From the Polypeptide Laboratory, Department of
Medicine and
Department of Biochemistry, McGill University,
Montreal, Quebec, H3A 2B2, Canada and the ¶ Department of
Biochemistry, Emory University, Atlanta, Georgia 30322
Received for publication, September 25, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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Ubiquitin-specific processing proteases (UBPs)
are characterized by a conserved core domain with surrounding divergent
sequences, particularly at the N-terminal end. We previously cloned two
isoforms of a testis UBP, UBP-t1 and UBP-t2, which contain identical
core regions but distinct N termini that target the two isoforms to different subcellular locations (Lin, H., Keriel, A., Morales, C. R., Bedard, N., Zhao, Q., Hingamp, P., Lefrancois, S., Combaret, L.,
and Wing, S. S. (2000) Mol. Cell. Biol. 20, 6568-6578). To determine whether the N termini also influence the
biochemical functions of the UBP, we expressed UBP-t1, UBP-t2, and the
common core domain, UBP core, in Escherichia coli. The
three isoforms cleaved branched triubiquitin at >20-fold faster rates
than linear diubiquitin, suggesting that UBP-testis functions as an
isopeptidase. Both N-terminal extensions inhibited the ability of
UBP-core to generate free ubiquitin when linked in a peptide bond with
itself, another peptide, or to small adducts. The N-terminal extension of UBP-t2 increased the ability of UBP-core to cleave branched triubiquitin. UBP-core removed ubiquitin from testis ubiquitinated proteins more rapidly than UBP-t2 and UBP-t1. Thus, UBP enzymes appear
to contain a catalytic core domain, the activities and specificities of
which can be modulated by N-terminal extensions. These divergent N
termini can alter localization and confer multiple functions to the
various members of the large UBP family.
The ubiquitin-proteasome pathway of protein degradation is a major
mechanism for intracellular protein catabolism (reviewed in Refs.
1-3). Proteins destined to be degraded through the
ubiquitin-proteasome pathway are first covalently ligated with
ubiquitin, a 76-amino acid peptide. This reaction involves the
sequential action of three enzymes. Ubiquitin is first activated by
ubiquitin-activating enzyme
(E1)1 (4) and is then
transferred to a specific cysteine residue of one of a family of
ubiquitin-conjugating enzymes (E2s) (5). Although some E2s can transfer
ubiquitin to substrates directly in vitro, most E2s support
ubiquitin conjugation to substrates by interaction with one of the many
ubiquitin protein ligases (E3s) (6-8). These E2/E3 enzymes form an
isopeptide bond between the C terminus of ubiquitin and the In addition to the families of enzymes involved in conjugation of
ubiquitin, a very large family of deubiquitinating enzymes has recently
been identified from various organisms (reviewed in Refs. 11-13).
These enzymes have several possible functions. First, they may have
peptidase activity and cleave the products of ubiquitin genes.
Ubiquitin is encoded by two distinct classes of genes. One is a
polyubiquitin gene, which encodes a linear polymer of ubiquitins linked
through peptide bonds between the C-terminal Gly and N-terminal Met of
contiguous ubiquitin molecules (14). Each copy of ubiquitin must be
released by precise cleavage of the peptide bond between Gly-76-Met-1
of successive ubiquitin moieties (15). The other class of ubiquitin
genes encodes ubiquitin C-terminal extension proteins, which are
peptide bond fusions between the C-terminal Gly of ubiquitin and
N-terminal Met of the extension protein (16-18). To date, the
extensions described are ribosomal proteins consisting of 52 or 76-80
amino acids (19, 20). These ubiquitin fusion proteins are processed to
yield ubiquitin and the corresponding C-terminal extension proteins (21). Second, deubiquitinating enzymes may have isopeptidase activities. When a target protein is degraded, deubiquitinating enzymes
can cleave the polyubiquitin chain from the target protein or its
remnants (22-24). The polyubiquitin chain must also be disassembled by
deubiquitinating enzymes during or after proteolysis by the 26 S
proteasome, regenerating free monomeric ubiquitin (25, 26). In this
way, deubiquitinating enzymes can facilitate the ability of the 26 S
proteasome to degrade ubiquitinated proteins. Third, deubiquitinating
enzymes may hydrolyze ester, thiolester, and amide linkages to the
carboxyl group of Gly-76 of ubiquitin (27-29). Such nonfunctional
linkages may arise from reactions between small intracellular compounds
such as glutathione and the E1-, E2-, or E3-ubiquitin thiolester
intermediates. Fourth, deubiquitinating enzymes may compete with the
conjugating system by removing ubiquitin from protein substrates,
thereby rescuing them from degradation or any other function mediated
by ubiquitination. Thus generation of ubiquitin by deubiquitinating
enzymes from the linear polyubiquitin and ubiquitin fusion proteins and
from the branched polyubiquitin ligated to proteins should be essential
for maintaining a sufficient pool of free ubiquitin. Many
deubiquitinating enzymes exist, suggesting that these deubiquitinating
enzymes recognize distinct substrates and are therefore involved in
specific cellular processes (11-13). Although there is recent evidence
to support such specificity of these deubiquitinating enzymes (30-32),
the structure-function relationships of these enzymes remain poorly studied.
