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A Method for Assaying Deubiquitinating Enzymes

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摘要:comAMethodforAssayingDeubiquitinatingEnzymesJaeIlLee,SeungKyoonWoo,KeunIlKim,KyungChanPark,SungHeeBaek,YungJoonYoo1,andChinHaChung*DepartmentofMolecularBiologyandResearchCenterforCellDifferentiation,CollegeofNaturalSciences,SeoulNationalUniversity,Seoul15......

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Biol. Proc. Online 1(1), 92-99
Biological Procedures Online · Vol. 1 No. 1 · July 20, 1998 · www.biologicalprocedures.com
A Method for Assaying Deubiquitinating Enzymes
Jae Il Lee, Seung Kyoon Woo, Keun Il Kim, Kyung Chan Park, Sung Hee Baek, Yung
Joon Yoo1, and Chin Ha Chung*
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural
Sciences, Seoul National University, Seoul 151-742, Korea; 1Department of Life Science, Kwangju
Institute of Science and Technology, Kwangju 506-303, Korea. *To whom correspondence should be
addressed. E-mail: chchung@plaza.snu.ac.kr


ABSTRACT
A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled
ubiquitin-fused aNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the
tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under
a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by
simple measurement of the radioactivity released into acid soluble products. Using this assay protocol,
we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their
specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the
assay protocol should be useful for identification and purification of eukaryotic deubiquitinating
enzymes cloned and expressed in the cells.
INTRODUCTION
Ubiquitin (Ub) is a highly conserved 76 amino acid protein found in all eukaryotic cells (1,2). Ub is
covalently ligated to a variety of intracellular proteins through an isopeptide linkage. Ubs by themselves
or that have already been conjugated to proteins may also be ligated to additional Ub molecules to form
branched poly-Ub chains. This ubiquitination has been implicated in the regulation of diverse cellular
processes, such as selective protein breakdown, cell cycle regulation, and stress response (3-5). In
addition, protein ubiquitination in vivo is a dynamic, reversible process that is under control responding
to external stimuli, such as heat shock and starvation (6-8). Therefore, the enzymes that proteolytically
remove Ub from Ub-protein conjugates should be of importance in maintaining the steady-state levels
of free Ub for its diverse cellular functions.
Ubs are encoded by two distinct gene classes. One is a poly-Ub gene that encodes a poly-protein of
tandemly repeated Ubs (9,10). The other encodes a fusion protein in which a single Ub is linked to a
ribosomal protein consisting of 52 or 76-80 amino acids. The transient association of Ub with the
ribosomal proteins has been suggested to promote their incorporation into ribosomes (11). Therefore,
proteolysis at the peptide bonds between Ub and the extension proteins is required for generation of
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ISSN: 1480-9222
J.I. Lee et al.
 

