Journal of Biochemistry Advance Access published June 26, 2015
Form : Regular Paper Field : Biochemistry, Enzymology
EXPRESSION, PURIFICATION AND ENZYMATIC CHARACTERIZATION OF A RECOMBINANT HUMAN UBIQUITIN-SPECIFIC PROTEASE 47
Hattori* and Hideaki Kakeya*
Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, Japan
*Address correspondence to: Akira Hattori, Ph.D. Department of System Chemotherapy and Molecular Sciences Graduate School of Pharmaceutical Sciences, Kyoto University Sakyo, Kyoto 606-8501, Japan Tel.: +81-75-753-9267; Fax: +81-75-753-4591 E-mail: [email protected]
Hideaki Kakeya, Ph.D. Department of System Chemotherapy and Molecular Sciences Graduate School of Pharmaceutical Sciences, Kyoto University
© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 1
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Jinhua Piao, Aika Tashiro, Minako Nishikawa, Yutaka Aoki, Eiko Moriyoshi, Akira
Sakyo, Kyoto 606-8501, Japan Tel.: +81-75-753-4524; Fax: +81-75-753-4591 E-mail: [email protected]
Running title : Characterization of recombinant human USP47
repeat-containing
protein;
DUB,
de-ubiquitinating
enzyme;
GST,
glutathione-s-transferase; GrB, granzyme B; NEM, N-ethylmaleimide; Ub, ubiquitn; UCH, ubiquitin C-terminal hydrolases; USP, ubiquitin-specific protease.
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Abbreviations : AMC, aminoacyl-4-metheylcoumaryl-7-amide; β-TrCP, β-transducin
Summary In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and
with a molecular mass of approximately 146 kDa on SDS-PAGE. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. A assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N-ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.
Key words: Ubiquitin, De-ubiquitinating enzyme, Ubiquitin-specific protease, C19 cysteine peptidase family
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purified to homogeneity. The purified protein was shown to be a monomeric protein
It is widely recognized that the ubiquitin (Ub) conjugation system is involved in a variety of cellular processes such as proteasome-dependent degradation of unnecessary proteins, membrane trafficking, transcription and DNA repair (1, 2). Ub is a 76-amino acid-long polypeptide, the primary structure of which is highly conserved in eukaryotes. Ub has a four-stranded mixed parallel/antiparallel β-sheet
structure is termed “the β-grasp fold” and shared among Ub-like molecules, although no significant similarity among their primary sequences is found. The C-terminal Gly residue of Ub is attached to the ε-amino group of a Lys residue of its target protein via a cascade of three separate biochemical reactions catalyzed by a Ub-activating enzyme (E1 enzyme), Ub-conjugating enzymes (E2 enzyme), and Ub ligases (E3 enzyme) (6-10). Subsequently, ubiquitinylation recurs on an internal Lys residue(s) (e.g., Lys48 and Lys63) of the Ub attached to the target protein through the Ub conjugation system, which, in turn, forms polyUb chains. A Lys48-linked polyUb chain acts as a signal for degradation by the proteasome (2, 11). A Lys63-linked polyUb chain serves as a scaffold activating signal transduction cascades involved in several cellular events, such as the interleukin-1β and tumor necrosis factor signaling pathways (2, 12, 13). Growing evidence has recently demonstrated that Lys11-linked polyUb chains have a non-degradative role in, for example, an NF-κB-activating scaffold in tumor necrosis factor signaling as well as a degradative role in cell cycle progression and cellular protein quality control mediated by endoplasmic reticulum associated protein degradation (12, 14, 15). Moreover, recent studies have uncovered that polyUb chains generated through the α-amino group of the N-terminal Met residue of Ub, referred to as linear polyUb
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with two α-helices located on its concave side (3-5). This characteristic tertiary
chains, are also involved in the interleukin-1β and tumor necrosis factor-induced activation of NF-κB (15, 16). Ub modification is a reversible process balanced by the action of de-ubiquitinating enzymes (DUBs) (17-19). DUBs specifically liberate Ub from substrates conjugated with mono Ub and/or a Lys48- or Lys63-linked polyUb chain.
precursor genes, i.e., the human Ub B gene (NM_018955.2), the human Ub C gene (NM_021009) and the human ribosomal protein S27a gene (NM_002954). Over 90 DUB genes are encoded in the human genome.