Deubiquitinating enzymes can be divided broadly on the basis of
sequence homology into two classes, the ubiquitin-specific processing
protease (UBP or USP, also known as type 2 ubiquitin C-terminal
hydrolase (type 2 UCH)) and the UCH, also known as type 1 UCH)
(12, 13). UCH (type 1 UCH) enzymes hydrolyze primarily C-terminal
esters and amides of ubiquitin (27) but may also cleave ubiquitin gene
products and disassemble polyubiquitin chains (30). They have in common
a 210-amino acid catalytic domain, with four highly conserved blocks of
sequences that identify these enzymes. They contain two very conserved
motifs, the CYS and HIS boxes. Mutagenesis studies revealed that the
two boxes play important roles in catalysis (33, 34). Some UCH enzymes
have significant C-terminal extensions (12, 32). The functions of the
C-terminal extensions are still unknown but appear to be involved in
proper localization of the enzyme (24, 32). The active site of these UCH enzymes contains a catalytic triad consisting of cysteine, histidine, and aspartate and utilizes a chemical mechanism similar to
that of papain (33, 34).
UBP (type 2 UCH) enzymes are capable of cleaving the ubiquitin gene
products (21) and disassembling polyubiquitin chains after hydrolysis
(15). It appears that there is a core region of about 450 amino acids
delimited by CYS and HIS boxes. Many of these isoforms have N-terminal
extensions and a few have C-terminal extensions (12). In addition,
there are variable sequences in the core region of many of the
isoforms. The functions of these divergent sequences remain poorly
characterized. Recently, we identified UBP-t, an UBP enzyme that is
primarily expressed in the testis as two isoforms with the same core
region (347 residues) but distinct N termini. The N-terminal extension
of UBP-t1 has 49 residues and that of UBP-t2 has 271 residues. The
divergent N termini were found to target distinct subcellular
compartments (35). UBP-t1 is located primarily in the nucleus, whereas
UBP-t2 is found primarily in a perinuclear location and can be
associated with the centrosome. To evaluate whether in addition these N
termini have functions in substrate specificity, we have tested the
abilities of the two isoforms as well as the common core domain to
cleave natural and semi-synthetic ubiquitin substrates.
Preparation of Recombinant UBP-core, UBP-t1, and UBP-t2--
To
express full-length UBP-t1 and UBP-t2 and only the common core region
of UBP-t1 and UBP-t2 (UBP-core), DNA fragments encoding the proteins
were amplified by polymerase chain reaction and subcloned into the
bacterial expression vector pET11-d (Novagen). Plasmids were sequenced
to confirm accurate amplification and cloning and then transformed into
Escherichia coli BL21 (DE3). After induction of expression
with 1 mM
isopropyl-1-thio-
The concentrations of active UBP-core, UBP-t1, and UBP-t2 were
determined by inhibitor titration with ubiquitin aldehyde (24). The
enzymes were preincubated at 37 °C with different amounts of
ubiquitin-aldehyde for 5 min, and then the isopeptidase activities were
assayed with 2 µM 125I-labeled branched
triubiquitin (Ub3) as described below.
