(Received for publication, January 17, 1995; and in revised form, June 2, 1995)
From the
A new method for assaying ubiquitin C-terminal hydrolases was
developed using a I-labeled
ubiquitin-
NH-MHISPPEPESEEEEEHYC as substrate. Since the peptide
portion was almost exclusively radiolabeled, the enzymes could be
assayed directly by simple measurement of the radioactivity released
into acid-soluble products. Using this assay protocol, we identified at
least 10 ubiquitin C-terminal hydrolase activities from the extract of
chick skeletal muscle, which were tentatively named UCHs 1 through 10.
Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6
behaved as a dimer of 27-kDa subunits. The apparent molecular masses of
the other partially purified UCHs ranged from 35 to 810 kDa as
determined under a nondenaturing condition. Muscle UCHs, except UCH-1,
were activated dramatically by poly-L-Lys but with an unknown
mechanism. All of the UCHs were sensitive to inhibition by
sulfhydryl-blocking agents such as iodoacetamide. In addition, all of
the UCHs were capable of releasing free ubiquitin from a
ubiquitin-
NH-carboxyl extension protein of 80 amino acids and from
ubiquitin-
NH-dihydrofolate reductase. Five of the enzymes, UCHs 1
through 5, were also capable of generating free ubiquitin from
poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove
ubiquitin that had been ligated covalently by an isopeptide linkage to
a ubiquitin(RGA)-
NH-peptide, the peptide portion of which consists
of the 20 amino acids of the calmodulin binding domain of myosin light
chain kinase. These results suggest that the 10 UCH activities isolated
from chick skeletal muscle appear to be distinct from each other at
least in their chromatographic behavior, size, and substrate
specificity.
Ubiquitin is a highly conserved and the most widely distributed
eukaryotic polypeptide(1) . This 76-amino acid protein is
ligated covalently to a variety of intracellular proteins by a family
of ubiquitin-conjugating enzymes, called E2s(2, 3) . An isopeptide linkage is formed
between the C-terminal Gly residue of ubiquitin and the -amino
group of the Lys residue(s) of proteins. Ubiquitins by themselves or
which have already been conjugated to proteins may also be ligated to
additional ubiquitin molecules to form branched polyubiquitin by the
linkage between the
-amino group of Lys-48 of one ubiquitin and
the C terminus of the other. This ubiquitination has been implicated in
the regulation of a variety of cellular processes such as selective
protein breakdown(4, 5) , cell cycle
regulation(6, 7, 8, 9) , and stress
response(10, 11, 12) . In addition, the
dynamic nature of the ubiquitin-protein conjugate pool has been
demonstrated in vivo by microinjection (13, 14) and immunochemical
techniques(15, 16) . These studies have shown that the
reversible ubiquitination of proteins is under the control of external
stimuli such as heat shock and starvation. Therefore, the enzymes that
remove ubiquitins proteolytically from ubiquitin-protein conjugates
should be of importance in maintaining the steady-state levels of free
ubiquitin for a variety of its cellular functions.
In all eukaryotic cells, ubiquitins are encoded by two distinct classes of gene, neither of which encodes the monomeric form of ubiquitin (17, 18, 19) . One is a polyubiquitin gene that encodes a polyprotein of up to 100 uninterrupted, in tandem repeated ubiquitins through peptide bonds between the C-terminal Gly and N-terminal Met of contiguous ubiquitin molecules. The other encodes a fusion protein of which a single ubiquitin is linked to a ribosomal protein consisting of 52 or 76-80 amino acids. The transient association of ubiquitin with the ribosomal proteins has been suggested to promote their incorporation into ribosomes(20) . Therefore, proteolysis at the peptide bonds between ubiquitin and carboxyl extension proteins is required to generate ribosomal proteins for ribosome biogenesis as well as free ubiquitins.