They are structurally classified
into five families: Ub C-terminal hydrolases (UCHs), Ub-specific proteases (USPs), Machado-Joseph disease proteases (MJDs) otubain proteases (OTUs) and JAB1/MPN/Mov34 motif metalloenzymes (JAMMs). USPs form the C19 family within clan CA of cysteine protease in MEROPS database, which is the largest gene family comprising over 50 members (17-19) USP47 [EC 3.4.19.12] is a novel member belonging to the C19 USP family. Its biological and physiological functions as well as enzymatic properties remain unclear. For example, although all active site residues are conserved, it was shown that recombinant human USP47 did not reveal any Ub-liberating activity toward Ub-β-galactosidase, an N-terminal Ub fusion β-galactosidase (20). On the other hand, another group detected Ub aminoacyl-4-metheylcoumaryl-7-amide (AMC) hydrolytic activity in partially purified recombinant human USP47 ( USP7 > USP8 > USP47. It was,
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recombinant USP47 virus, Sf9 cells (7.5 × 107 cells) were collected and lysed in PBS
nevertheless, demonstrated that USP47 is able to liberate a Ub moiety in a dose-dependent manner (Fig. S1A). Neither the primary structure similarity within the catalytic core domain nor the domain composition of those DUBs was also found to be correlated with the hydrolyzing profile towards the fluorescent DUB substrates (Fig. S2).
fusion protein, Ub-GrB. When Ub-GrB (~48kDa) was incubated with USP47, a mature GrB (~40kDa) was efficiently generated (Fig. 3A). However, in contrast to what was observed for LRGG-AMC and Ub-AMC, the mobility shift of Ub-GrB into GrB by UCH-L3, USP7, and USP8 was less apparent than that by USP47. The Ub-liberating activity of DUB by monitoring the Ac-IETD-AMC hydrolysis activity of the DUB-producing mature GrB was also demonstrated. As shown in Fig. 3B, although Ub-GrB showed no hydrolytic activity toward Ac-IETD-AMC, removal of a Ub moiety by each DUB resulted in a remarkable increase in fluorescence intensity in a dose- and time-dependent manner, which was significantly correlated with the Ub-GrB/GrB-converting activity of each DUB (Fig. S1B). The Ub-GrB hydrolytic activity of a USP47 mutant was also investigated by the GrB reporter DUB assay (Fig. 3B). In this mutant enzyme, the potential nucleophile Cys109 was replaced with Ser (Cys109Ser USP47). As expected, the substitution severely abrogated the Ub-releasing activity of USP47. These results strongly indicated that USP47 is indeed a cysteine protease with Ub-releasing activity. The optimal pH at which human USP47 can employ Ub-AMC as a substrate was determined. Figure 4 shows that USP47 was active above pH 6.0. The activity reached a plateau at pH 7.0–10.0, suggesting that human USP47 has a broad
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Next, the DUB activity of USP47 was tested by employing the N-terminal Ub
optimal pH.
DUB Activity of a Recombinant Human USP47 toward PolyUb Substrates Next, the DUB activity of USP47 toward isopeptide-linked Ub dimers was investigated (Fig. 5). USP47 cleaved an isopeptide bond between the carboxyl group
Ub. The enzyme showed limited DUB activity toward Met1-, Lys27-, Lys29-, and Lys33-linked di-Ubs. These results suggest that USP47 has a marginal specificity toward the isopeptide linkage between two Ub molecules, indicating the involvement of the enzyme in various Ub-regulated cellular processes. The DUB activity of USP47 toward polyUb chains was also tested. As shown in Figure 6, USP47 processed Lys48- and Lys63-linked polyUb chains into a monomeric Ub in a time-dependent manner. The kinetics of the generation of a monomeric Ub was clearly shows that USP47 cleaved both Lys48- and Lys63-linked polyUb chains much more efficiently compared to USP7.
Effects of Protease Inhibitors and Ub Derivatives on USP47 Activity The effects of various known protease inhibitors on the enzymatic activity of USP47 were examined. As shown in Fig. 7A, NEM, a Michael acceptor which alkylates-reactive thiol groups of cysteine proteases, inhibited the Ub-GrB processing activity of USP47 with an IC50 value of = 921 ± 287 nM, although NEM inhibited USP7 more effectively (IC50 = 8.11 ± 2.94 nM). Even though the catalytic core structure of the USP family DUBs has been shown to have a papain-like architecture, both DUBs (i.e. USP47 and USP7) were not susceptible to E-64, which
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of Gly76 of one Ub and the ε-amino group of Lys6, Lys11, Lys48 or Lys63 of another
is an irreversible inhibitor of papain-like cysteine proteases up to 100 µM.
