From the Departments of Pharmaceutical Chemistry and
Biochemistry and Biophysics, University of California, San
Francisco, California 94143-0446 and the ¶ Department of
Chemistry, Genomics Institute of the Novartis Research
Foundation, San Diego, California 92121
Received for publication, January 8, 2003, and in revised form, February 21, 2003
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ABSTRACT |
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We describe here biochemical characterization of
the 20 S proteasome from the parasitic protozoan Trypanosoma
brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven Proteasomes are multisubunit protein complexes responsible for the
programmed degradation of protein substrates involved in a number of
diverse cellular processes, including cell cycle progression, transcriptional regulation, and antigen presentation by major histocompatibility complex class I molecules (1, 2). The 20 S
proteasome core particle
(CP)1 in eukaryotes is a
barrel-shaped structure made up of a stack of four rings of proteins,
each containing seven distinct protein subunits. The inner two rings
contain Eukaryotic proteasomes possess multiple peptidase activities that are
classified into three major categories based upon the primary amino
acid residues found at the site of hydrolysis. The "chymotrypsin-like" activity cleaves substrates after hydrophobic residues and is thought to be the primary activity of the complex required for initial attack on protein substrates. The
"trypsin-like" activity cleaves substrates after basic residues and
the "peptidyl-glutamyl peptide hydrolytic" (PGPH) or
"caspase-like" activity cleaves after acidic residues (13, 14).
These primary hydrolytic activities have been linked through
mutagenesis and inhibitor studies to three catalytically active Although the primary protease activities of the proteasome have been
classified based on the amino acid residues found at the site of amide
bond hydrolysis (P1 position), extensive inhibitor (21), substrate
(22), and structural (23) studies have confirmed the importance of
extended substrate recognition by the proteasome. In particular,
variations at the P4 position of a substrate or inhibitor have a
profound effect on recognition by individual Trypanosoma brucei is a parasitic protozoan that is the
causative agent of African sleeping sickness. Resistance to commonly used anti-trypanosomal chemotherapeutics presents a potentially serious
problem for most parts of sub-Saharan Africa (25, 26). Several unique characteristics of proteasome-mediated protein degradation in T. brucei have been recently reported. First,
mouse ornithine decarboxylase expressed in T. brucei
together with a rat antizyme was found to be highly stable (27),
whereas this same protein complex is rapidly degraded by the 26 S
proteasome in mammalian cells (28). Second, down-regulation of
expression of each of the 7 In the present study, we describe the subunit composition and
biochemical properties of the T. brucei proteasome.
Surprisingly, although five of the seven T. brucei Cultures and 20 S Proteasome
Purification--
T. brucei 427 strain procyclic form cells
were cultured in Cunningham's medium supplemented with
heat-inactivated fetal calf serum at a final concentration of 10%
(32). Purification of the 20 S proteasome from T. brucei
procyclic form cells was carried out as previously described (33). The
human 20 S proteasome, purified from outdated human blood by previously
described methods (34), was a kind gift from Prof. Martin Rechsteiner
of the University of Utah.
Subunit N-terminal Sequence Determination--
Two-dimensional
protein gel analysis of the purified T. brucei 20 S
proteasome was carried out as previously described (31). The separated
subunit proteins in the gel were stained with Coomassie Blue, and each
protein spot was subjected to Edman degradations for N-terminal
sequence determination at the Protein Core Facility, Howard Hughes
Medical Institute, Columbia University, College of Physicians and
Surgeons, New York, NY.
Competition Assay of Fluorogenic Substrate
Hydrolysis--
4-Hydroxy-3-nitrophenyl-Leu-Leu-Leu-vinylsulfone
(NP-L3-VS), Tyr-Leu-Leu-Leu-vinylsulfone
(YL3-VS), and
4-hydroxy-3-nitrophenol-Leu-Leu-Asn-vinylsulfone (NP-L2N-VS) were synthesized and iodinated with
125I as described (21, 35). Leupeptin
(acetyl-Leu-Leu-DL-Arginal) was purchased from Boehringer
Manheim. Fluorogenic peptides
succinyl-Leu-Leu-Val-Tyr-4-methyl-7-aminocoumarin Suc-(LLVY-MAC) and
Gly-Gly-Arg-4-methyl-7-aminocoumarin (GGR-MAC) were purchased from
Sigma. For peptidase assay, 1 µg of the purified 20 S proteasome from
T. brucei was suspended in 100 µl of the peptidase assay
buffer; 30 mM Tris-HCl, pH 7.8, 5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol. Individual vinylsulfone derivatives or leupeptin were
each added to the proteasome-containing peptidase assay buffer at
varying concentrations and incubated at 25 °C for 45 min prior to
adding 1 µl of a 5 mM stock solution of fluorogenic peptide substrate. The assay mixture was further incubated at 25 °C
for 60 min, and the enzyme-catalyzed reaction was stopped by 100 µl
of 2% SDS. The fluorescence in the mixture was measured with a
fluorometer at an excitation wavelength of 360 nm and an emission
wavelength of 460 nm.