A number of the
genes encoding ubiquitin C-terminal hydrolases (UCHs) ()have
been cloned from Saccharomyces cerevisiae. Miller et
al.(21) cloned the gene for a YUH1 protease in S.
cerevisiae which releases ubiquitin from its linear C-terminal
conjugates to relatively short peptides. Varshavsky and co-workers (22, 23) cloned three different genes for yeast
ubiquitin-specific proteases named UBP1, UBP2, and UBP3, which also
hydrolyze linear ubiquitin conjugates irrespective of the size of their
C-terminal polypeptides. Papa and Hochstrasser (24) demonstrated that the yeast DOA4 (UBP4)
gene and the human tre-2 oncogene encode deubiquitinating
enzymes, both of which can release ubiquitin molecules that are
conjugated to proteins by
NH-peptide bonds or
NH-isopeptide
linkages. These studies with yeast containing at least five different
deubiquitinating enzymes imply the existence of a variety of
uncharacterized UCHs also in higher eukaryotic organisms.
A family
of UCHs, named L1, L2, L3, and H2, has been identified from bovine calf
thymus using a small ubiquitin C-terminal adduct (i.e. ubiquitin-O-CH
) as a
substrate(25) . Of these, L3 has been shown to be a mammalian
homolog of the yeast YUH1 protease(26) . However, only two of
the enzymes (L1 and L3) have been purified so far, partly because of
the difficulty and/or insensitivity of the available assay methods.
Therefore, a simple and sensitive assay method is of necessity for
facilitating the purification of UCHs, for studying more systematically
the diversity of the enzymes in a single source, and for comparison of
the properties of the enzymes from different sources.
Rechsteiner
and co-workers have constructed ubiquitin-NH-peptide extensions
containing ``PEST'' sequences(27, 28) . In
the present study, for assaying UCHs, we used one of the ubiquitin
extensions, of which the peptide portion (MHISPPEPESEEEEEHYC) can be
almost exclusively radioiodinated and is short enough to be released as
acid-soluble products upon hydrolysis of the ubiquitin-peptide by UCHs.
Using this assay protocol we show that the extracts of chick skeletal
muscle contain at least 10 UCHs that appear to be distinct from each
other in their chromatographic behavior, size, and substrate
specificity, and we purify one of them to apparent homogeneity.
To prepare ubiquitin-NH-protein conjugates,
Ub(A)-P-MLCK20 was radioiodinated using chloramine T(34) . The
oocyte extracts (0.6 mg) were incubated with 0.4 µg of the
I-labeled Ub(A)-P-MLCK20, 10 µg of free ubiquitin,
and the ATP-regenerating system for 2 h at 30 °C and then for 10
min at 55 °C. Precipitates were removed by centrifugation, and the
heat-stable supernatant fraction containing
ubiquitin-
NH-conjugates of Ub(A)-P-MLCK20 was used as a substrate
for UCHs.
Figure 1:
Time-dependent hydrolysis of I-labeled Ub-PESTc by purified YUH1. Reaction mixtures
contained 0.3 µg of YUH1 and 1 µg of
I-labeled
Ub-PESTc (1-2
10
cpm/µg) in 100 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM DTT, and 5%
glycerol. The mixtures were incubated for various periods at 37 °C
in the absence (
) and presence of 1 mM iodoacetamide (IAA,
). After incubation, the radioactivity released as
acid-soluble products was determined as described under
``Experimental Procedures.''
The 26 S protease complex has been reported to have
deubiquitinating activity in the presence of ATP(38) .
Therefore, we examined whether purified 26 S and/or 20 S proteosomes
can also convert I-labeled Ub-PESTc into acid-soluble
product. Since the casein-degrading activity of the 20 S proteosome is
known to be activated by poly-L-Lys or fatty
acids(39, 40) , the assays were performed in the
presence and absence of poly-L-Lys. Little or no hydrolysis of
the substrate was observed with either the 26 or 20 S proteosome
whether or not ATP was supplied (Table1). However, we
fortuitously found that poly-L-Lys could markedly increase the
Ub-PESTc-hydrolyzing activities of YUH1 and the extracts of chick
skeletal muscle. Therefore, the assays for separation of the UCH
activities in the muscle extract were performed in the presence and
absence of poly-L-Lys.