ribosomal proteins for ribosome biogenesis as well as of free Ubs.
Deubiquitinating enzymes (DUBs) are known to consist of a large protein family in eukaryotes. For
example, the budding yeast has 17 genes for DUBs (4). Moreover, so far more than 60 full-length DUB
sequences have been identified in eukaryotes (12). However, comprehensive searches for DUBs,
particularly in mammalian cells, were hampered due to the lack of rapid and efficient methods for
assaying the enzymes. We have recently reported that 125I-labeled Ub-PESTc serves as an excellent
substrate for the sensitive and quantitative assay of various DUBs (13,14). Using this assay, we have
also isolated a number of novel DUBs in chick skeletal muscle and yeast (13, 15-18). Here we describe
the detailed protocol for assaying DUBs using 125I-labeled Ub-PESTc. We also compare the sensitivity
of this method to that using a fluorogenic peptide substrate, carbobenzoxy-LRGG-7-amido-4-
methylcoumarin (Cbz-LRGG-AMC), that has also been used as a substrate for DUBs (19).
MATERIALS AND METHODS
Materials
Yeast Ub hydrolase-1 (YUH1) was purified as described previously (20). The purified Ub-specific
protease-6 (yUBP-6) in yeast and cUBP41, Ub C-terminal hydrolase-1 (cUCH-1), cUCH-6, and cUCH-
8 in chick skeletal muscle were prepared as described (13, 15-18). Ub-PESTc was purified from an E.
coli strain AR13 carrying pNMHUB-PESTc as described by Yoo et al. (21). Ub-aldehyde was prepared
as described (13). Cbz-LRGG-AMC was kindly provided by Dr. K. Tanaka (Tokyo Metropolitan
Institute of Medical Science, Japan).
Radioiodination of Ub-PESTc
Ub-PESTc was radiolabeled with Na125I using IODO-BEADS (Pierce) by following the procedure
recommended by the manufacturer and Markwell (22). One IODO-BEAD was incubated in 0.2 ml of
0.1 M Tris-HCl (pH 7) in a microfuge tube for 5 min at room temperature. After incubation, 0.2 mg of
the purified Ub-PESTc and 200 mCi of Na125I were added to the tube and further incubated for the next
15 min. The radioiodinated Ub-PESTc (i.e., the liquid portion) was then removed and subjected to gel
filtration on a Sephadex G-10 column equilibrated with the Tris buffer to remove free iodine. Upon the
iodination procedure, approximately 85% of 125I was incorporated into Ub-PESTc.
Assay for hydrolysis of 125I-labeled Ub-PESTc
Reaction mixtures (final 0.1 ml) contained proper amounts of the purified DUBs, 10-20 mg of 125Ilabeled
Ub-PESTc, 0.1 M Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol, and 5% (v/v) glycerol.
After incubation of the mixtures for appropriate periods at 37 °C, the reaction was terminated by adding
50 ml of 40% (v/v) trichloroacetic acid and 50 ml of 1.2% (w/v) bovine serum albumin. The samples
were vortexed and centrifuged for 10 min at 10,000 x g using a microfuge, and aliquots (0.1 ml) of the
resulting supernatants were counted for radioactivity using a gamma-counter (13).


 

Assay for hydrolysis of Cbz-LRGG-AMC
Reaction mixtures (final 0.1 ml) contained appropriate amounts of the purified DUBs, 0.2 mM Cbz-
LRGG-AMC, 0.1 M Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol, and 5% (v/v) glycerol.
After incubation of the mixtures for various periods at 37 °C, the reaction was terminated by adding 0.1
ml of 1% (w/v) SDS and 0.8 ml of H2O. Release of free AMC by the enzyme reaction was determined
by measuring its fluorescence at 380 nm (excitation) and 440 nm (emission) (23). Proteins were
quantified as described by Bradford (24).
Gel electrophoresis
Polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol was performed using
Tris-Tricine buffer as described by Schägger and von Jagow (25). The discontinuous slab gels
contained 4, 10 and 16% polyacrylamide to improve resolution of small proteins. The sample buffer
contained 150 mM Tris-HCl (pH 6.8), 1.5% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 0.002% (w/v)
bromophenol blue and 7% (v/v) glycerol. After electrophoresis, the gels were stained with Coomassie
blue R-250 or subjected to autoradiography.
RESULTS AND DISCUSSION
Rechsteiner and coworkers (21) have constructed Ub-aNH-peptide extensions containing "PEST"
sequences. Of these, Ub-PESTc contains a peptide extension of 18 amino acids, which carries a single
tyrosine residue that can be radioiodinated and is short enough to be released as an acid-soluble product
when incubated with DUBs. Moreover, they have reported that the recombinant Ub-PESTc protein can
be easily purified by heating, such as at 85 °C, because fusion of the short peptide to Ub does not alter
the heat resistance of Ub molecule. In addition, they have demonstrated that Ub-PESTc appears
correctly processed to yield free Ub upon incubation with the chromatographic fractions of rabbit
reticulocytes. Therefore, we chose Ub-PESTc for labeling with 125I and hence for using the labeled
protein as a substrate for the assay of DUBs. Overall procedure for the enzyme assay is summarized in
Fig. 1.