Labeling of 20 S Proteasome with Iodinated Peptide
Vinylsulfones--
Purified T. brucei 20 S proteasome (1-3
µg) was suspended in 100 µl of TSDG (10 mM Tris-HCl, pH
7.4, 25 mM KCl, 10 mM NaCl, 1 mM
MgCl2, 0.2 mM EDTA, 1 mM
dithiothreitol, 20% glycerol). 125I-YL3-VS,
125I-NP-L3-VS, and
125I-NP-L2N-VS were each added at 1.8 × 104 Bq/ml. The labeling reaction was carried out at
25 °C for 60 min and then stopped by adding 25 µl of 5× Laemmli
SDS-PAGE sample buffer to the reaction mixture (21). For competition
experiments, 1-3 µg of the purified T. brucei 20 S
proteasome was preincubated with the unlabeled inhibitors (as
indicated) under the identical conditions as for the labeling
experiment. 125I-YL3-VS was then added to the
reaction mixture at 1.8 × 104 Bq/ml. Protein labeling
proceeded at 25 °C for 60 min and was then stopped by adding 25 µl
of 5× SDS sample buffer as before. The radiolabeled protein was
fractionated and analyzed by 12% SDS-PAGE and autoradiography.
Substrate Library Kinetic Screening--
The purified
recombinant T. brucei 11 S regulator protein PA26 was
prepared as previously described (31). Purified T. brucei 20 S proteasome (24 nM for assays without PA26, 2.4 nM for assays with 640 nM PA26) in a buffer
containing 60 mM Tris-HCl, pH 7.5, 10 mM KCl,
and 5 mM MgCl2 was preincubated at 37 °C for
10 min to allow for association of the 20 S proteasome with PA26. The preincubated proteasome was then added to the substrate libraries, which were synthesized using methods described previously (22). The
substrate concentration was 15 nM/substrate/well in the
one-position fixed library at 6859 substrates/well (Cys was excluded
and Met was replaced by the isosteric non-natural amino acid norleucine (Nle)). In the two-position fixed library, the substrate concentration was 0.25 µM/substrate/well at 361 substrates/well (Cys
was excluded and Met was replaced by Nle). Fluorescence was monitored
for 30 min at Single Substrate Kinetics of the Human and T. brucei
Proteasome--
The following substrate sequences were designed
to be selective substrates for the Analysis of Biochemical Properties of the Subunits of T. brucei 20 S Proteasome--
Samples of purified T. brucei 20 S
proteasome were resolved by two-dimensional gel electrophoresis and
stained with Coomassie Blue (data not shown). The identity of each
protein spot on the stained gel was previously determined by mass
spectrometric analysis (31). The
molecular masses and pI values of each of the identified subunit
proteins were estimated by their positions in the two-dimensional gel.
These values were compared with the molecular weight and isoelectric
point (pI) of each of the
Among the
N-terminal sequence analysis of each of the 14 identified protein spots
indicated that all the
Among the Active Site Labeling of the T. brucei 20 S Proteasome
Labeling of purified T. brucei 20 S proteasome with
125I-YL3-VS revealed modification of only two
subunits of apparent molecular masses of 22.7 and 24.0 kDa (Fig. 2).
The identity of these two subunits was confirmed as the
Affinity labeling of the T. brucei proteasome with the
related peptide vinylsulfone, 125I-NP-L3-VS
resulted in modification of only the Inhibitor Specificity Profiles of the Primary Catalytic
Inhibition of Peptidase Activities of the T. brucei 20 S
Proteasome--
The T. brucei 20 S proteasome is capable of
hydrolyzing a variety of short fluorogenic peptide substrates. Of the
many commercial substrates tested, GGR-MAC was identified as the
most efficient substrate (28) followed by Suc-LLVY-MAC as the next best
substrate. Several other substrates such as IIW-MAC, PFR-MAC, and
AAF-MAC underwent only limited hydrolysis (28), and no hydrolysis of AFK-MAC or YVAD-MAC was detectable. This limited substrate analysis suggests an unusual specificity profile favoring Arg and hydrophobic residues at the P1 position of a substrate. To investigate the potential contributions of Substrate Specificity of T. brucei 20 S Proteasome and Its 11 S Regulator Complex--
To further elaborate the substrate
specificity of the T. brucei 20 S proteasome and to compare
the profile to that of the human enzyme, kinetic screening was
performed using a P1-P4 diverse, positionally scanned library of
peptide substrates. Such libraries have been developed to sequentially
analyze the specificity of proteolytic enzymes by measurement of
kinetic constants for individual library members with a single fixed
amino acid at a single position in the substrate. Library data can then
be used to construct optimal peptide substrates that correlate with the
optimal or specific primary sequences on protein substrate. Substrate
scanning of the T. brucei 20 S proteasome indicates that the
major activity of the complex is for cleavage after P1-leucine residues
with additional minor activities preferring a P1-hydrophobic amino acid
such as Met, Ala, Val, and Tyr and polar or basic amino acids Arg and
Gln (see Figs. 4 and 6). In agreement with the labeling and inhibitor
results, there is little PGPH activity detected in the one-position
fixed library. T. brucei 20 S proteasome was also analyzed
in the presence of the 11 S regulator. As anticipated, a dramatic
increase in the existing peptidase activities was observed upon
addition of the 11 S regulator complex (Fig. 4). In addition, the
cleavage after P1-Glu, Asp, His, and Lys, which were undetectable in 20 S proteasome, were induced by formation of the 11 S-20 S proteasome
complex (Fig. 4) suggesting broadening of substrate specificity as a
result of the complex formation. A control experiment with the 11 S
regulator alone showed negligible background activity.