In
addition, leupeptin, which competitively inhibits serine and cysteine proteases with trypsin-like activity, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, showed no inhibitory activity against either of the DUBs up to 1 mM. Moreover, the metal chelator EDTA had a negligible effect on the DUB activities of
revealed only a marginal inhibition of USP7 (~40% inhibition at 1 mM) (data not shown). The effects of various divalent cations on the DUB activity of USP47 were also examined (Fig. S3). Of the metal ions tested when the Ub-releasing activity of USP47 was monitored using the Ub-GrB reporter assay, only Zn2+ inhibited USP47 activity at 1 mM. The inhibitory effect of Zn2+, which is possibly attributed to the formation of a Zn2+-thiolate complex with the catalytic triad, was observed in a dose-dependent manner (IC50 = 46.6 ± 1.1 µM) and reached a maximum at 100 µM. The susceptibility of USP47 to Ub derivative inhibitors was further examined. As shown in Fig. 7B, USP7 was much more susceptible to Ub aldehyde (IC50 = 69.2 ± 34.4 nM), a competitive DUB inhibitor, than USP47 (IC50 = 249 ± 31 nM). An irreversible Ub derivative, Ub vinylsulfone, showed only marginal inhibition of the USP7 DUB activity and was barely effective for USP47 in the concentration range tested. These differences in the susceptibility toward Ub-based inhibitors between USP47 and USP7 correlate well with the differences in hydrolytic activity toward the mono Ub derivative substrate Ub-AMC. These results also revealed that the inhibitor profile of USP47 is different from that of USP7 as in the case of their substrate specificities.
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USP7 and USP47 up to 1 mM, while another metal chelator, 1,10-phenanthroline,
In conclusion, we report the detailed characterization of the physicochemical and enzymatic properties of recombinant human USP47 for the first time, providing valuable insights into our understanding of the patho-physiological function of the enzyme. Our in vitro investigation demonstrated that USP47 capably releases a Ub moiety from Lys48-linked polyUb chains. It is well known that the covalent
degradation (2, 11). DUBs that process Lys48-linked Ub chains are shown to be involved in the stabilization of a variety of proteins such as the stabilization of p53 and MDM2 by USP7 (24, 25). Recent reports proposed that USP47 degrades Lys48-linked polyUb chains formed by the CHIP Ub E3 ligase on nascent DNA polymerase β and the katanin p60 subunit in the cytoplasm (23, 26). Very recently, the involvement of USP47 in E-cadherin homeostasis was also reported (27). In brief, a portion of USP47 forms a complex with a minus end-directed kinesin motor KIFC3, and is recruited to the cell adhesion junctions. USP47 removes Lys48-linked polyUb chains conjugated on the cytoplasmic region of E-cadherin by E3 ligase Hakai, which protects E-cadherin from polyUb chain-induced cleavage at a cytoplasmic juxtamembrane region followed by its internalization and lysosomal degradation.
Taking these findings together, it is plausible that USP47 plays a role in the stabilization of DNA polymerase β, the katanin p60 subunit and E-cadherin via its DUB activity. We also showed that USP47 efficiently liberates a Ub moiety from Lys63-linked polyUb chains. This implies that the enzyme has physiological functions other than a role in protein degradation. It is therefore important to further investigate the physiological functions of USP47 by exploring, for example, its substrates with Lys63-linked polyUb chains. On the other hand, Peschiaroli et al.