Preparation and Use of Antibodies against the Core Region of
UBP-t1 and UBP-t2--
Antibodies specific for the core region of
UBP-t1 and UBP-t2 were prepared by immunizing rabbits with Freund's
adjuvant mixed with UBP-core bearing an N-terminal His6
tag. Antibodies were affinity-purified by passing crude anti-serum over
an Affi-Gel 10 (Bio-Rad) column coupled to glutathione
S-transferase fused to the same UBP-core sequence.
To detect UBP-core, UBP-t1, and UBP-t2, protein samples were resolved
by SDS-PAGE on 10% acrylamide gels and transferred to nitrocellulose
membranes. Membranes were probed with anti-UBP-core antibody followed
by incubation with horseradish peroxidase-protein A and detection by
chemiluminesence (ECL, Amersham Pharmacia Biotech).
Iodination of Proteins--
The chloramine-T method was used to
label ubiquitin, diubiquitin, and triubiquitin with Na125I.
Ub-PESTc was labeled with Na125I using Iodobeads (Pierce).
Unincorporated 125I was removed by passing the reaction
products over a Sephadex G-25 column.
Ubiquitin Peptidase and Isopeptidase Assays--
To measure the
abilities of UBP-core, UBP-t1, and UBP-t2 to cleave some natural
substrates and semi-synthetic ubiquitin derivatives, the different
enzymes were incubated in a total volume of 20 µl as follows: 100 mM Tris, pH 7.5, 1 mM DTT, 1 mM
EDTA, 1 mg/ml bovine serum albumin or ovalbumin, 2 µM
125I-labeled substrate. After incubating at 37 °C for
various times, the reactions were quenched as indicated below. For the
substrate Ub-PESTc, the reaction was terminated by adding 100 µl of
2.5% bovine serum albumin and 1 ml of 20% (w/v) trichloroacetic acid. After incubation on ice for 30 min, the samples were centrifuged, and
the resulting supernatants were counted for their radioactivities to
detect the PESTc peptide bearing the iodinated tyrosine. For the linear
diubiquitin (Ub2) or branched Ub3 substrates,
the reaction was stopped with Laemmli sample buffer containing
2-mercaptoethanol, and the products were resolved by SDS-PAGE on 20%
acrylamide gels and detected by autoradiography. After detection by
autoradiography, the monoubiquitin (Ub) band and Ub2 band
(in the assay with Ub3) were excised from the dried gel and
counted. The rate of cleavage of peptide bonds for linear
Ub2 substrate was calculated as half of the Ub produced in
this reaction. The rate of cleavage of isopeptide bonds for branched
Ub3 substrate was calculated as (2/3 (Ub
To compare the abilities of these enzymes to cleave the substrates
Ub-PESTc, linear Ub2, and branched Ub3,
equivalent amounts of these enzymes (38 nM) were incubated
with 2 µM substrate. The initial rates obtained with the
different enzymes were compared. To compare the abilities of UBP-core,
UBP-t1, and UBP-t2 to interact with the Ub-PESTc substrate, the
apparent Km values for this substrate were
determined. Variable concentrations of the substrate were assayed in
reactions as described above. The products were monitored by removing
aliquots from the reaction at various times. Initial velocities were
calculated from the time courses and used in double-reciprocal plots
(Lineweaver-Burk) to determine apparent Km values
and maximal velocities.
To determine the dissociation constants for the inhibition of UBP-core
by nonhydrolyzable diubiquitin, reactions were conducted using
ubiquitin-AMC as substrate at five or more different concentrations of
dimers. The dissociation constant was calculated by a non-linear curve-fitting analysis using fitting function v = vi + (vo
To evaluate the abilities of these enzymes to remove ubiquitin from
testis ubiquitinated proteins, rat testis extracts were prepared by
homogenizing the tissues in 5 vol/g of wet weight of 0.25 M
sucrose, 50 mM Tris, pH 7.5 (at 4 °C), 5 mM
N-ethylmaleimide (to inactivate endogenous isopeptidases), 1 mM EDTA, and protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, and 10 µg/ml
leupeptin). The extracts were clarified by centrifugation at 3000 × g for 20 min at 4 °C and then frozen at Preparation of UBP-core, UBP-t1, and UBP-t2--
To characterize
the biochemical properties of UBP-t1 and UBP-t2 as well as their common
core region, UBP-core (Fig.