By following the protocol, we determined the activity of YUH1 by measuring its ability to release
radioactive PESTc into an acid-soluble form from 125I-labeled Ub-PESTc. The purified enzyme was
incubated with the radioiodinated substrate for various periods in the absence and presence of Ubaldehyde,
which is known as a specific inhibitor of DUBs (26). Table 1 shows that the acid-soluble
radioactivity increases in an incubation time-dependent fashion and this increase can be completely
blocked by the treatment of Ub-aldehyde. Since YUH1 as well as other DUBs are known to specifically
cleave the carboxyl side of the C-terminal Gly residue of Ub, the acid-soluble products should represent
the PESTc peptide released from the Ub-peptide extension.
Table 1. Hydrolysis of 125I-labeled Ub-PESTc by the purified YUH1.
 

Incubation period  (min)

Radioactivity (cpm)released into

without

acid-soluble form

with Ub-aldehyde

0 29 31
20 1,235 112
40 2,481 147
60 3,957 220


125I-labeled Ub-PESTc (20 mg) was incubated with YUH1 (0.1 mg) at 37 °C for various periods in the presence
and absence of 1 mg of Ub-aldehyde. After incubation, radioactivity in the acid-soluble fraction was counted
using a gamma-counter.
To validate further the assay method, 125I-labeled Ub-PESTc was incubated with increasing amounts of
YUH1 for 30 min at 37 °C. The samples were then subjected to polyacrylamide gel electrophoresis in
duplicate under denaturing conditions. After electrophoresis, one of the gels was stained with
Coomassie R-250. Fig. 2A shows that the intensity of a protein band corresponding to the size of Ub
increases upon incubation with increasing amounts of YUH1.


Fig. 2. SDS-polyacrylamide gel electrophoresis of the products generated by incubation of YUH1 and 125Ilabeled
Ub-PESTc. The radioiodinated substrate (10 mg) was incubated in the absence (lane b) and presence of
 


0.1 mg (lane c), 0.2 mg (lane d), and 0.4 mg (lane e) of the purified YUH1 for 30 min at 37 °C. The samples were
then electrophoresed in duplicate on a discontinuous gels containing SDS and 2-mercaptoethanol. After
electrophoresis, one of the gels was stained with Coomassie R-250 (A), and the other was directly exposed on
an X-ray film (B). Lane a contains 10 mg of unlabeled Ub as a control.
In this gel, however, we could not find the band corresponding to the PESTc peptide, which might have
been diffused out during the staining and destaining process. Therefore, the other gel was covered with
a Saran wrap and directly exposed on an X-ray film. Upon the autoradiography, we were able to detect
the band of the PESTc peptide, whose intensity also increased upon incubation with increasing amount
of YUH1. In addition, this increase in the band intensity was approximately proportional to the increase
in the release of acid-soluble radioactivity (data not shown). Furthermore, the amino acid sequence of
the acid-soluble product determined by Edman degradation after separation from undigested 125Ilabeled
Ub-PESTc by gel filtration was shown to be identical with the N-terminal sequence of PESTc
(see Fig. 8 of ref. 13). Thus, it is clear that the acid-soluble radioactivity represents PESTc released
from 125I-labeled PESTc by the action of YUH1.


Surprisingly, however, the band intensity of Ub upon the autoradiography was far lower than the others, despite the fact that Ub itself has a single tyrosine residue at the 59th position that can also be labeled by 125I. Typically, chloramine T is used for radioiodination of Ub molecules. In our studies, we used IODO-BEADS, in which chloramine T isimmobilized on non-porous polystyrene beads.Perhaps, the tyrosine residue in Ub is not accessible to electrophilic idodine species (i.e., I+), which are produced by the immobilized chloramine T, due to structural barrier, unlike that in PESTc, that is fused to the flexible Cterminal region of Ub. In any event, we could obtain 125I-labeled Ub-PESTc, in which PESTc was almost exclusively radioiodinated. This fortuitous finding allowed us to determine rapidly the activity of YUH1 as well as of other DUBs and to quantify precisely cleavage products by simple measurement of the radioactivity released into the acid-soluble products.

Using the assay method, we have previously purified several novel DUBs from chick skeletal muscle and yeast, including cUBP41 (15), cUCH-1 (16), cUCH-6 (13), cUCH-8 (17), and yUBP-6 (18), using conventional chromatographic procedures. To compare the activities of these DUBs against 125I-Ub-PESTc, the same amount of each enzyme was incubated with the substrate for various periods. Fig. 3 shows that the hydrolytic rates differ markedly from each other.

