seven identical active
sites, located at equal distances from each other (8), unlike the
eukaryotic particle which contains seven different
-subunits and
seven different
-subunits, and has three active sites with different
specificities (12). Therefore, the factors determining product size and
rate of proteolysis should be easier to elucidate and the data easier
to interpret with archaeal particle.
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EXPERIMENTAL PROCEDURES |
Materials--
The Thermoplasma proteasome was
expressed in Escherichia coli and purified as described
previously (10). Recombinant human IGF was a kind gift of Dr. W. Prouty
(Eli Lilly, Indianapolis, IN), bovine
-lactalbumin and
-casein,
alkaline phosphatase from E. coli, Leu-enkephalin amide,
substance P, and oxidized A- and B-chains of bovine insulin
were purchased from Sigma. Synthetic peptides SIINFEKL,
YPHPARIGL, TYQRTRALV, and YSDEDMQTM were kindly provided by Dr. K. Rock
(University of Massachusetts Medical Center, Worcester, MA). All other
peptides were from Bachem AG (Switzerland).
For use as substrates, IGF and lactalbumin were unfolded by reduction
of disulfide bonds and carboxymethylation as described previously (10).
The IGF, lactalbumin, and casein were exhaustively, reductively
methylated (10) to prevent the extent of the reaction of the undegraded
molecule with fluorescamine, and the modified polypeptide purified by
HPLC prior to use. To denature alkaline phosphatase, it was oxidized
with performic acid (25). The concentrations of modified IGF,
lactalbumin, and casein were determined by UV absorption at 280 nm.
Calculations of the extinction coefficient for each protein were based
on the contents of Tyr and Trp residues in each molecule (26). The
concentration of alkaline phosphatase was measured by the Lowry method,
because Tyr and Trp are destroyed or modified during performic acid
treatment.
Degradation of Protein Substrates and "Two Rates"
Method--
The protein substrates were incubated with proteasomes at
54 °C in 50 mM Bis-tris propane buffer, pH 7.5. Substrates incubated without proteasomes and proteasomes incubated
without substrate served as controls. At different times, aliquots were
taken from the reaction mixtures and mixed with an equal volume of
0.4% trifluoroacetic acid to stop the reaction. The concentration of
the newly formed amino groups was measured by reaction with
fluorescamine at pH 6.8 as described previously (10). Another aliquot
was run on the C18 HPLC column (Vydac peptide and protein,
0.46 × 25 cm, 10 µm) to separate products from the undegraded
protein substrate. The column was equilibrated with 0.06%
trifluoroacetic acid and eluted with a gradient of buffer B (80%
acetonitrile, 0.05% trifluoroacetic acid) at a flow rate of 1.5 ml/min. A stepwise increase in buffer B concentration to 40-50%
(depending on the substrate) was used in the region where peptide
products were eluted, followed by isocratic elution for 2 min, and a
gradient of 10% buffer B/min for 2 min was used in the region where
the undegraded protein was eluted. Such a gradient allowed us to
decrease the time required for analysis and the volume of pooled
peptides. In some experiments (Table II and Fig. 6), the pooled
peptides were collected for further analysis. (Precipitation with
trichloracetic acid could not be used here because a significant
fraction of peptides was found to precipitate together with the
undegraded substrate.) The amount of undegraded protein was measured by
integration of its HPLC UV absorbance peak at three different
wavelengths (214, 230, and 280 nm).
To obtain kinetic constants, the concentrations of amino groups and the
area of the substrate peaks were plotted against the incubation time.
The rates of substrate disappearance and of product accumulation were
then determined from the slopes of these plots, which were linear under
conditions used here. Less than half of the initial amount of the
substrate was degraded at the end of incubation. To ensure this
linearity, the initial substrate concentrations were at least 2-fold
greater than the concentrations at which Vmax
was reached (500 µM for IGF, 90 µM for
lactalbumin, 25 µM for casein, and 12 µM
for alkaline phosphatase). The number of cuts in a polypeptide was
calculated by dividing the rate of product accumulation by the rate of
substrate consumption. The mean length of the products (in residues)
was obtained by dividing the length of the protein by the number of
cuts plus one.