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attachment of a Lys48-linked polyUb chain to proteins results in their proteasomal
identified USP47 as an associated protein with β-TrCP, an F-box protein comprising the SCF E3 Ub ligase complex that catalyzes the formation of Lys48-linked polyUb chains on its substrates such as Cdc25A, β-catenin, NFκB, and IκB (22). Although they showed that the N-terminal domain of USP47 directly and specifically binds to the WD-repeat region of β-TrCP which is important for the interaction with the
in the removal of Ub from β-TrCP substrates. Therefore, it is interesting to investigate non-catalytic functions of USP47 in the regulation of cell proliferation and survival through the interaction with β-TrCP. USP47 shows the highest sequence similarity with USP7 among DUBs. They also share a common structural feature in that; they have an N-terminal papain-like catalytic core and a non-catalytic long C-terminal region containing multiple Ub-like domains (Fig.S2A). Recently, in an effort to explore the roles of the C-terminal Ub-like domains of USP7, it was revealed that two Ub-like domains located at the C-terminal tail are necessary for the rearrangement of its catalytic triad, which, in turn, promotes the affinity of the enzyme for Ub and increases its activity (28). In addition to the investigation of USP47 as a novel DUB capable of releasing a Ub moiety from both Lys48- and Lys63-linked polyUb chains, a difference in the enzymatic properties of USP47 versus USP7 was revealed in this
in vitro study. Despite the significant homology in DUB domains of USP47 and USP7 (Fig.S2B), we found a higher susceptibility of USP7 for Ub derivatives with a small C-terminal chemical adduct (i.e. Ub-AMC, Ub vinylsulfone and Ub aldehyde) than USP47. Because a deletion of the C-terminal region of USP7 led to a marked decrease in the affinity to Ub-AMC (28), it is plausible that this region can induce a conformational
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β-TrCP substrate, no evidence has been provided indicating that USP47 is involved
change in the S1 pocket required for the accommodation of those mono Ub-like molecules. Results obtained in this study also strongly suggest that USP47 may play
distinct roles in Ub-mediated cellular processes via DUB activity. It is, therefore, important to understand the catalytic mechanisms of USP47 and clarify the differences in the regulatory mechanism between USP47 and USP7, leading to a
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better comprehension of their functional differences.
Acknowledgements This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Ministry of Health, Labor and Welfare of Japan (MHLW). Downloaded from http://jb.oxfordjournals.org/ at University of California, San Diego on September 2, 2015
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and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate
Figure Legends Figure Figure 1.
Purification and physicochemical characterization of a recombinant
human USP47 expressed in Sf-9 cells. A., SDS-PAGE of purified recombinant human USP47.
Recombinant USP47 (1 µg) was run on an 8% SDS-PAGE gel, and
stained with Coomassie brilliant blue. B., Gel filtration analysis of recombinant
G3000SW; 7.8 × 300 mm; Tosoh, Tokyo) was carried out with a mobile phase of 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM Na2SO4 at a flow rate of 0.5 mL/min.
The elution profiles were monitored by ultraviolet absorbance at 280 nm.
The molecular size markers (GE Healthcare) used were thyroglogulin (669 kDa, 1), catalase (235 kDa, 2), aldolase (158 kDa, 3), ovalbumin (69 kDa, 4) and chymotrypsin (25 kDa, 5).
Figure 2.
DUB activity of recombinant human USP47 toward synthetic
fluorogenic substrates. Recombinant DUBs (10 µg/mL) were incubated with 50 µM LRGG-AMC (A) or 2 µM Ub-AMC (B) at 37 °C for 1 h. Each reaction was conducted at 37 °C in 50 mM Tris-HCl pH 8.0, 10 mM DTT, and 100 mM NaCl. The results represent the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments.
Figure 3.
DUB activity of recombinant human USP47 toward Ub-GrB.
Recombinant DUB (10 µg/mL) was incubated with Ub-GrB (10 µg/mL) at 37 °C for 1 h in 50 mM Tris-HCl pH 8.0, 10 mM DTT and 100 mM NaCl. A., Following the reaction, samples were separated by SDS-PAGE and mobility shift of GrB was visualized with Lumitein. The relative amounts of mature GrB to the input Ub-GrB
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USP47. Size exclusion chromatography of recombinant USP47 (1 µg) (TSKgel
were quantified by scanning densitometry using a Typhoon 9410 scanner (Ex, 488 nm; Em, 610 nm). B., Following DUB treatment, GrB activity was measured using Ac-IETD-AMC (50 µM, 37 °C, 30 min) as a GrB substrate. The results represent the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments.
The pH profile of recombinant human USP47.
Each reaction was
conducted at 37 °C for 1 h. Ub-AMC (1 µM) was incubated with USP47 (20 µg/mL) in various buffers. Each point represents the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments.
Figure 5.
Cleavage of di-Ub by recombinant USP47.