Figure 2:
Separation of UCH activities in the
extract of chick skeletal muscle using a DEAE-Sepharose column. The
muscle extract (4.2 g of protein) was chromatographed on a
DEAE-Sepharose column (2.5 20 cm) as described under
``Results.'' Fractions of 20 ml were collected at a flow rate
of 150 ml/h, and aliquots (20 µl each) of them were assayed for
their ability to hydrolyze
I-labeled Ub-PESTc in the
absence (
) and presence of 0.1 mg/ml poly-L-Lys (
).
Incubations were performed for 30 min at 37 °C. The slashed
line shows the NaCl gradient, and the dotted line indicates the protein profile.
The
dialyzed proteins were loaded again onto the same DEAE-Sepharose column
but equilibrated with buffer A (i.e. without NaCl). After
collecting the flow-through fractions, proteins bound to the column
were eluted with 0.25 M NaCl (Fig.3A). The
UCH activity seen in the flow-through fractions was tentatively named
UCH-1 since it remained as a single peak upon further chromatographic
trials on other columns (see below). The fractions under the bar in Fig.3A were pooled, dialyzed against buffer A,
and loaded onto a heparin-Sepharose column (2.5 16 cm)
equilibrated with the same buffer. After collecting the flow-through
fractions, proteins bound to the column were eluted with 0.4 M NaCl (Fig.3B). The heparin flow-through fractions
were pooled, added with NaCl to a final concentration of 0.15 M, and loaded onto a Mono Q column (0.5
5 cm)
equilibrated with buffer A containing 0.15 M NaCl (Fig.3C). Proteins bound to the column were then
eluted with a linear gradient of 0.15-0.35 M NaCl. Two
peaks of UCH activity were eluted and named UCH-2 and UCH-3 in the
order of their elution. The heparin-bound fractions in Fig.3B were pooled, dialyzed against buffer A
containing 50 mM NaCl, and applied to a HiLoad-Q column (2.6
10 cm) equilibrated with the same buffer. Proteins bound to the
column were then eluted with a linear gradient of 0.05-0.4 M NaCl (Fig.3D). A symmetric peak of activity
(UCH-4) was eluted at a NaCl concentration of 0.12 M, in
addition to multiple peaks of UCH activity eluting at higher salt
concentrations (UCH-5s). Because of their unstability no further
attempts were made to separation the latter activity peaks.
Figure 3:
Separation of UCH activities in peak I
obtained from the first DEAE-Sepharose column. The active fractions
under peak I from the first DEAE-column (see Fig.2) were pooled
and added with solid ammonium sulfate to 55% saturation. The
precipitated proteins were dialyzed against buffer A and separated
further by chromatography on DEAE-Sepharose (panel A),
heparin-Sepharose (panel B), Mono Q (panel C), and
HiLoad-Q columns (panel D) as described under
``Results.'' Hydrolysis of I-labeled Ub-PESTc
was then assayed by incubating appropriate amounts of the column
fractions for 0.5-2 h at 37 °C in the absence (
) and
presence of 0.1 mg/ml poly-L-Lys (
). The slashed
lines show the NaCl gradients, and the dotted lines indicate the protein profiles. The numbers on the top of
each activity peak represent the individuals or mixtures of the UCHs
in peak I from the first DEAE-Sepharose
column.
Fractions under peak II from the first DEAE column (see Fig.2) were pooled, dialyzed against buffer A, and
chromatographed on a heparin-Sepharose column (1 10 cm)
equilibrated with the same buffer. After collecting the flow-through
fractions, proteins bound to the column were eluted with a linear
gradient of 0-0.6 M NaCl. At least three peaks of UCH
activity were eluted from the column (Fig.4A): the
first one in the flow-though fractions (UCH-6), the broad second peak
eluting from about 30 to 350 mM NaCl, and the third eluting at
about 0.5 M NaCl (UCH-10). The fractions under the bar (i.e. the broad second peak) were pooled, concentrated by
ultrafiltration, and subjected to chromatography on a Superose-6 column
(1
30 cm) equilibrated with buffer A. As shown in Fig.4B, the sample was again separated into three
distinct peaks (UCH-7, UCH-8, and UCH-9 in the order of their elution).
Except UCH-5s, all other UCH activities could not be separated further
by other columns so far tested, including hydroxylapatite,
phosphocellulose, and gel filtration columns (data not shown).