"Acid Hydrolysis" Method--
The substrates were incubated
with proteasomes until 30-50% was degraded. The products were
separated from the undegraded substrate by HPLC on the C18
column, pooled, lyophilized, and redissolved in water. Free amino
groups in the pool (i.e. the amount of peptides) were
measured by the reaction with fluorescamine in 0.2 M
phosphate buffer (pH 6.8) (10). These peptides were then hydrolyzed
completely to amino acids with 6 M HCl in sealed ampoules
for 24 h at 108 °C, and the amount of amino acids after hydrolysis was measured by the fluorescamine assay in 0.2 M
borate buffer (pH 8.6). A standard mixture of amino acids, treated in the same fashion as the samples, was used for calibration of the amino
acid assay. Then, the mean size was determined by dividing the molar
amount of amino acids found after acid treatment by the molar amount of
peptides before the treatment.
Size-exclusion Chromatography of Degradation
Products--
Size-exclusion chromatography was performed on
polyhydroxyethyl aspartamide column (0.46 × 20 cm, Poly LC,
Columbia, MD), using a HP1090 chromatographer (Hewlett-Packard). The
mobile phase was 0.2 M sodium sulfate, 25% acetonitrile,
pH 3.0 (adjusted with phosphoric acid), and the flow rate was 0.125 ml/min. To determine the apparent molecular mass of the peptides
eluted, the column was calibrated each time before use with 8-10
standard peptides in the 550-3500-Da range. The pool of proteasome's
products (the same as in acid hydrolysis method) containing 5-10 nmol
of peptides was dissolved in 50 µl of the mobile phase and loaded
onto the column. Fractions (0.5 min) were collected, and the molar
amount of peptides in each fraction was measured with the fluorescamine assay as described above. The corresponding fraction of the control mixture, in which the substrate was incubated without proteasome, was
also run on the size-exclusion column. No fluorescamine-reactive material was found in the fractions of this run.
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RESULTS |
Number of Cuts Made in Substrate Molecules--
To ensure rapid
hydrolysis and to eliminate possible complications due to substrate
folding, three substrates (alkaline phosphatase, IGF, and lactalbumin)
were denatured prior to study, as described above. The other substrate
studied, casein, had little or no tertiary structure and did not
require denaturation to be degraded rapidly by the proteasome. To
determine the number of cuts made by the proteasome in these different
polypeptides, we measured the rate of disappearance of substrate
molecules and the rate of appearance of the new amino groups during the
same incubation. Denatured IGF, lactalbumin, casein, and alkaline
phosphatase were incubated at 53 °C and pH 7.5 with highly purified
recombinant Thermoplasma proteasomes. Initial substrate
concentrations were high enough to ensure a constant rate of
degradation during the entire incubation period. At different times,
aliquots were removed, and one portion was analyzed by HPLC to
determine the amount of substrate consumed (by measuring the amount of
undegraded substrate by integration of its peak area), another portion
was used to determine the amount of peptides produced by assaying them
as the number of new primary amino groups generated that react with
fluorescamine. As found previously (10), the rates of accumulation of
peptide products and of the disappearance of the substrates paralleled
each other. The ratio of the amount of new products generated to the
amount of protein molecules degraded did not change with time,
indicating a processive mechanism. Moreover, the products once released
by the proteasome did not get cleaved again at later times under these
incubation conditions, where there was a large molar excess of the
substrate.
If the proteasome makes n cuts in a protein molecule and the
products generated do not undergo further cleavages, there should always be n-fold more new amino groups than substrate
molecules consumed. Therefore, the number of cuts made per protein can
be determined by dividing the rate of peptide product accumulation by
the rate of substrate disappearance (Fig.
1). This value increased with the length
of the substrate, ranging from 11 cuts in IGF, which is 70-residues
long, to 71 cuts in alkaline phosphatase, which contains 471 residues
(Table I). The demonstration that a large
number of cuts are made in a single substrate molecule, together with
the previous finding that the proteasome does not release the substrate
until all these cuts are made (10), is clear evidence of a highly
processive mechanism for protein degradation (Fig.
2).

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Fig. 1.
Two methods to determine the mean size of the
peptides generated by the proteasome and the number of cuts in a single
polypeptide. See "Experimental Procedures" for further
details.
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Table I
Kinetic parameters of protein degradation by the proteasome
The number of protein molecules degraded and peptide bonds cut by a
single proteasome particle per min were determined by division of the
corresponding cleavage rate (as described under "Experimental
Procedures") by the proteasome concentration measured by the methods
of Bradford or Lowry with similar results. The number of cuts in the
molecule made (column 5) was determined by dividing the number peptide
bonds cleaved (column 4) by the number of substrate molecules degraded
(column 3). See Fig. 1 for an explanation of the calculations.