Fig. 3. Hydrolysis of 125I-labeled Ub-PESTc by various DUBs. The same amount (0.1 mg) of the purified YUH1 (m), yUBP6 (D), cUCH-1 (t), cUCH-6 (l), cUCH-8 (s), or cUBP41 (n) was incubated with 20 mg of 125I-Ub-PESTc at 37 °C for various periods. After incubation, the release of PESTc was determined as described under Materials and Methods.
 

 


Among the DUBs, cUCH-6 hydrolyzed Ub-PESTc most rapidly. The Km values for the enzymes were
also different, ranging from 5 to 65 mM (e.g., 5.1 mM for cUCH-6 and 64.5 mM for yUBP-6.)
Ub ends with the amino acid sequence of -RLRGG76. Based on the C-terminal sequence, Stein and
coworkers (19) have synthesized a variety of fluorogenic peptides by conjugating AMC to the Ctermini
of the peptides and used them as substrates for determination of the specificity of isopeptidase
T, which exists in all mammalian cells and hydrolyzes the isopeptide linkages of poly-Ub chains (27).
Of these, Cbz-LRGG-AMC was used in the present studies to compare its sensitivity to the purified
DUBs to that of 125I-Ub-PESTc. Table 2 shows that all of the DUBs have at least three order higher
specific activities against 125I-Ub-PESTc than those against Cbz-LRGG-AMC, indicating that the assay
method using 125I-Ub-PESTc is much more sensitive for determining the activities of DUBs than that
using the fluorogenic peptide substrate. In addition, we have recently found that the extracts of E. coli
by themselves are capable of releasing AMC from the peptide substrate (data not shown), implying that
E. coli contains a protease(s), which specifically cleaves off AMC from the peptide substrate. On the
other hand, the same extracts could not hydrolyze 125I-Ub-PESTc at all. Thus, the protease(s) is likely
to interfere with the assay for overproduced eukaryotic DUBs in E. coli cells, when Cbz-LRGG-AMC
was used as a substrate. Furthermore, extracts prepared from most of eukaryotic cells also contain a
protease(s) that rapidly cleaves Cbz-LRGG-AMC but not 125I-Ub-PESTc. Thus, the assay method using
125I-Ub-PESTc should be appropriate for identification of DUBs in eukaryotic cells and their clones
expressed in E. coli. In fact, we were able to identify and partially purify at least 10 different DUBs
from the extract of chick skeletal muscle (13) and to purify several novel chick DUBs cloned and
expressed in E. coli (15,28).
Table 2. Comparison of the specific activities of various DUBs against Cbz-LRGG-AMC to those against 125Ilabeled
Ub-PESTc.
 


The amounts of the enzymes used were 0.1 mg and 5 mg for assaying the hydrolysis of 125I-Ub-PESTc and Cbz-
LRGG-AMC, respectively. Incubations were performed at 37 °C for various periods to obtain initial velocity for
each DUB. The specific activities of the enzymes were expressed as mol 125I-PESTc released into acid-soluble
products or AMC released into the solution per min per mg protein.
Recently, Stein and coworkers (29) have developed a new assay method for DUBs based on the
substrate Ub C-terminal AMC (Ub-AMC). They showed that the rate constants (kc/Km) for the
hydrolysis of Ub-AMC are 104- and 107-fold over those for the cleavage of Cbz-LRGG-AMC for
J.I. Lee et al.
 


isopeptidase T andUCH-L3 respectively. UCH-L3 is a 26 kDa Ub C-terminal hydrolase (30) isolated
from rabbit reticulocytes. However, it has not yet been tested whether Ub-AMC is susceptible to any
protease in bacterial or eukaryotic cells other than DUBs.
ACKNOWLEDGMENTS
We are grateful to Dr. Martin Rechsteiner (University of Utah) for providing E. coli strain AR13
carrying pNMHUB-PESTc, for which Ub-PESTc was purified. This work was supported by grants
from Korea Science and Engineering foundation through Research Center for Cell Differentiation,
Lotte Foundation, and Korea Ministry of Education (BSRI-97-4415).
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