1A), the proteins were
expressed in E. coli. Since expressed proteins were susceptible to degradation, purification of the various enzymes was
undertaken to remove degradation products (Fig. 1B).
UBP-core was purified to apparent homogeneity, as estimated by
Coomassie Blue-staining of the protein on a polyacrylamide gel. UBP-t1
and UBP-t2 were enriched to levels of specific activity that were 17- and 27-fold greater, respectively, than in the crude bacterial lysates.
Control lysates not expressing enzymes did not show activity in these
assays, and the activities of all isoforms were found to be highly
susceptible to inhibition by ubiquitin aldehyde, and therefore, they
were quantitated by titration with this inhibitor.
N-terminal Extensions Inhibit the Peptidase Activity of
UBP-core--
Since a key function of many deubiquitinating enzymes is
to process the products of ubiquitin genes, we tested whether UBP-t1 and UBP-t2 can generate free ubiquitin from a fusion protein with another peptide, as in the case of the model substrate Ub-PESTc. UBP-core readily cleaved the
To test whether these enzymes may be involved in processing the linear
polyubiquitin that arises from the polyubiquitin genes, we tested the
activities of these enzymes against linear Ub2. UBP-t1,
UBP-t2, and UBP-core all had low abilities to generate free ubiquitin
from Ub2. Interestingly, as occurred for Ub-PESTc, the
initial velocities of UBP-t1 and UBP-t2 to cleave this Abilities of UBP-core, UBP-t1, and UBP-t2 to Cleave Ubiquitin Fused
to Small Adducts--
It has been postulated that ubiquitin may become
linked to small thiols and amines in the cell arising from nucleophilic
attack of these compounds on the reactive thiol ester linkage between ubiquitin and E1, E2s, or some E3s. Deubiquitinating enzymes may play
an important role in regenerating ubiquitin from these nonproductive adducts. Indeed, the deubiquitinating enzymes UCH-L1 and UCH-L3 can
efficiently hydrolyze these small thiols and amines from ubiquitin in vitro. To test the potential function of our UBP enzymes
to hydrolyze such compounds, Ub ester and N
Ubiquitin is conjugated to proteins through an isopeptide bond (through
the Branched Ub3 Is Preferentially Cleaved by UBP-t1 or
UBP-t2 Isoforms--
Most biological functions of ubiquitin are
mediated by the linkage of the C-terminal glycine of ubiquitin in an
isopeptide bond with the
Although ubiquitin moieties joined by Gly-76-Lys-48 linkages appear to
be most efficient at targeting for proteasomal-mediated degradation,
linkages of Gly-76 to other lysine residues of ubiquitin have been
observed. To evaluate whether these enzymes show preference for
specific linkages, the ability of nonhydrolyzable ubiquitin dimer
analogs joined in various linkages (38) to inhibit these enzymes was
tested. These dimer analogs were synthesized by using dichloroacetone
to cross-link ubiquitin containing a terminal cysteine to another
ubiquitin in which individual lysines have been mutated to cysteine. To
determine inhibitory constants easily and accurately, a sensitive
continuous fluorometric assay using ubiquitin-AMC as substrate was
employed (Table III). At the low concentrations of ubiquitin-AMC used, only UBP-core and UBP-t2 had
significant activities, and so UBP-t1 was not tested. The apparent
Km of ubiquitin-AMC for UBP-core was similar to that
of Ub-PESTc. However, in contrast to Ub-PESTc, where the affinity for
UBP-t2 decreased 1 order of magnitude compared with UBP-core, the
affinity of ubiquitin-AMC actually increased approximately 3-fold. When
the ubiquitin dimer analogs were tested, diubiquitin linked by lysine
63 was found to be most inhibitory. However, the differences between
the inhibition constants of the analogs were relatively small,
suggesting that there is no significant preference of the enzymes for a
particular linkage. Furthermore, even for the lysine 63-linked analog,
the Ki value was relatively high
(10 UBP-core, UBP-t2, and UBP-t1 Can Remove Ubiquitin from
Endogenous Testis Proteins--
Since the various isoforms could
disassemble a short branched polyubiquitin chain, we tested whether
they had isopeptidase activity against a broader spectrum of
ubiquitinated proteins in a testis extract. The UBP-core and UBP-t2
enzymes deubiquitinated ubiquitin from high molecular weight
ubiquitinated proteins, but the UBP-t1 enzyme had relatively low
activity (Fig. 5). This is consistent
with the observation that UBP-t1 was the least efficient among the
three isoforms in cleaving branched triubiquitin (Fig. 4). UBP-core was
most active, suggesting that the N termini impose varying degrees of
substrate specificity on the isopeptidase activity. Without the
addition of the enzymes, the levels of ubiquitinated proteins were
stable, confirming that endogenous deubiquitinating enzymes had been
inactivated by the presence of N-ethylmaleimide in the
homogenization buffer. The activities of the UBP isoforms were
inhibited by ubiquitin aldehyde, indicating that these cleavages were
unlikely to be due to any contaminating bacterial proteases.