Therefore, these activities appear to represent distinct UCHs in chick
skeletal muscle at least in their chromatographic behavior.
Figure 4:
Separation of UCH activities in peak II
obtained from the first DEAE-Sepharose column. The active fractions
under peak II from the first DEAE-column (see Fig.2) were
pooled, dialyzed against buffer A, and subjected to chromatography on
heparin-Sepharose (panel A) and Superose-6 columns (panel
B) as described under ``Results.'' Hydrolysis of
Ub-PESTc was then assayed by incubating appropriate amounts of the
column fractions for 0.5-1 h at 37 °C in the absence ()
and presence of 0.1 mg/ml poly-L-Lys (
). The slashed
line shows the NaCl gradient, and the dotted lines indicate the protein profile. The numbers at the top of each activity peak represent the individuals or mixtures of
UCHs in peak II from the first DEAE-Sepharose
column.
Figure 5:
Separation of UCH-6 using phenyl-Superose
and Superose-12 columns. The UCH-6 preparation obtained from the
heparin-Sepharose column (see Fig.4A) was subjected to
three successive chromatographies on hydroxylapatite, phenyl-Superose (panel A) and Superose-12 columns (panel B) as
described under ``Results.'' Hydrolysis of Ub-PESTc was then
assayed by incubating appropriate amounts of the column fractions for
30 min at 37 °C in the absence () and presence of 0.1 mg/ml
poly-L-Lys (
). The size markers used are: a,
alcohol dehydrogenase (150 kDa); b, bovine serum albumin (66
kDa); c, carbonic anhydrase (29 kDa); d, cytochrome c (12.4 kDa). The slashed line shows the ammonium
sulfate gradient, and the dotted lines indicate the protein
profile. Active fractions from the Superose-12 column were pooled, and
an aliquot (7 µg) of the pooled sample was subjected to
electrophoresis on a 12% polyacrylamide slab gel containing SDS and
2-mercaptoethanol followed by staining with Coomassie Blue R-250 (panel C).
To determine
the sizes of the other partially purified muscle UCHs under
nondenaturing conditions, each of them was subjected to gel filtration
on either a Superose-6 or Superose-12 column (1 30 cm) that had
been equilibrated with buffer A containing 50 mM NaCl.
Fractions of 0.5 ml were collected and assayed for their ability to
cleave
I-labeled Ub-PESTc in the presence of 0.1 mg/ml
poly-L-Lys. Apparent sizes of the UCHs were then estimated by
running standard size markers on the same gel filtration columns, and
they are as follows: UCH-1, 35 kDa; UCH-2, 50 kDa; UCH-3, 45 kDa;
UCH-4, 100 kDa; UCHs 6, 9, and 10, 54 kDa; UCH-7, 810 kDa; UCH-8, 200
kDa. However, the subunit sizes of the muscle UCHs, except UCH-6,
remain to be determined until after each of them was purified to near
homogeneity.
Figure 6:
Effect of poly-L-Lys on the
hydrolysis of I-labeled Ub-PESTc by purified UCH-6. Left panel, in the absence (
) and presence of 0.1 mg/ml
poly-L-Lys (
), the activity of purified UCH-6 (20 ng)
was assayed by incubation with 1 µg of Ub-PESTc at 37 °C for
various periods. Right panel, UCH-6 activity was assayed as
above but by incubating the enzyme for 30 min in the presence of
increasing amounts of poly-L-Lys.
Since the peptide portion of Ub-PESTc contains 7 Glu and 2 His
residues and therefore should be highly negative at the assay
condition, it appeared possible that the stimulatory effect of
poly-L-Lys is due simply to its ability to neutralize the
negative charge. To test this possibility, the enzyme assays were
performed in the presence of a variety of polyionic agents. As shown in Table3, poly-L-Arg stimulated the hydrolysis of I-labeled Ub-PESTc by UCH-6 nearly as well as
poly-L-Lys. However, other polycations, including histone,
spermine, and putrescine, showed little or no effect on enzyme
activity. Moreover, aninoic poly-L-Glu showed no effect. These
results suggest that the stimulatory effect of poly-L-Lys on
the Ub-PESTc-degrading activity of UCH-6 and the other UCHs in muscle
is not due to simple charge effect. Purified UCH-6 was maximally active
between pH 7 and 7.5 and nearly inactive at pH below 6 and above 9.