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Fig. 2.
The number of cuts made by the 20 S
proteasome in a protein depends on its length. The number of cuts
was determined by the two rates method (open circles) and by
the acid hydrolysis method (closed rectangles) (Fig. 1). The
proteins are: 1, IGF; 2, lactalbumin;
3, casein; and 4, alkaline phosphatase.
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Time Necessary to Degrade Different Protein Molecules--
This
highly processive mechanism suggests that the proteasome should require
more time to degrade a longer polypeptide than a short one, provided
that it moves along the substrate at a relatively constant rate. These
measurements of the rate of disappearance of the different substrates
(Table I) allowed us also to determine the time needed to degrade a
substrate molecule, because these experiments were performed at
Vmax. The number of protein molecules degraded
per min by a 20 S particle was calculated by dividing the rate of
substrate disappearance by the molar concentration of the proteasome
(Table I). The reciprocal therefore represents the time that the
proteasome takes to degrade one substrate molecule, assuming it
degrades only one substrate molecule at a time.
The time required to degrade each of these polypeptides was a
characteristic feature of the substrate. As shown in Fig.
3, the time for degradation by the
proteasome depended on the polypeptide's length. At 53 °C, the
enzyme required 10 s to digest one IGF molecule (70 residues, 11 cuts) and 50 s to degrade one casein molecule (209 residues, about
20 cuts). When casein was modified by fluorescein isothiocyanate, the
proteasome still took approximately 1 min to degrade it. Thus, with
smaller polypeptides, there was almost a linear relationship between
substrate length and duration of the degradative process. In contrast,
almost 5 min were required to digest alkaline phosphatase (471 residues, 71 cuts). The disproportionately long time required for
degradation of alkaline phosphatase suggests that there are additional
rate-limiting steps in the digestive process aside from peptide bond
cleavage, such as the unfolding of residual secondary structure or
disassembly of substrate aggregates, which may slow substrate entry
into the central chamber. However, this preparation of alkaline
phosphatase appeared to contain another conformational form, which had
a lower affinity for the proteasome but was degraded severalfold
faster. It was impossible to measure the time required for degradation
of this form because it was insoluble at high concentrations.
Therefore, all measurements were done on the slowly degraded form of
alkaline phosphatase, which remained soluble at
Vmax. To study additional unfolded proteins as
substrates with lengths between casein and alkaline phosphatase we
tried to study rhodanese, glyceraldehyde-3-phosphate dehydrogenase and
-subunit of tryptophan synthase. However, at 53 °C (where Thermoplasma enzymes are quite active), these polypeptides
were insoluble. Another polypeptide tested (ovalbumin) remained
soluble, but was a poor substrate in vitro even after
denaturation.

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Fig. 3.
The time needed by the 20 S proteasome to
degrade a polypeptide depends on its length. The time for
degradation was calculated as the inverse of the number of protein
molecules degraded/min at Vmax. It was presumed
that only one polypeptide is degraded by a proteasome particle at a
time. The proteins are: 1, IGF; 2,
lactalbumin; 3, casein; 4, alkaline
phosphatase; and 5, fluorescein isothiocyanate-casein.
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Mean Size of the Peptide Products--
The finding that the number
of cuts made in a polypeptide is proportional to its length implies
that the length of the peptides generated by the proteasome is similar
for the different proteins. We have developed two simple methods to
determine the mean size of these peptide products. In the first, which
we term the two rates method, the mean size of the products was
calculated by dividing the number of amino acids in the protein by the
number of bonds cut plus one (Fig. 1). For example, if a protein
containing 100 amino acid residues is cut at 9 sites to yield 10 pieces, their average length is 10 residues. The mean size of products found by this approach was similar with the four different substrates. The values obtained ranged from 6 for IGF to 11 for casein with an
average length of 8 for the four proteins (Table
II). These differences, although small,
in the mean sizes of peptides generated from different proteins were
found reproducibly (Table II). The standard errors on these values in
Table II represent the range of the mean sizes obtained in independent
experiments, rather than the variation in length about the mean (see
below).