In summary, the UBP core region containing the conserved elements of
the UBP family of enzymes and delimited by the CYS and HIS boxes
contains ubiquitin-specific protease activity. Thus we provide the
first direct evidence that this core region represents a functional and
quite possibly a structural domain also. Interestingly, this core
domain appears to preferentially cleave isopeptide rather than peptide
linkages (compare rates in Figs. 3 and 4), thus suggesting that the UBP
family functions primarily in either the regeneration of ubiquitin from
polyubiquitin chains produced after the action of the 26 S proteasome
or in the editing or rescuing of ubiquitinated protein substrates
before the action of the 26 S proteasome. Indeed, the isoforms were
capable of removing ubiquitin from endogenous testis proteins (Fig. 5).
The lower Ki values for the ubiquitin dimers
compared with ubiquitin alone (Table III) would also be consistent with
preferential binding of branched polyubiquitin to the core region of
the enzyme.
We have previously shown that the N-terminal extensions of UBP-t1 and
UBP-t2 serve to localize the enzyme to different compartments of the
cell (35). It should be noted that UBP-t2 is localized in a perinuclear
pattern resembling that of the proteasome and is also the most active
on branched polyubiquitin conjugates. It is intriguing to speculate
that these indicators may suggest a role for UBP-t2 in metabolism of
branched polyubiquitin at or near the proteasome. Our data demonstrate
clearly and for the first time that these divergent N termini can also
modulate the activity of the core domain. Interestingly, the activity
can be both positively and negatively affected by the extension
depending on the particular substrate. For the substrates for which
measures of affinity could be determined, the parameters obtained were relatively high (in the micromolar range). Rates of cleavage were also
relatively low. Thus, there are probably specific substrates for these
enzymes, and the N-terminal extensions may play roles in recognizing
these specific ubiquitinated proteins. Indeed, without N-terminal
extensions, UBP-core efficiently removed ubiquitin from endogenous
proteins. However, with N-terminal extensions, UBP-t2 did this at a
slower rate, and UBP-t1, hardly at all (Fig. 5). Thus, it is quite
possible that the N-terminal extensions are positioned near the S1'
site, and the different effects of the UBP-t2 extension on affinity for
ubiquitin-AMC and ubiquitin-PESTc would support this model (Tables I
and III). In addition, given their precise localization in the cell,
the enzymes may be co-localized with their substrates in specific
compartments. Such co-localization would permit privileged delivery of
substrates to the enzymes and diminish dependence of access to the
enzymes on factors such as affinity constants and diffusion rates.
Finally, the observations on UBP-testis in this and our previous work
(35) indicate that the numerous distinct N-terminal extensions of UBP
enzymes could permit specific spatial, temporal, and kinetic modulation
of UBP function and allow these enzymes to mediate quite precise
functions in the ubiquitin-dependent proteolytic pathway.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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-amino
group of the side chain of lysine residues of the target protein. A
branched polyubiquitin chain is then formed on the protein through the
ligation of additional monomers of ubiquitin to the side chain of a
lysine residue of the previous ubiquitin in successive rounds of
ubiquitination (5). The covalent attachment of a polyubiquitin chain to
proteins generally acts as a signal for their degradation by a
multisubunit protease, the 26 S proteasome (9, 10).