Analysis of the extended substrate specificity (positions P2, P3, and
P4) of the 20 S proteasome indicates that the preference at these sites
is similar to that of P1 site, preferring aliphatic and hydrophobic
amino acids (Fig. 4). A similar broadening of activity in the 11 S
regulator-20 S proteasome complex to accept polar and charged amino
acids was also observed for the extended P2, P3, and P4 positions
regardless of the amino acid residue found at the P1 position (Fig.
4).
A comprehensive view of the interdependence for the P1 to P4 diversity
space was assessed by employing a two-position fixed library for
substrate analysis of both the core 20 S and the 11 S-20 S complexes
(Fig. 5). Overall, the resulting activity
profiles mirror those seen in the single fixed position library. The
peptidase activity of 20 S proteasome is clustered in the hydrophobic
region of substrate residues, again suggesting that hydrophobic amino acids at P1 to P4 facilitate peptide hydrolysis (Fig. 5A).
The profile of the 11 S regulator-proteasome complex becomes
considerably broader to include the acidic as well as the basic amino
acids, and the importance of the extended binding sites up to P4 is
demonstrated (Fig. 5B). Upon closer examination of the
substrate specificity profiles, several unique features of the
individual peptidase activities emerge. For example, proline is
tolerated at the P2 position, irrespective of the amino acid at P1.
Conversely, a proline at the P3 position is only tolerated well if P1
is an acidic amino acid: aspartic acid or glutamic acid. Another
notable feature of the dependences between sites that is observed by
this data is the change in P2 and P3 selectivity for cleavage after large hydrophobic P1 amino acids upon 11 S cap binding. In the presence
of the 11 S cap, the 20 S protease cleaves more efficiently substrate
P1-hydrophobic amino acids with basic amino acids in the P2 and P3
positions, whereas in the absence of the 11 S cap, basic amino acids
are clearly disfavored.
Finally, the profiles of T. brucei and human 20 S
proteasomes resulting from screening against the one-position fixed
library were compared (Fig. 6). The
results indicate that, although the profiles of hydrophobic amino acids
from P1 to P4 appear similar between the two, the human 20 S proteasome
shows clear recognition of Glu, Asp, His, and Lys at these positions,
whereas T. brucei 20 S proteasome does not. However, one
distinguishing additional activity observed in the T. brucei proteasome over the human proteasome is for cleavage after
glutamine (Fig. 6). To test the magnitude of these differences, single
substrates were designed and Michaelis-Menten kinetics performed for
both the T. brucei and the human 20 S proteasomes. The
results show that the overall kinetics of substrates is less efficient
for the T. brucei proteasome for cleavage of substrates with
P1-Leu, P1-Asp, and P1-Arg, as observed by decreased
kcat/Km (Table
III). In contrast to this observation,
the activity is dramatically increased for cleavage by the T. brucei proteasome for substrates with P1-glutamine as predicted
from the substrate specificity libraries (Table III). These differences
in activity may indicate differences in the active sites between the
T. brucei and the human proteasomes that could be exploited
for therapeutic intervention of trypanosomiasis. The potential
significance of this difference will be discussed below.
In the present investigation, we perform detailed biochemical
analysis of the T. brucei proteasome. Analysis of subunit
composition of the complex indicated that five out of the seven
These data support the conclusion that evolution of the seven
originally identical Alignment of the sequences of T. brucei Labeling of the proteolytically active A similar finding was also made from a kinetic screen of T. brucei 20 S proteasome using one-position fixed fluorogenic
substrate libraries (Fig. 4). The activity profiles for the P1, P2, P3, and P4 positions appear quite similarly oriented toward primarily hydrophobic residues, and P3 appears to have the most pronounced effect
on substrate suitability. Similar results were also obtained for the
human 20 S proteasome (Fig. 6). Comparison between the proteasome from
human and T. brucei revealed several activities that appear
to be specific to the T. brucei proteasome, namely cleavage after P1-glutamine substrates. Indeed, a single substrate designed with a glutamine at P1 showed a 12-fold increase in the activity of T. brucei proteasome versus the human
proteasome. These differences in activities and specificities may be
the initial step into elucidating the different structural requirements
for the T. brucei proteasome and the human proteasome and
open a window of opportunity for therapeutic drug design.