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Table II
Mean size of peptides generated by the proteasome
The values in the table are means ± S.E. of at least three
experiments (for the acid hydrolysis method) or two experiments (for
the two rates method). In the two rates method, the mean size was
determined by dividing the number of amino acids in a protein by the
number of cuts plus one (see Table 1 and Fig. 1). In the acid
hydrolysis method, the pooled products were hydrolyzed to amino acids,
and mean peptide size was obtained by dividing the amount of amino
acids after acid hydrolysis by the amount of peptides before it (Fig.
1).
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To obtain an independent measure of the mean sizes of these peptide
products, another approach was developed, which we call the acid
hydrolysis method. The peptides generated by the proteasomes were
separated from the undigested substrate by HPLC on a reverse-phase column. The amount of peptides produced was measured with
fluorescamine, they were then hydrolyzed completely to amino acids by
acid treatment, and the amount of amino acids was measured with
fluorescamine. If an individual peptide containing n
residues is hydrolyzed completely, the molar amount of amino acids
produced should be n-fold greater than the amount of
peptides present initially. Therefore, dividing the amount of amino
acids after acid hydrolysis by the amount of peptides before this
treatment gives the length of peptide, and with a mixture of peptides,
this approach gives the weighted average of the lengths of peptides,
i.e. their mean size (Fig. 1).
The values obtained by this method for the mean sizes of the peptides
generated from the different substrates (Table II) ranged between 7 and
9 residues and resembled closely the values obtained by the two rates
method for the same substrate (Table II). Thus, with proteins that
differ in length by 7-fold and more than 20-fold in the time required
for their degradation, the mean product length was close to 8 residues,
although slightly smaller values were consistently found with IGF (6 to
7 residues) and longer values with casein (9-11 residues). This
finding of a mean size of 8 residues is consistent with the suggestion
that a major determinant of product size is the distance between active
site threonines, which corresponds to 8 residues of an extended
polypeptide chain. However, the demonstration of similar mean sizes
does not imply that individual peptide products are of uniform lengths,
as would be expected if the proteasome cleaves substrates according to a molecular ruler model (8, 16).
Size Distribution of the Peptide Products--
To understand how
proteins are digested within the proteasome's central chamber and to
investigate the metabolic fates of peptides generated, it is essential
to determine the actual size distribution of the peptides produced from
a specific substrate. Two approaches were used to characterize the
lengths of products generated during degradation of IGF. In the first
one, these peptides were separated by HPLC on a C18-reverse
phase column (Fig. 4A), and
all peaks detected with UV were collected individually. The molar
amounts of the peptides in each peak were then measured with the
fluorescamine assay. These measurements demonstrated that individual
peptides appeared in nonstochiometric amounts, and that the molar
amounts of the majority of individual products were less than the molar
amount of substrate degraded. Thus, although the proteasome always
degraded IGF molecules processively to peptides of small size, it did
not cut all IGF molecules at identical places. As a result, the
particle generated at least 50 different products (as judged from the
number of distinct HPLC peaks), while cleaving on the average eleven
bonds in each IGF molecule (Table I).

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Fig. 4.
Distribution of peptides generated by
proteasomes from IGF. A, HPLC separation of a digest. IGF
was incubated with Thermoplasma proteasomes for 4 h as
described under "Experimental Procedures." At this time ~45% of
the substrate was consumed. Peptides were separated on a
C18 (5 µm) column using the buffer system described under
"Experimental Procedures." The flow rate was 0.75 ml/min, and the
gradient of acetonitrile was from 0 to 8% in 60 min, from 8 to 28% in
100 min, and from 28 to 36% in 20 min. UV absorption at 214 nm was
used for detection of peptides. The large peak, eluting at 180 min,
corresponds to the undegraded substrate. B, analysis of the
size distribution of the products by mass spectrometry. All
UV-detectable peaks of the HPLC run and the fractions between them were
collected, and the amounts of the peptides present were determined by
reaction with fluorescamine at pH 6.8 (10). 27 major peaks comprising
75% of total pool of peptides were subjected to matrix-assisted
laser-desorbtion/ionization mass spectroscopy. In addition, six peaks,
which appeared to be impure by mass spectroscopy, were sequenced. The
minor peaks, which together comprised 25% of the total amount of
products were not analyzed. C, distribution of the products
by molecular weight obtained by size-exclusion chromatography. The
results of the experiment demonstrated in Fig. 6 were graphed against
the molecular weight on a linear scale. The amount of peptides in each
fraction was determined with fluorescamine.