MATERIALS AND METHODS
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-D-galactopyranoside for 2 h at
28 °C, cells were harvested from 800 ml of culture and rinsed with
PBS, and the cell pellets were frozen at
20 °C. Subsequent
manipulations were on ice or at 4 °C. The frozen pellets were
resuspended in
Ub2)) + Ub2 produced in this reaction. To test
the abilities of these UBP enzymes to cleave ubiquitin ester and
ubiquitin linked to lysine by the
or
amino groups, they were
incubated with these substrates (16 µM) separately in 10 µl of 50 mM Tris, pH 7.6, 3 mM DTT, 5 mM MgCl2 at 37 °C for 20 or 30 min. The
reactions were quenched with 40 µl of 0.1 N HCl and then
injected onto a 4.6 × 250-mm C8 column equilibrated and eluted
with 60% acetonitrile in 25 mM perchlorate to resolve the
free ubiquitin product from the substrates (29). Fluorometric assays
used ubiquitin-AMC as the substrate (37). In a typical assay, 80 µl
of assay buffer (50 mM Tris, pH 7.8, 100 µg/ml ovalbumin,
10 mM DTT) containing 6.6 nM enzyme were added
to a 100-µl cuvette. When present, diubiquitin analogs were added,
and after a 5-min preincubation at 37 °C to achieve thermal
equilibrium, ubiquitin-AMC was added to a final concentration of 40 nM. Reaction progress was monitored by the increase in
fluorescence emission at 450 nm (
ex = 355 nm) that resulted from the cleavage of AMC from the substrate.
vf)/(1 + I/Ki) where
vo is the uninhibited rate, vf is
the rate at high concentration of inhibitor, and Ki
is the inhibition constant. In all cases inhibition was fully
competitive, and vf = 0.
80 °C
until analysis. Before assaying, the excess N-ethylmaleimide
in the samples was neutralized by adding DTT to a final concentration of 5 mM. Aliquots (50 µg) of the protein were then
incubated with equal amounts of each of the UBP enzymes (38 nM) for various times at 37 °C. The reaction was stopped
with Laemmli sample buffer containing 2-mercaptoethanol, and the
proteins were resolved by SDS-PAGE on 10% acrylamide gels and
transferred to nitrocellulose membranes. The ubiquitinated proteins
were detected by Western blotting with anti-ubiquitin antibodies
(Sigma) followed by protein A coupled to horseradish peroxidase and a
chemiluminescent detection method (ECL, Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
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ABSTRACT
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Fig. 1.
A, schematic of the UBP-t1,
UBP-t2, and UBP-core isoforms. UBP-t1 and UBP-t2 consist of identical
core regions containing the conserved motifs of the UBP family (CYS and
HIS boxes are indicated) but distinct N termini. B, the
three isoforms were expressed in E. coli, and the crude
extracts were purified sufficiently to remove partially degraded forms.
UBP-t1 and UBP-t2 were analyzed by immunoblotting with anti-UBP-core
antibodies. UBP-core was analyzed by SDS-PAGE followed by staining with
Coomassie Blue.
-peptide bond between ubiquitin and the
PESTc extension, indicating that the core region itself has activity
and therefore likely represents a functional domain. However, both
UBP-t1 and UBP-t2 had very low activities (<20% that seen with
UBP-core) with this substrate (Fig. 2).
Thus the N-terminal extensions inhibited the hydrolytic activity
inherent in the core domain. To test whether the N-terminal extensions of UBP exert this inhibitory effect by decreasing the abilities of the
UBP core to interact with Ub-PESTc, we measured apparent Km values of Ub-PESTc for UBP-core, UBP-t1, and
UBP-t2. Indeed, UBP-t2 and UBP-t1 had greater than 10-fold higher
Km values with this substrate than did UBP-core
(Table I). However, the
Vmax value of UBP-t2 was similar to that of the
UBP-core enzyme, indicating that the N-terminal extensions of UBP can
negatively influence binding of the core domain to the substrate
without significantly affecting catalytic function.
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Fig. 2.