The results from both the inhibitor and substrate studies suggest a
general rule that the amino acid in the P1 position alone does not
determine the substrate specificity of the 20 S proteasome. Rather, it
is the extended peptide sequence that dictates the specificity.
Furthermore, the similar preference for hydrophobic residues among P1,
P2, P3, and P4 positions suggests that it is the overall hydrophobic
property of the peptide that makes a good substrate for the 20 S
proteasome. The qualification for a good substrate may not only be
determined by recognition of specific P1 residues among the individual
The enhanced and broadened activity profiles exhibited by the 11 S
regulator (PA26)-T. brucei 20 S proteasome complex indicate that, in addition to the hydrophobic amino acids, charged and polar
amino acids, such as Glu, Asp, His, Lys, and Arg, at the P1, P2, P3,
and P4 positions of the substrate are also capable of improving the
kinetics of substrate digestion (Figs. 4 and 5). With the addition of
the 11 S regulator (PA26), it appears that a more diverse subset of
peptides is able to access to the catalytic chamber of the complex.
This finding, coupled with the potential activation of Overall, a fairly thorough structure-activity analysis of the
- and seven
-subunits. Of the seven
-type subunits, five contain
pro-sequences that are proteolytically removed during assembly, and
three of them are predicted to be catalytic based on primary sequence.
Affinity labeling studies revealed that, unlike the mammalian
proteasome where three
-subunits were labeled by the affinity
reagents, only two
-subunits of the T. brucei proteasome
were labeled in the complex. These two subunits corresponded to
2
and
5 subunits responsible for the trypsin-like and
chymotrypsin-like proteolytic activities, respectively. Screening of a
library of 137,180 tetrapeptide fluorogenic substrates against the
T. brucei 20 S proteasome confirmed the nominal
1-subunit (caspase-like or PGPH) activity and identified an overall
substrate preference for hydrophobic residues at the P1 to P4 positions
in a substrate. This overall stringency is relaxed in the 11 S
regulator (PA26)-20 S proteasome complex, which shows both appreciable
activities for cleavage after acidic amino acids and a broadened
activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage
after P1-Gln that is minimally observed in the human counterpart. These
results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical
divergence from the human enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-type subunits, whereas the outer two rings contain
-type
subunits (1, 3, 4). The
rings play mainly a structural role and
have been shown to be important initiators of proteasome assembly (5).
The primary proteolytic chamber of the CP contains six active sites resulting from three catalytically active subunits in each of the two
inner
rings (6). Entry of substrate into the catalytic chamber is
assisted by binding of a 19 S regulatory "cap" complex at the top
and bottom of the 20 S CP (1, 2). This regulatory complex contains six
ATPase subunits and 11 non-ATPase subunits and is required for
the ATP-dependent degradation of ubiquitinated proteins (2,
7, 8). Additionally, substrate-binding properties of the CP can be
altered by binding of an 11 S regulator complex, resulting in
enhanced proteolytic activity and production of extended peptide
substrate required for major histocompatibility complex class
I-mediated antigen presentation (9-11). The human 11 S regulator protein has also recently been shown to alter the cleavage pattern and
substrate specificity of the CP (12).
subunits:
1,
2, and
5 (15). These studies have assigned the
chymotryptic activity to
5, the tryptic activity to
2, and the
PGPH activity to
1 (16). Hydrolysis by the proteasome is catalyzed
by activation of a bound water molecule by the free N-terminal
threonine residue found on all catalytically active
subunits (17).
These N-terminal threonine residues are generated by proteolytic
cleavage of extended precursor proteins during proteasome assembly,
which serves as the primary mechanism to prevent pre-mature activation
of incompletely assembled complexes (18). In addition, a lysine
residue at position 33 of the mature
-subunit protein is
essential for catalysis (19, 20).
subunits (21, 22).
Furthermore, recent observations suggested that the primary proteolytic
activities of the mammalian proteasome might be linked through
allosteric interactions in a bite-chew model (24). However, it is not
yet clear if this substrate-induced regulation of hydrolysis may in
fact result from multiple substrate binding sites on a single catalytic subunit.
-subunits, 7
-subunits, the 6 ATPase
subunits, and the 11 non-ATPase subunits of the 26 S proteasome
by RNA interference in T. brucei leads to intracellular
accumulation of ubiquitinated proteins and blocked cell growth (29,
30). Third, the 11 S regulator PA26-20 S proteasome complex
constitutes the predominant complex in T. brucei (31), yet a
down-regulation of PA26 expression by RNA interference results in no
detectable phenotype in the insect form of T. brucei (29).