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The molecular weights of the peptides present in the 27 major peaks
were determined by mass spectroscopy. Some peaks, which appeared to be
impure by mass spectrometry, were analyzed by microsequencing to
establish the nature and relative amounts of the peptides present. The
great majority of these peptides ranged in molecular mass from below
500 to 1300 Da, and relatively few were detected in the 1300-2000-Da
range (Fig. 4B). Together these peaks comprised 75% of
total molar amount of peptides generated. Assuming that the average
molecular mass of an amino acid in IGF is 116 Da, the majority of the
products were between 4 and 12 residues long, although one major
peptide of 413 Da on sequencing appeared to be a tripeptide. The mean
molecular mass of analyzed peptides for this distribution was 800 Da (7 residues), which equals the mean length obtained by the acid hydrolysis
method (Table II). However, peptides of this size, 7 ± 1 residues, were not a predominant species, and the size distribution was
highly asymmetric. Peptides of 5 residues and shorter were most
abundant and the abundance of the products tended to decrease as their
size increased.
This HPLC-mass spectrometry approach was not applicable to the longer
substrates. Since there were more than 50 peptides generated from IGF
(Fig. 4A), it seemed likely that almost 100 would be generated from lactalbumin and several hundreds from casein and alkaline phosphatase. Even if such a large number of peptides could be
resolved on HPLC, it would be impractical to analyze these products
individually, as was done with IGF-derived peptides. Moreover, analysis
of IGF degradation products could not include the least abundant 25%
peptides. Therefore, an additional method to analyze the size
distribution of peptides was developed, in which product length was
assayed using size-exclusion chromatography on polyhydroxyethyl
aspartamide HPLC column. This column had been reported to fractionate
peptides in the range of 500-10,000 Da, which should include the great
majority of proteasome products, and has been used to separate by size
peptides generated from casein by trypsin (27). In initial control
studies, the retention times of 13 randomly selected synthetic peptides
with very different sequences were found to be highly reproducible and
to show a linear dependence on the logarithm of their molecular weights
(Fig. 5).

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Fig. 5.
Calibration curve for polyhydroxyethyl
aspartamide size-exclusion column. Standards used: 1,
t-butoxycarbonyl-IEGR-7-amido-4-methylcourmarin (Amc); 2,
succinyl (Suc)-AAF-Amc; 3,
Suc-ALPF-p-nitroanilide; 4, Leu-enkephalin amide
(5 amino acid residues); 5, Suc-LLVY-Amc; 7,
SIINFEKL (8 residues); 8, YPHPARIGL (9 residues);
9, TYQRTRALV (9 residues); 10, YSDEDMQTM (9 residues); 11, substance P (11 residues); 12,
oxidized A-chain of insulin (21 residue); and 13, oxidized
B-chain of insulin (30 residues). The typical peak width of these
peptides was 0.6 min.
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To analyze the sizes of the proteasome products, these peptides were
first separated from the undegraded protein substrate by HPLC on a
reverse phase column, as described above. The combined products were
then fractionated on the size-exclusion column. The molar amounts of
the peptides in each fraction were determined by the fluorescamine
assay and graphed against the elution time (Fig.
6). The resulting curves were highly
reproducible (see, for example, the two separate runs for casein) and
differed with each substrate. However, the general pattern was similar
for the peptides generated from all four proteins. With the longer
substrates, casein and alkaline phosphatase, the abundance of peptides
fit closely a normal distribution when graphed against the logarithms of their molecular weights (Fig. 6), i.e. against the
retention time on the size exclusion column, which is proportional to
the log(molecular weight). In other words, on such a logarithmic scale, the amounts of peptides generated by proteasome seem to be normally distributed, indicating a log-normal distribution. With IGF and lactalbumin, the abundance of peptides of different sizes also appeared
to fit a log-normal distribution, but with greater deviation, probably
because of the smaller sampling size (Fig. 6).

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Fig. 6.
Size-exclusion chromatography of peptides
generated from different proteins. Protein substrates from which
peptides were generated are indicated on each plot. 62.5-µl (0.5 min)
fractions were collected, and the molar amount of the peptides in each
fraction was measured with the fluorescamine assay and normalized to
the total amount of peptides eluted from the column. Results of two experiments with two different preparations of peptides generated from
casein are shown. Molecular weights were obtained from the calibration
curve shown in Fig. 5.