Hydrolysis of 125I-labeled
Ub-PESTc by UBP-t1, UBP-t2, and UBP-core enzymes. UBP-t1
(filled circles), UBP-t2 (open circles), and
UBP-core (filled triangles) (all at 38 nM) were
incubated in the presence of 2 µM
125I-Ub-PESTc. Aliquots of the reaction mixtures were
removed at the indicated times, and the substrate and ubiquitin product
were precipitated with trichloroacetic acid. After centrifugation, the
supernatant containing the soluble PESTc peptide product was
counted.
Kinetic constants of ubiquitin-PESTc for UBP-t1, UBP-t2, and
UBP-core
-peptide bond
were lower than that of UBP-core (Fig.
3). This confirms the previous
observation (Fig. 2) that N-terminal extensions inhibit the ability of
the core region to cleave linear peptide fusions. All of the above
results would suggest that both UBP-t1 and UBP-t2 do not generate free
ubiquitin from ubiquitin gene products. They also do not appear to be
involved in processing the precursor forms of the ubiquitin-related
proteins SUMO1 (sentrin) or NEDD8, as none of the three isoforms were
able to cleave proSUMO1 or proNEDD8 (data not shown). Thus the core
region alone has activity, but the N-terminal extensions limit that
activity presumably by imposing constraints and selectivity on the
ability of substrates to access the core domain.
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Fig. 3.
Hydrolysis of 125I-labeled linear
Ub2 by UBP-t1, UBP-t2, and UBP-core enzymes. UBP-t1
(filled circles), UBP-t2 (open circles), and
UBP-core (filled triangles) (all at 76 nM) were
incubated in the presence of 2 µM
125I-labeled linear Ub2. Aliquots of the
reaction mixtures were removed at the indicated times and resolved by
SDS-PAGE on 20% acrylamide gels. After detection by autoradiography,
the monoubiquitin bands were excised from the gel and counted. The
concentration of peptide bonds cleaved by the enzymes is equal to half
of the amount of monoubiquitin produced.
-Ub-L-lysine
were used as substrates. UBP-core had higher activity in hydrolyzing
the ester and lysine from ubiquitin than both UBP-t1 and UBP-t2 (Table II). This indicates that the N-terminal
extensions of both UBP-t1 and UBP-t2 inhibit the ability of the core
region to hydrolyze small thiols and amines from ubiquitin and further
supports the role of these extensions in imposing substrate
selectivity.
Rates of hydrolysis of ubiquitin derivatives by UBP-t1, UBP-t2, and
UBP-core
-amino group of lysine). So it was of interest to examine
whether UBP-t1 and UBP-t2 could cleave Ub-
-amino lysine derivatives.
When N
-Ub-L-lysine was used as such a model isopeptidase substrate, UBP-core still had higher activity than UBP-t1 or UBP-t2 against the
-isopeptide bond (Table II). Thus, at least for a small
leaving group, UBP-core does not discriminate between isopeptide and
peptide linkages. This is similar to UCH enzymes tested to date that also do not appear to discriminate between these two model
substrates (30).
-amino group of the side chain of lysine
residues of the protein substrate. Recognition of ubiquitinated
proteins for degradation by the 26 S proteasome generally requires the presence of a polyubiquitin chain on the protein substrate in which
each ubiquitin is linked to each other via these isopeptide linkages.
To evaluate whether UBP-core, UBP-t1, and UBP-t2 can potentially
disassemble such a polyubiquitin chain, Ub3, a branched triubiquitin chain linked via isopeptide bonds between the
-amino group of lysine 48 in one ubiquitin molecule and the C-terminal of
another ubiquitin molecule, was used as a substrate. All three enzymes
had activity against this substrate, and the rates of cleavage were
similar to that for Ub-PESTc but 1-2 orders of magnitude higher than
that seen for linear Ub2 (Fig.
4). Thus, the UBP core domain appears to
prefer branched rather than linear ubiquitin polymers as substrate. The
higher rate of cleavage of triubiquitin compared with diubiquitin was
not due to the extra moiety of ubiquitin in the former, as diubiquitin
did not accumulate over time in the reactions with triubiquitin as
substrate (data not shown). In addition, UBP-t2 had higher activity
than UBP-core. This indicates that the N-terminal extension of UBP-t2
not only suppresses peptidase activity but enhances isopeptidase
activity. In contrast, UBP-t1 had lower activity than UBP-core in
cleaving branched Ub3 (Fig. 4). This indicates that the
N-terminal extensions can modulate the intrinsic activity of the core
domain both positively and negatively.