Figure 7:
Hydrolysis of I-labeled
Ub-PESTc by partially purified UCHs from muscle extract and
electrophoretic analysis of their products. Aliquots (2-10 µl
depending on their specific activities) of partially purified UCHs were
incubated with 5 µg of Ub-PESTc and 0.1 mg/ml poly-L-Lys
for 2 h at 37 °C. After incubation, the mixtures were heated for 10
min at 85 °C and centrifuged for 10 min at 12,000
g to remove precipitates. The supernatants were then electrophoresed
in duplicate on discontinuous slab gels containing SDS and
2-mercaptoethanol as described under ``Experimental
Procedures.'' One of the gels was stained with Coomassie Blue
R-250 (panel A), and the other gel was exposed directly to
x-ray film for autoradiography (panel B). Lane a indicates 5 µg of unlabeled free ubiquitin; lane b,
I-labeled Ub-PESTc incubated alone; lane y,
incubated with 0.4 µg of purified YUH1; lanes 1-10,
incubated with partially purified UCHs from muscle extract. The arrowhead indicates the cleavage product of the PESTc
peptide.
To
determine whether the muscle UCHs indeed specifically cleave the
NH-peptide bond between ubiquitin and PESTc, the incubation
mixtures were prepared as above and loaded onto a Sephadex G-50 column
equilibrated with the assay buffer. Fractions of 1 ml were collected
and counted for their radioactivity. As shown in Fig.8, a new
peak of radioactivity which corresponds to a size of about 2 kDa was
generated by UCH-1 and UCH-6 with a concomitant reduction in the
radioactivity peak of
I-labeled Ub-PESTc. The fractions
with high radioactivity under the second peak were pooled and subjected
to Edman degradation. The N-terminal 9-amino acid sequence of the
peptide products was MHISPPEPE, which is identical to that of the PESTc
peptide. We performed the same experiments for all other UCHs except
UCH-2 and UCH-5s and obtained identical results (data not shown). These
results clearly indicate that the UCHs from chick skeletal muscle
specifically cleave the
NH-peptide bond between ubiquitin and the
PESTc peptide.
Figure 8:
Separation of the PESTc peptides generated
by partially purified UCH-1 and UCH-6 and determination of their
N-terminal amino acid sequences. I-Labeled Ub-PESTc (20
µg) was incubated for 3 h at 37 °C without (
) and with 360
µg of partially purified UCH-1 (
) or 8 µg of UCH-6
(
). After incubation, the samples were loaded on a Sephadex G-50
column (1
40 cm) equilibrated with buffer A. Fractions of 1 ml
were collected and counted for their radioactivity. The new
radioactivity peaks generated by incubation with the UCHs were pooled
and subjected to Edman degradation for determination of their
N-terminal amino acid sequence as described under ``Experimental
Procedures.'' The resulting 9-amino acid sequence is shown at the top of the radioactivity peaks.
Figure 9:
Hydrolysis of Ub-CEP80 and
Ub-DHFR by UCH-6 and YUH1. Purified UCH-6 (0.5 µg; lanes
c) and YUH1 (1 µg; lanes d) were incubated with 5
µg of Ub-CEP80 (panel A) or Ub-DHFR (panel B) at
37 °C for 2 h. After incubation, the samples were subjected to
electrophoresis on discontinuous slab gels as described in the legend
of Fig.7. The proteins in the gels were stained with Coomassie
Blue R-250. Free ubiquitin (lanes a), Ub-CEP80 (lane b in panel A), and Ub-DHFR (lane b in panel
B), which were incubated by themselves, are also shown. The dots indicate the position where the purified enzymes
migrated. The Ub-DHFR preparation is contaminated by an unknown protein (arrowhead) having exactly the same size as DHFR (22 kDa).
Note that the intensity of the 22-kDa band increases upon hydrolysis of
Ub-DHFR by UCH-6.