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In contrast, when relative amounts of the products were graphed against
molecular weight on a linear scale (Fig. 4C), this distribution was clearly asymmetric. The decrease in amount of products
with increasing size is similar to the finding we obtained in the mass
spectroscopy analysis of the same IGF-derived peptides (Fig.
4B). In addition, both methods gave the same mean molecular masses of 800 Da (7 residues) for the products, which agreed well with
the mean size value obtained by the acid hydrolysis method.
As shown in Fig. 6, the sizes of the products varied widely, and a
fraction was shorter than 500 Da (4-5 residues), but the exact size of
the shortest peptides could not be determined, since it was impossible
to calibrate the size-exclusion column for peptides smaller than 500 Da. On the other hand, peptides reached up to 2300 Da (about 20 residues) when the shorter substrates, IGF and lactalbumin, were
degraded. With the longer substrates, casein and alkaline phosphatase,
some peptides ranged up to 5000 Da or ~45 residues. This broad range
in the sizes of products and the asymmetric log-normal distribution are
not consistent with the proteasome's cleaving proteins according to a
molecular ruler mechanism.
Another informative way to analyze the data on size distribution of
products (Fig. 6) was by calculating the cumulative frequency of
peptides of a given size, i.e. the fraction of all peptides having a molecular weight equal or less than the peptide of interest (Fig. 7). Fig. 7A demonstrates
that such curves for the products of IGF degradation obtained by mass
spectrometry and size-exclusion chromatography were practically
indistinguishable. Thus, these two different methods yielded a very
similar or identical size distribution of peptide products. Plotting
the data from the gel filtration column for products from the other
polypeptide substrates (Fig. 7B) also showed a smooth
increase in cumulative frequency with increasing peptide size. The lack
of an inflection point in these cumulative frequency plots is clearly
not consistent with a normal distribution (28) and consistent with a
log-normal distribution. This type of analysis also clearly
demonstrates that proteasomes generate a significant fraction of
products shorter than pentapeptides. The fraction of these small
peptides ranged from 15% of the products generated from lactalbumin up
to 40% of the IGF-derived peptides. With all substrates, 40-50% of
the products fell between 6 and 10 residues.

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Fig. 7.
Cumulative frequency curves for peptides
generated by proteasome. The curves were obtained by
transformation of the mass spectrometry and size-exclusion
chromatography data demonstrated on Figs. 4B and 6. For each
point, the fraction of peptides with molecular masses of those at this
point and lower was calculated. The scale for peptide length was
obtained by dividing the molecular mass scale by 115 Da, the average
molecular mass for an amino acid residue. The arrow
indicates the average mean size (Table II). A, cumulative
frequency curve for peptides generated from IGF, as determined by two
different methods. B, curves for four different substrates
obtained by size-exclusion chromatography.
|
|
Of the peptides generated from IGF and lactalbumin, all were shorter
than 20 residues (2300 Da). However, a small fraction (<10%) of the
peptides generated from the longer proteins, casein and alkaline
phosphatase, were between 20 and 30 residues long. Some peptides
(<5%) generated from casein even appeared to be longer than 30 residues. This high value, however, may be an artifact due to the
unusual primary sequence of casein, in which 15% of all residues are
prolines. Prolines can impose conformational constraint on peptides
leading to increased hydrodynamic radii and anomalous behavior on the
size-exclusion column. In either case, some of the products are of
approximately the same length or are longer than in oligopeptide
substrates used in previous studies of proteasome function (18,
20-23).
Although the weighted average size of peptides generated by the
proteasome appeared to be 8 (Table II), less than 20% of the products
had lengths between 7 and 9 residues. This size was expected to
predominate based on the molecular ruler model in which the distance
between the adjacent active sites was the primary determinant of
product size.
The mean size of the products calculated from the size distribution was
in good agreement with the results of both chemical methods for
determining mean product size (Table II). Our earlier findings on the
mean size of the products (Table II) also suggested that, with larger
substrates, the peptides generated tended to be larger than with
smaller substrates. When the cumulative frequency of peptides generated
from these different proteins were compared, the peptides from alkaline
phosphatase appeared consistently slightly longer than those generated
from IGF but slightly shorter than those from casein by 1 to 2 residues. These findings indicate that the small apparent differences
found in the mean product size of products from these three proteins
(Table II) represent real differences. Thus, the sequence and length of
a protein substrate can influence the fraction of large peptides
generated by the proteasome.