Thus, it is not clear how the various forms of proteasome in T. brucei are regulated for coordinated protein degradation, cell
cycle progression, and development of T. brucei. A detailed
biochemical analysis of the proteasome from T. brucei may
help to explain how this enzyme complex differs from the human homolog.
Such information is likely to help find ways to specifically target
this essential cellular component of protein breakdown for therapeutic gain.
-subunits undergo apparent
post-translational modifications via proteolytic removal of a portion
of the N termini, only two show proteolytic activity as measured by
labeling with irreversible inhibitors. The profile of peptidase
activities in the T. brucei 20 S proteasome suggests a
stringent substrate specificity for the
1 and the
2 subunit and a
broad substrate specificity for the
5 subunit. These profiles became
considerably less selective and included an increase in the
caspase-like activity when the PA26 heptamer rings bound the 20 S CP,
suggesting an 11 S regulator-induced structure-function change of the
-subunits. Comparison of overall substrate specificity of the
T. brucei and human proteasome suggests that subtle
differences exist that may aid in the design of specific inhibitors of
this essential protease complex in trypanosome.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ex of 380 nm and
em of 450 nm in a Molecular Devices Gemini XS microtiter plate reader. The
purified human 20 S proteasome was used to screen the substrate
libraries by the same procedures (22).
1,
2, and
5
subunits for both the human and the T. brucei proteasome:
acetyl-EPFD-7-amino-4-carbamoylcoumarin (acc),
acetyl-norleucine(n)RnR-acc, and acetyl-HHSL-acc. An additional substrate was designed based on the substrate specificity for cleavage
after P1-Gln observed in the T. brucei proteasome,
acetyl-YWTQ-acc. The purified 20 S proteasomes from human and T. brucei at a concentration of 20 nM were preincubated
for 10 min in the assay buffer containing, 60 mM Tris-HCl,
pH 7.5, 10 mM KCl, and 5 mM MgCl2.
Substrates were added at multiple concentrations, from 0.00001 to 1.75 mM (depending on solubility) to the preincubated enzyme,
and substrate hydrolysis was monitored for 90 min at
ex
of 380 nm and
em of 450 nm in a Molecular Devices Gemini
XS microtiter plate reader. Data were fit using non-linear regression
to the Michaelis-Menten equation and values for
kcat, Km, and
kcat/Km were determined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and the
-subunits of T. brucei
20 S proteasome calculated based on sequences of the full-length
cDNAs encoding each protein (see footnote 2 and Tables I and
II).2
Comparison of calculated molecular weights with those experimentally
estimated molecular masses among T. brucei 20 S proteasomal
subunits
Comparison of calculated pI values with those experimentally estimated
values among T. brucei 20 S proteasomal subunits
-subunits, there is little discrepancy between the
calculated molecular weights and the experimentally estimated molecular
masses and only minor differences between the calculated and
experimentally determined pI values for all subunits except
1
(Tables I and II). This subunit has a calculated pI of 9.24 but an
experimentally determined pI of 6.1. The molecular basis for this
discrepancy remains unclear. On the other hand, five of the seven
-subunits demonstrated significantly lower molecular masses by
two-dimensional gel when compared with their calculated molecular
weights from the encoding cDNAs (Table I). These subunits,
1,
2,
5,
6, and
7, also showed significant differences between their calculated and experimental pI values (Table II).
-subunits with the exception of
3 have
blocked N termini. The experimentally determined N-terminal sequence of
3 was identical to that of the predicted sequence, indicating no
post-translational modification at its N terminus. Despite having
blocked N termini, the
2,
4,
5,
6, and
7 subunits had
very similar predicted and measured molecular weights and pI values,
suggesting a lack of significant modification of their N termini.
However, a major post-translational modification of
1 may have
occurred, resulting in a blocked N terminus, a decreased molecular
mass, and a dramatic decrease in its pI value (from 9.24 to 6.1). The
precise structural changes involved in this post-translational
modification remain to be elucidated.
-subunits, only
4 was found blocked at its N terminus.
This N-terminal modification does not result in a major change in
either its molecular weight or its pI (Tables I and II). Sequencing of
3 indicated that its N terminus matched that predicted by the
encoding cDNA, whereas the remaining five
-subunits each lost a
portion of their N termini (Fig. 1).
N-terminal sequencing of these subunits identified N-terminal
truncation of 53 residues from
1, 29 residues from
2, 46 residues
from
5, 41 residues from
6, and 5 residues from
7. The
molecular weights and pI values derived from the corresponding
truncated cDNAs agree well with those observed from the
two-dimensional gel (Tables I and II). Among the five truncated
-subunits,
1 and
2 have an N-terminal amino acid sequence of
TTI, whereas
5 has the sequence TTTL, both of which are typical of
the N termini of catalytically active
-subunits (19, 20).
Furthermore, there is a conserved catalytic lysine residue at position
33 of each of these three matured
proteins (Fig.