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Fig. 4.
Hydrolysis of 125I-labeled
branched Ub3 by UBP-t1, UBP-t2, and UBP-core enzymes.
UBP-t1 (filled circles), UBP-t2 (open circles),
and UBP-core (filled triangles) (all at 38 nM)
were incubated in the presence of 2 µM
125I-Ub3. Aliquots of the reaction mixtures
were removed at the indicated times and resolved by SDS-PAGE on 20%
acrylamide gels. After detection by autoradiography, the Ub and
Ub2 bands were excised from the gel and counted separately.
The concentration of isopeptide bonds cleaved by the enzymes was
calculated as 2/3([Ub] [Ub2]) + [Ub2].
7 M range) compared with an
enzyme such as isopeptidase T in which diubiquitin linked by lysine 48 inhibits with a Ki in the range of
10
8 M (38). It is conceivable
that UBP-t2 shows enhanced activity against branched Ub3
due to the presence in this isoform of a binding site for an additional
ubiquitin moiety. To evaluate this possibility, the
Ki values of monoubiquitin and the dimers for
UBP-core and for UBP-t2 were compared to see if the dimer Ki values were lower for the UBP-t2 isoform. The
ratios of Ki for UBP-core to Ki
for UBP-t2 were similar for ubiquitin monomer and for all of the
dimers, suggesting that the N-terminal extension in UBP-t2 does not
confer an additional binding site for ubiquitin (Table III).
Kinetic constants of ubiquitin-AMC for UBP-core and UBP-t2 and
inhibition of these isoforms by ubiquitin and nonhydrolyzable ubiquitin
dimer analogs
View larger version (30K):
[in a new window]
Fig. 5.
UBP-testis isoforms can remove
ubiquitin from endogenous ubiquitinated proteins in testis
extracts. Testis extracts were prepared by homogenizing testes in
50 mM Tris, pH 7.5, containing 5 mM
N-ethylmaleimide to inactivate endogenous deubiquitinating
enzymes. Soluble fractions were obtained by centrifugation. After
neutralization of excess N-ethylmaleimide with DTT, aliquots
were incubated in the presence and absence (Control) of
UBP-t1, UBP-t2, or UBP core (all at 38 nM). At the
indicated times, aliquots of the reactions were quenched with sample
buffer and analyzed by immunoblotting with anti-ubiquitin antibodies.
Ubal indicates reactions incubated for 30 min in the
presence of Ub aldehyde (0.5 µM), an inhibitor of the UBP
enzymes. A, representative immunoblot. B,
quantitation of five separate reactions. Shown are means ± S.E.
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ACKNOWLEDGEMENT |
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We are grateful to Cecile Pickart for supplying us with triubiquitin.
![]() |
FOOTNOTES |
---|
* This work was supported by Medical Research Council of Canada Grant MT14700 (to S. S. W) and National Institutes of Health Grant GM30308 (to K. D. W.).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.
§ Recipient of a studentship from the Royal Victoria Hospital Research Institute and Department of Medicine.
** Recipient of a studentship award from the Fonds de la Recherche en Santé du Québec.
Recipient of a Senior Chercheur Boursier salary award from the
Fonds de la Recherche en Santé du Québec.
§§ To whom correspondence should be addressed: Polypeptide Laboratory, McGill University, Strathcona Anatomy and Dentistry Bldg, 3640 University St., Room W315, Montreal, Quebec, Canada, H3A 2B2. Tel.: 514-398-4101; Fax: 514-398-3923; E-mail: simon.wing@mcgill.ca.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M008761200
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ABBREVIATIONS |
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The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzymes; E3, ubiquitin protein ligases; UBP, ubiquitin-specific processing protease; UCH, ubiquitin C-terminal hydrolase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; Ub, ubiquitin; Ub2, diubiquitin; Ub3, triubiquitin; AMC, amidomethylcoumarin; Mes, 4-morpholineethanesulfonic acid.
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