We also examined whether partially purified muscle
UCHs can generate free ubiquitin from other ubiquitin-NH-extension
proteins. Each of the enzyme preparations was incubated with Ub-CEP80
and Ub-DHFR as above, heated at 85 °C for 10 min, and centrifuged
at 12,000
g for 10 min. The heat-stable supernatant
fractions were then subjected to electrophoretic analysis as above. All
of the muscle UCHs were capable of generating ubiquitin molecules from
both Ub-CEP80 and Ub-DHFR (data not shown). Without
poly-L-Lys, Ub-CEP80 was hydrolyzed at approximately the same
rates as Ub-PESTc by the muscle UCHs, except UCH-6, by which Ub-PESTc
was cleaved about 10-fold more rapidly than Ub-CEP80. In addition, at
least 2-5-fold higher amounts of the enzymes were required for
generation of the same amounts of free ubiquitin from Ub-DHFR as that
from Ub-PESTc. When similar experiments were performed with His-tagged
diubiquitin, only five of the enzyme preparations, UCHs 1 to 5s, could
release free ubiquitin from the substrate (data not shown).
To
determine whether any of the UCHs can hydrolyze the isopeptide linkage,
partially purified UCHs were incubated with the ubiquitin-conjugates of I-labeled Ub(A)-P-MLCK20, in which the C-terminal Gly
residue of ubiquitin was replaced by Ala, and a Pro residue was
inserted to the N terminus of MLCK20 for preventing the action of the
enzyme preparations on the
NH-peptide bond between ubiquitin and
the extension peptide. As shown in Fig.10, only UCH-1 and UCH-7
were capable of removing ubiquitin from monoubiquitinated
Ub(A)-P-MLCK20. However, little hydrolysis of di- or triubiquitinated
Ub(A)-P-MLCK20 was observed by the enzyme preparations or by other
muscle UCHs. These results suggest that UCH-1 and UCH-7 may cleave off
only the ubiquitin molecules that are linked directly to proteins by
isopeptide bonds. Unlike the hydrolysis of Ub-PESTc by UCHs 2 to 10,
however, poly-L-Lys showed little or no stimulatory effect on
the cleavage of any of Ub-CEP80, Ub-DHFR, His-tagged diubiquitin, or
the monoubiquitinated Ub(A)-P-MLCK20 (data not shown). Thus, it remains
unclear how poly-L-Lys can specifically activate the
Ub-PESTc-degrading activities of YUH1 and the muscle UCHs, except
UCH-1.
Figure 10:
Cleavage of isopeptide linkages in
ubiquitinated Ub(A)-P-MLCK20 by partially purified muscle UCHs. I-Labeled Ub(A)-P-MLCK20 was ubiquitinated on its
NH-Lys group as described under ``Experimental
Procedures.'' Muscle UCHs were incubated with the ubiquitinated
Ub(A)-P-MLCK20 at 37 °C for 4 h. After incubation, the samples were
subjected to electrophoresis on discontinuous gels as described in Fig.7. The resulting gels were then exposed to x-ray films for
autoradiography. The arrowheads from top to bottom indicate tri-, di-, and monoubiquitinated
Ub(A)-P-MLCK20.
In the present studies, a simple method was developed for
assaying UCHs using I-labeled Ub-PESTc as a substrate.
When the substrate was incubated with purified YUH1 or chick muscle
extract, the
I-labeled PESTc peptide portion was readily
released into acid-soluble products. Of interest was the finding that
most of the radioactivity in the substrate could be recovered from the
acid-soluble fraction upon incubation for prolonged periods or with
increasing amounts of the enzymes (see Fig.1and Table 1). These results indicate that radioiodination of Ub-PESTc
under a mild condition, such as using IODO-BEADS, occurs almost
exclusively to the PESTc peptide portion (most likely at the 17th Tyr
residue), despite the fact that ubiquitin itself also contains a Tyr
residue. This finding allows us to assay UCH activities rapidly and to
quantify precisely the cleavage products. Furthermore, the assay
protocol was validated by electrophoretic analysis of the cleavage
products and by N-terminal sequencing of the PESTc peptide. Therefore,
by simple measurement of radioactivity of the PESTc peptide released
into the acid-soluble fraction we were able to identify at least 10
chromatographically distinct UCHs in chick muscle extracts and to
purify one (UCH-6) of them to apparent homogeneity.