 |
DISCUSSION |
Mode of Protein Degradation--
The present findings and related
observations (10) clearly show that the proteasome digests proteins in
a fundamentally different manner from the great majority of proteolytic
enzymes. Generally, an endoprotease or exoprotease dissociates from its substrate after each cleavage. In contrast, the proteasome degrades proteins in a highly processive manner (10), i.e. the enzyme makes multiple cuts in a protein and converts it to oligopeptides before attacking the next substrate molecule. For example, the archaeal
proteasome made about 70 cleavages in degrading alkaline phosphatase
(Fig. 2, Table I) and converted it into peptides ranging from about 4 to 30 residues in length. Further evidence for a highly processive
mechanism was the finding that the number of cuts in a polypeptide and
the time needed to degrade it increase with the length of the
substrate. Interestingly, as the length of the substrate increased, the
number of peptide bonds cleaved per min decreased (Table I). With the
longer substrates more time is probably necessary for a cleavable bond
on the substrate to reach the active site(s). For polypeptides shorter
than 200 amino acids, the time required to degrade them was directly
proportional to their length, but increased steeply and nonlinearly
with alkaline phosphatase, a 471-residue protein. Perhaps the alkaline
phosphatase, although initially denatured, retained more secondary
structure or reversibly aggregated; either mechanism would retard its
diffusion into the central chamber or proteolytic attack.
This highly processive degradation to small peptides must mean that the
protein substrate remains associated with the proteasome until the
degradative process is complete. Such a mechanism would appear to be
highly advantageous to the cell, since it ensures rapid elimination of
proteins targeted for degradation and prevents the accumulation of
partially degraded proteins, which could be highly toxic. Presumably,
this behavior is due to the particle's complex architecture. To reach
the active sites in the proteasome's central chamber, substrates must
first traverse the narrow opening in the
-ring, the outer chamber,
and finally the opening in the
-ring. These multiple chambers
probably help prevent the premature release of substrates until the
products are small enough to exit the particle.
Rates of Protein Degradation--
Under the present conditions
(53 °C, pH 7.5), which were chosen to ensure high activity of the
Thermoplasma proteasome, the particle took about 50 s
to degrade casein and 4.5 min to digest alkaline phosphatase. At the
optimal growth temperature for this organism (60 °C), degradation of
casein occurred approximately 20-25% faster, but the proteasome still
took approximately 40 s to degrade casein (not shown). These
values were calculated on the assumption that at
Vmax only one polypeptide molecule was degraded
at a time by the proteasome. However, it is unclear whether one
proteasome particle degrades only 1 or 2 substrate molecules at a time.
Two Nanogold-modified insulin molecules were found associated with each
end of a single proteasome (24). If 2 molecules can be degraded
simultaneously, the time required for degradation of a single
polypeptide should be twice as long as values shown here (Fig. 3).
In either case, the times necessary to digest proteins appear
surprisingly long. If these rates are similar to the rates in vivo, then it would suggest that the proteasome may take a longer time to degrade a protein than the ribosome takes to synthesize it. In
E. coli, the ribosome requires about 10 s to synthesize a protein the size of casein and 24 s for one the size of alkaline phosphatase, which are much shorter than the times for proteolysis measured here. The eukaryotic proteasome (12) may well digest proteins
at different rates, since it contains only three active sites with
distinct specificities, and within the 26 S complex, its function is
linked to ATP hydrolysis, which may enhance the rate of protein
degradation.
Size of Degradation Products--
Using either of the new methods,
the average size of the products of the proteasome was found to be 8 residues (Table II). Similar sized peptides were generated from
proteins that varied 7-fold in length, had markedly different
sequences, and differed up to 25-fold in the times required for their
degradation. Small differences, however, in the mean size were seen
repeatedly with different substrates (Table II), apparently because
some longer peptides were generated from the longer polypeptides (Fig.
6). An average size of 8 residues indicates that the proteasome cuts only 10-15% of the peptide bonds present in the full-length protein. In vivo, the remaining 85-90% of the peptide bonds must be
cleaved by other proteolytic enzymes, which remain to be identified.
Since peptides of this size cannot be found in the cytosol, they must be rapidly digested and tend to be short-lived in cell
extracts.2 Thus,
intracellular proteolysis seems to require the concerted activity of
the 20 S complex and multiple endo- and exopeptidases.