2). The mature
6 and
7 subunits, on
the other hand, lack both the catalytic Thr-1 and Lys-33
residues (Fig. 2), thus suggesting that
1,
2, and
5 are the
only catalytically active subunits of the T. brucei 20 S
proteasome.
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Fig. 1.
Sequence alignment of the N terminus of
T. acidophilum proteasome
-subunit (Ta-
) with
the N-terminal regions from the five truncated T. brucei
-subunits. The sequences removed from the
proteins during maturation are in lowercase, whereas the
N-terminal sequences of the matured proteins are in capital
letters. Numbers to the left of the T. brucei sequences indicate numbers of truncated residues.
Bold letters under an asterisk indicate the important
residues Thr-1 and Lys-33, respectively.
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Fig. 2.
Labeling of the catalytically active
-subunits in T. brucei 20 S
proteasome with irreversible inhibitors. The
125I-vinylsulfones used in the individual labeling
experiments are indicated at the top of each
lane.
-Subunits--
Peptide vinylsulfone derivatives have been shown to
function as irreversible inhibitors of the three primary peptidase
activities of the mammalian and yeast 20 S proteasomes (21). These
small molecule inhibitors act by formation of a covalent bond with the catalytic Thr-1 hydroxyl residues in each of the active
subunits. Because inhibition results in permanent modification of the active subunits, the radiolabeled version of these compounds can be used for
biochemical analysis of each of the proteasome active sites. The
peptide vinylsulfones, 125I-NP-L3-VS and
125I-YL3-VS, can be used to label the
1,
2, and
5 as well as the
-interferon-inducible subunits
1i
(LMP-2),
2i (MECL-1), and
5i (LMP-7) of the human 20 S proteasome
(21). However, these compounds label the active sites with different
relative intensities based on binding affinity, with YL3-VS
having a dramatically increased activity for the
2 and
2i
subunits (21).
5 and the
2 subunits, respectively, by mapping of the labeled proteins by
two-dimensional gel electrophoresis (data not shown). Surprisingly,
this radiolabeled vinylsulfone failed to label the T. brucei
1 subunit (24.7 kDa), predicted to be catalytically
active based on primary sequence and homology to the mammalian
1
subunit. This result suggests that the
1 either has a dramatically
altered substrate specificity compared with the mammalian 20 S
proteasome and is not sensitive to labeling or it is catalytically
inactive. Interestingly, even upon addition of the PA26 cap complex to
the 20 S core, the
1 subunit still failed to label with the
general affinity probe (data not shown).
5 subunit, indicating an
extended substrate specificity similar to that observed in the
mammalian enzyme. Substitution of P1 Leu for Asn in
125I-NP-L3-VS results in an affinity probe,
125I-NP-L2N-VS, that labels almost exclusively
the
2 subunit of the T. brucei proteasome (Fig. 2). This
strict substrate specificity differs from the human enzyme and suggests
potentially divergent functional properties of T. brucei proteasome.
-Subunits of the T. brucei Proteasome--
The general probe
125I-YL3-VS can be used to assess the binding
of other classes of small molecule inhibitors to the proteasome active
sites. To investigate the specificity in labeling
subunits of the
T. brucei 20 S proteasome, YL3-VS,
NP-L3-VS, NP-L2N-VS, and leupeptin were each
tested in competing with 125I-YL3-VS labeling
of
2 and
5. As summarized in Fig.
3, NP-L3-VS competes
effectively only against labeling of
5 (IC50 = 7 µM), whereas NP-L2N-VS competes moderately
only against labeling of
2 (IC50 = 22.5 µM). These results are in perfect agreement with the
labeling specificity profiles generated by direct labeling. Leupeptin,
a specific inhibitor of the trypsin-like activity in mammalian 20 S
proteasome (21), strongly inhibits the labeling of
2 with a roughly
estimated IC50 below 1 µM (Fig.
3) but with little effect on the labeling
of
5. Together these results suggest that the
2 subunit possesses
trypsin-like peptidase activity, whereas the
5 subunit is
responsible for the chymotrypsin-like peptidase activity.
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Fig. 3.
Competition of unlabeled irreversible
inhibitors with 125I-YL3-VS labeling of
T. brucei 20 S proteasome. Identities of
unlabeled competing inhibitors are presented above each
panel with their concentrations stated (in micromolar) at the
top of each lane in SDS-PAGE.
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[in a new window]
Fig. 4.
Results from the one-position fixed scanning
library where the y-axis represents the rate of
substrate cleavage in relative fluorescence units over time per
nM T. brucei 20 S proteasome
(RFU/s/nM), and the x-axis represents the
fixed amino acid. The T. brucei 20 S is shown by the
black bars, and the T. brucei 20 S complexed to
PA26 is shown by the gray bars.