Of particular interest was the observation that poly-L-Lys markedly stimulated the Ub-PESTc-cleaving activities of purified YUH1 and the newly identified muscle UCHs, except UCH-1. Furthermore, treatment of other short polycationic agents, including spermine, putrescine, and histones, showed little or no effect on the activity of purified UCH-6 and the other partially purified enzymes, suggesting that the stimulatory effect of poly-L-Lys is not due to a simple charge interaction between the poly-L-Lys and the PESTc peptide. The 20 S proteosome, which is known to be a proteolytic core of the ATP/ubiquitin-dependent 26 S proteosome, has been suggested to be a latent enzyme since it degrades proteins such as casein only in the presence of poly-L-Lys or fatty acids(39, 40) . In this regard, we initially thought that the muscle UCHs might also be latent enzymes that could be activated under appropriate conditions. However, poly-L-Lys showed little or no stimulatory effect on the activity of any of the 10 muscle UCHs against Ub-CEP80, Ub-DHFR, His-tagged diubiquitin, or monoubiquitinated Ub(A)-P-MLCK20. Therefore, the mechanism by which poly-L-Lys stimulates the Ub-PESTc-cleaving activities of the muscle UCHs (except UCH-1) as well as of purified YUH1 remains unclear.
All of the UCHs identified in the present studies using I-labeled Ub-PESTc as a substrate were also capable of
releasing free ubiquitin from Ub-CEP80 and Ub-DHFR. Moreover, in the
absence of poly-L-Lys, Ub-CEP80 was hydrolyzed by the
individual UCH, except UCH-6, more or less at the same rate as
Ub-PESTc. Thus, muscle UCHs may play a role in the generation of both
ribosomal proteins and free ubiquitins. On the other hand, Ub-DHFR was
hydrolyzed by all of the muscle UCHs much less efficiently than
Ub-PESTc or Ub-CEP80. In addition, UCH-6 cleaved Ub-PESTc much faster
than Ub-CEP80. These findings suggest that the size and/or the tertiary
structure of extension proteins may affect the susceptibility of
ubiquitin-
NH-protein extensions to the muscle enzymes. In contrast
to the ubiquitin extension proteins, His-tagged diubiquitin was cleaved
by only five of the enzymes (UCHs 1 to 5). Therefore, the five UCHs may
also participate in the generation of free ubiquitin molecules from the
product of the polyubiquitin gene in muscle cells, although it has been
suggested that the processing of polyubiquitin may occur
cotranslationally in yeast(23) .
In addition to the
NH-peptide bond cleaving activity, UCH-1 and UCH-7 were also
capable of hydrolyzing the isopeptide linkage of monoubiquitinated
Ub(A)-P-MLCK20. These enzymes or other muscle UCHs, however, showed
little or no activity against di- or triubiquitinated Ub(A)-P-MLCK20.
Hershko and co-workers (41) have shown that the isopeptidase T
removes ubiquitins from high molecular weight, multiubiquitinated
protein conjugates but not from low molecular weight forms. They also
have demonstrated that the 26 S proteosome has inherent
deubiquitinating activity against adducts of which a single ubiquitin
is linked to
NH-Lys group of protein as well as against conjugates
containing multiple ubiquitins(38) . Therefore, UCH-1 and UCH-7
together with the 26 S proteosome may be involved in the release of
ubiquitin from the Lys residue of the end products generated by the
action of the isopeptidase T against the polyubiquitinated protein
conjugates for complete recycling of ubiquitin molecules.
However, determination of the activities against ubiquitinated Ub(A)-P-MLCK20 as well as against Ub-CEP80 and Ub-DHFR was done with partially purified muscle UCHs, which were separated from each other using Ub-PESTc as the substrate. Therefore, we could not exclude the possibility that these activities are due to other contaminated enzymes active against the substrates other than Ub-PESTc. Much effort on complete purification of the muscle UCHs and isolation of their cDNAs is required for detailed characterization of the enzymes, for clarification of identity of the enzymes with the previously reported UCHs from other sources, and ultimately for their functional analysis.