As shown by size-exclusion chromatography and mass spectrometry, the
sizes of peptides produced by the proteasome are broadly and
asymmetrically distributed around the mean. This pattern and the
results obtained by plotting cumulative frequencies of individual peptide sizes are not consistent with a Gaussian or normal distribution (e.g. in a cumulative frequency plot, a Gaussian
distribution would have shown an inflection point at 50%). However,
the product sizes seem to fit a log-normal distribution, such that the
relative amount of the peptides decreases as their length increases.
This fit was particularly strong with casein and alkaline phosphatase, presumably because of the larger number of peptides generated from
these substrates. It is noteworthy that (except for peptides generated
from casein) no product was longer than 30 residues, and in all cases,
at least 90% of the peptide products were shorter than 20 residues.
While peptides longer than 20 residues comprised fewer than 10% of the
peptides produced from casein and alkaline phosphatase, these products
actually contain 20-30% of the amino acids and mass of the original
proteins.
These findings therefore are not consistent with the idea that the
proteasome digests substrates according to a precise molecular ruler,
as had been first proposed (16) and as has been widely assumed (8, 12,
14, 15). On the contrary, octapeptides were not even a major species.
In fact, less than 15% of the peptides fell within the 7-9-residue
range, and products were not uniformly distributed about this size.
This size distribution is also not consistent with the limited data on
product size published previously (16, 18-23). Several studies (16,
20-23) had suggested that the peptides generated by proteasomes were
distributed more symmetrically and tightly about the mean. Several
reasons may account for these differences. 1) Short peptides are more
likely to be missed in eluates from reverse-phase column used in all
previous studies, because of their low UV absorption. By contrast, we
used size-exclusion column from which peptides were eluted as pools,
and not as individual peaks, and also fluorescamine, which detects
shorter peptides reliably and quantitatively. 2) The absolute amounts
of these products were not quantified previously. 3) Most prior studies used substrates containing less than 44 residues, which provide too
small a sampling size for accurate conclusions about the
size-distribution of the products of large proteins. In addition,
Wenzel et al. (16) did not find products longer than 14 residues, probably because the products were analyzed after a 24-h
incubation, when the proteins had been digested completely and when
peptides released from the proteasome are cleaved further by the
particle (20, 22, 23).
The mechanisms which determine the size of products and this log-normal
distribution are quite unclear. It was proposed that the main factor
determining product size (the molecular ruler) was the distance between
adjacent active sites, which corresponds to a 7- or 8-residue peptides
in an extended conformation (8, 16). Our finding of a mean product size
of 8 suggests that this distance is one factor influencing product
size. However, since less than 15% of the products were of this size,
other factors must also influence product size. The processive
cleavage of a polypeptide presumably results from the coordinated
action of multiple active sites. Huber and co-workers (8, 17) proposed that the substrate remains covalently attached to one active site threonine residue, while it is attacked by an active site on an adjacent subunit. Such a mechanism would generate predominantly 7-8-residue peptides. However, if cleavages are made by nonadjacent active sites, longer products would be generated. Peptides smaller than
7 residues are probably produced if the cleaved fragments diffuse to
and are cut by other active sites (18). The probability of such an
additional attack must depend on the peptide's sequence. Once
generated, the shorter peptides will have a higher chance to exit the
particle, especially since smaller peptides tend to bind less tightly
to proteolytic sites than longer ones (16). A final determinant of
product size may simply be the time that a fragment remains within the
-chamber (with longer occupancy, there should be more opportunities
for additional cleavages).
Presumably, similar factors determine product size in more complex
eukaryotic proteasomes, but it is impossible to extrapolate from these
data on the products of the archaeal particle to those of the 20 or 26 S eukaryotic proteasome that do not contain seven identical,
symmetrically distributed active sites. Analogous studies of product
size with eukaryotic proteasomes are also of particular interest
because of their role in generating the 8-9-residue peptides used in
MHC class 1 antigen presentation (13).
We are grateful to Drs. M. Zelen (Dana Farber
Cancer Institute) for advice in statistical analysis of data, E. Seemüller (Max-Planck Institute für Biochemie, Germany) for
providing proteasome clones, O. Coux for preparation of Fig. 1, R. Moerschell for assistance with HPLC experiments, O. Kandror, P. Zwickl,
V. Solomon, O. Coux, and H. C. Huang for thoughtful criticisms,
and A. Scott for assistance in the preparation of this manuscript.