2 and
5 subunits to hydrolysis of the
GGR-MAC and Suc-LLVY-MAC substrates, inhibition studies were performed
with the same set of inhibitors used for the competition studies in
Fig. 3. Hydrolysis of GGR-MAC was inhibited by each of the four
inhibitors with estimated IC50 values of 48 µM for YL3-VS, 40 µM for
NP-L3-VS, 63 µM for NP-L2N-VS,
and 1.5 µM for leupeptin and 33, 7, 77, and >100
µM, respectively, for the same inhibitors for hydrolysis
of Suc-LLVY-MAC (data not shown). Leupeptin therefore is the most
potent inhibitor of GGR-MAC hydrolysis and the most active competitor
for labeling of
2 (Fig. 3), indicating that the
2 subunit
catalyzes hydrolysis of the GGR-MAC substrate. Similarly,
NP-L3-VS is the most potent inhibitor of Suc-LLVY-MAC hydrolysis and labels only the
5 subunit. It is likely responsible for hydrolysis of the Suc-LLVY-MAC substrate.
View larger version (55K):
[in a new window]
Fig. 5.
Results from the two-position fixed
libraries, where the shade of the square
represents the RFU/s/nM and the x- and y-axes
represent the P1-, P2-, P3-, P4-amino acid. A, T. brucei 20 S; B, T. brucei 20 S complexed to
PA26.
View larger version (48K):
[in a new window]
Fig. 6.
Comparison of results from the one-position
fixed libraries for the 20 S proteasome from T. brucei
(black) and human (gray). The
y-axis represents the normalized activity (RFU/s), and the
x-axis represents the fixed amino acid.
Kinetic constants of human 20 S proteasome and T. brucei proteasome
against selected substrates
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits of T. brucei 20 S proteasome have N-terminal
segments that are proteolytically processed during maturation. Among
the five mature subunits, only
1,
2, and
5 have Thr-Thr at
their N termini and a Lys at position 33. Thus, they likely represent
the primary catalytically active subunits (19, 20).
Saccharomyces cerevisiae and mammalian 20 S
proteasomes undergo similar processing of these same five
-subunit precursors during assembly, and only
1,
2, and
5 are catalytically active in the mature enzyme
complexes (20, 36). It is yet unclear why the 20 S proteasome
of Thermoplasma acidophilum (37) utilizes only a
single chymotrypsin-like
-subunit and how this single
subunit evolved to produce seven different
-subunits found
in the eukaryotic 20 S proteasome. A close scrutiny of the
-subunits in T. brucei reveals that truncation
of the 33 N-terminal residues from the
3 subunit would
result in a threonine at position 1 and a lysine at position
33. Similarly, removal of the N-terminal 3 residues of
4 subunit
would result in a Thr-Thr-Ile sequence at the N terminus with a lysine
at position 33. However, processing of these subunits does not occur by
these predictions most likely due to the lack of a Gly residue directly
N-terminal to the threonine at the site of hydrolysis. This Gly residue
is highly conserved and is required for processing the
1,
2, and
5 precursors (38) (Fig. 1). Interestingly, removal of the 53 N-terminal residues from the
6 subunit would result in threonine at
position 1 and a lysine at position 33. The required Gly residue directly N-terminal to the predicted Thr1 is also found in this subunit, yet the actual truncation takes place between His-41 and
Pro-42, resulting in a catalytically inactive subunit. In the case of
the
7 subunit, removal of N-terminal five residues from the
precursor occurs, resulting in a product with a serine at position 1 and a lysine at position 34 (Fig. 2). This mature protein is apparently
enzymatically inactive even though serine has been shown to function in
place of the catalytic threonine in mutant
subunits of the yeast
proteasome (36).
-subunits may have taken place, preventing some
subunits from maturing to become active enzymes. Alternatively, some of
the subunits may have lost their catalytic activity through accumulated
random mutations. Only three
-subunits have retained the enzymatic
activity with varied substrate specificities. Preservation of this set
of three active
-subunits may be attributed to the three
indispensable peptidase activities that are required of the proteasomes
to carry out its necessary biological role in eukaryotes. It is
therefore surprising that the
1 subunit of the T. brucei
20 S proteasome has little PGPH activity. The trypsin-like
2 subunit
also demonstrates a limited profile of activity even though the basic
P1 substrate GGR-MAC is an effective substrate (28). However, the
dominant form of the proteasome in T. brucei is the 11 S
regulator (PA26)-20 S proteasome complex (31), which has a full
spectrum of enhanced and broad spectrum peptidase activities (Fig. 4).
Surprisingly, down-regulated expression of PA26 does not affect the
in vitro growth of the procyclic form of T. brucei (29), suggesting that other forms of the proteasome may
exist that can compensate for the lack of 11 S regulator complexes.
1 and
2 with
those from yeast and human fails to explain the lack of
1 activity and the restricted
2 activity in T. brucei 20 S
proteasome. All of the catalytic subunits share considerable sequence
identities from T. brucei to human with complete
conservation of all pivotal active site residues (39). Most likely,
additional structural requirements on the rest of protein backbone in
T. brucei 20 S proteasome also play essential roles (40).
This method of regulation is indirectly supported by the observations
that the
1 subunit becomes activated and
2 substrate specificity
is broadened upon addition of the 11 S regulator (PA26) complex (Figs.
4 and 5). Recently, the crystal structure of T. brucei 11 S
regulator (PA26)-yeast 20 S proteasome complex was determined at 3.2-Å
resolution (41). A major structural change is induced upon binding of
the 11 S regulator to the yeast 20 S proteasome. This change results in widening of the opening of the proteasome from a diameter of 13 to 32 Å, which may be accompanied by multiple structural changes within the
proteasomal channel that are not visible in this relatively low
resolution structure. Similar structural changes in T. brucei 20 S proteasome induced by PA26 heptamer binding could
bring about the observed functional activation in
1 and specificity
change in
2.
subunits of the T. brucei proteasome with covalent affinity probes showed a unique profile that is significantly different from that of the mammalian 20 S
proteasome (21). In particular, NP-L3-VS can label human
2 but not T. brucei
2, whereas NP-L2N-VS
can label human
5 but not T. brucei
5. It appears that
T. brucei
-subunits have an alternative substrate
specificity comparing with the corresponding human
-subunits. This
observation indicates that the T. brucei proteasome may have
distinct functional properties that are reflected in its substrate
specificity profiles. Furthermore, the labeling results indicate that
residues positioned at P4 and P1 are both critical for directing
substrates to the active subunits in T. brucei 20 S
proteasome, as was recently shown for the human 20 S proteasome
(12).
subunits but also by the ease of a substrate to enter the catalytic
chamber of a 20 S proteasome, which may involve an initial interaction
with the interior of an
-ring. In the case of T. brucei
and human 20 S proteasomes, hydrophobic peptides may be more capable of
entering the catalytic chamber to be digested.
1 and
2
through structural changes inside the chamber (see above), could
explain how the proteasome is regulated by addition of cap complexes to
the 20 S core. The recently reported crystal structure of 11 S
regulator (PA26)-yeast 20 S proteasome complex supports this mechanism
(41). The seven activation loops in the interior of the PA26 heptamer
ring are located around the chamber opening on top of the
-ring.
These loops may play a pivotal role in facilitating entrance of ionic
peptide into the catalytic chamber of proteasome. Further analysis
through site-directed mutagenesis of PA26 may provide some answer to
this postulation.
-subunits in T. brucei 20 S proteasome was conducted.
Their unique features, including the lack of
1 activity and the
restricted spectrum of
2 activity, were found all "normalized"
in T. brucei 11 S regulator (PA26)-20 S proteasome complex.
An 11 S regulator-induced structural change of the proteasomal
catalytic chamber was speculated to cause the functional change.
However, in view of the apparent failure of the irreversible inhibitors
in labeling the
1 subunit in the 11 S regulator-20 S proteasome
complex (preliminary data), we cannot yet rule out the possibility that
acidic substrates could be degraded by
2 and
5 to some extent due
to enhanced substrate accessibility to the catalytic chamber while
1
remains still inactive. The subsequent finding that all residues from P1 to P4 of the substrate were important determining factors of the
proteasomal activity profiles confirmed a previous similar observation
in human proteasome (12) that the overall property of the peptide may
be the major determinant of substrate suitability for proteasome. Most
interestingly, the strong preference for substrate with glutamine at
the P1 position by T. brucei proteasome has revealed a
major discrepancy from the human proteasome. It may provide a rare
opportunity for selective drug design against trypanosomiasis.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Professor Martin Rechsteiner of the University of Utah for the kind gift of purified human 20 S proteasome. We also thank Dr. Ziyin Li of the University of California at San Francisco for valuable assistance during the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI-21786.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF198386, AF148125, AF198387, AF169652, AF140353, AJ131148, AF169651, AJ131043, AJ130820, AF169653, AF226673, AF226674, AF148124, and AF290945.
§ To whom correspondence should be addressed. Tel.: 415-476-1321; Fax: 415-476-3382; E-mail: ccwang@cgl.ucsf.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M300195200
2
The GenBankTM database accession
numbers of the DNA sequences encoding the 14 subunits of T. brucei 20 S proteasome are as follows: 1, AF198386;
2,
AF148125;
3, AF198387;
4, AF169652;
5, AF140353;
6,
AJ131148;
7, AF169651;
1, AJ131043;
2, AJ130820;
3,
AF169653;
4, AF226673;
5, AF226674;
6, AF148124; and
7,
AF290945.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CP, core particle; VS, vinyl sulfone; NP, 4-hydroxy-3-nitrophenol; MAC, 4-methyl-7-aminocoumarin; PGPH, peptidyl-glutamyl peptide hydrolytic; RFU, relative fluorescence unit(s); acc, 7-amino-4-carbamoylcoumarin; n and Nle, norleucine.
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