Recombinant USP47
(15 µg/mL) was incubated with di-Ub (5 µg/mL) in 50 mM Tris-HCl pH 8.0, 0.05% CHAPS, and 10 mM DTT at 37 °C. Following incubation, samples were separated by SDS-PAGE on a 16% tricine-glycine gel, and were visualized with Lumitein. The gel image was obtained by scanning using a Typhoon 9410 scanner (Ex, 488 nm; Em, 610 nm). Similar results were obtained in three separate experiments.
Figure 6.
Digestion
of
Lys48-
and
Lys63-linked
polyUb
chains
by
recombinant USP47. Lys48- or Lys63-linked polyUb chain (4 µg/mL) was incubated with USP47 (A) or USP7 (B) (40 µg/mL) in 50 mM Tris-HCl pH 8.0, 10 mM DTT, and 100 mM NaCl. Digested Ub chains were separated by SDS-PAGE, followed by Western blot analysis with anti-Ub mAb. C., Kinetic comparison of the
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Figure 4.
USP47-catalyzed (closed square) and the USP7-catalyzed (closed circle) hydrolysis of polyUb chains. The relative amount of the generated Ub monomer to that of 16 h-incubated sample (∞) was plotted at each time point.
Figure 7.
Susceptibility of recombinant USP47 toward various protease
were examined in the presence of cysteine protease inhibitors (A) or Ub derivatives (B). DUB-mediated GrB activity was measured by using Ac-IETD-AMC (50 µM, 37 °C, 30 min). Residual activity was calculated as a percentage of the activity in a vehicle-treated control. The results represent the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments. Ub-VS, Ub vinylsulfone; Ub-CHO, Ub aldehyde.
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inhibitors and Ub derivatives. DUB activities of USP47 (upper) and USP7 (lower)
Manuscript Submitted to JB
Page 28 of 38
DECLARATION OF FUNDING This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Ministry of Health, Labor and Welfare of Japan (MHLW). Downloaded from http://jb.oxfordjournals.org/ at University of California, San Diego on September 2, 2015
CONFLICT OF INTEREST None declared.
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http://mc.manuscriptcentral.com/oup/jb
Figure 1 Piao JH et al.
A kDa 200 116 97.4 66 45
31 21
Absorbance ( 280 nm )
B (mAU) 2.0
USP47 1
23
4
5
1.5 1.0 0.5 0 0
9 10 11 12 13 14 15 16 17 18 19 20 21 22 Retention Time ( min )
Relative Fluorescent Unit (x104)
LRGG-AMC Hydrolytic Activity 4
3
2
1
0 Ub-AMC Hydrolytic Activity
A Relative Fluorescent Unit (x104)
Figure 2 Piao JH et al.
B 12
10
8
6
4
2
0
Ub-GrB Hydrolytic Activity (%) 70
60
50
40
30
20
10
A
0 – Relative Fluorescent Unit (x105)
IETD-AMC Hydrolytic Activity
Figure 3 Piao JH et al.
Ub-GrB
B
GrB
6
5
4
3
2
1
0 –
Relative Fluorescent Unit (x104)
Ub-AMC Hydrolytic Activity
Figure 4 Piao JH et al.
10 8 6 Acetate buffer
4
Phosphate buffer
2
Tris-HCl buffer Glycine-NaOH buffer
0
5
6
7
8
pH
9
10
Figure 5 Piao JH et al.
Met1 0 15 30 60 120 0
Lys6 15 30 60 120 0
Lys11 15 30 60 120 0
Lys27 15 30 60 120 (min)
dimer monomer
Lys29 0 15 30 60 120 0
Lys33
Lys48
Lys63
15 30 60 120 0 15 30 60 120 0 15 30 60 120 (min)
dimer monomer
Figure 6 Piao JH et al.
A
USP47 Lys48-linked polyUb chain 0
3
Lys63-linked polyUb chain
10 20 30 60 120 ∞ (min)
5
Ub6 Ub5 Ub4 Ub3
0
3
5 10 20 30 60 120 ∞ (min)
Ub6 Ub5 Ub4 Ub3
Ub2
Ub2
Ub1
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Form : Regular Paper Field : Biochemistry, Enzymology
EXPRESSION, PURIFICATION AND ENZYMATIC CHARACTERIZATION OF A RECOMBINANT HUMAN UBIQUITIN-SPECIFIC PROTEASE 47
Hattori* and Hideaki Kakeya*
Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, Japan
*Address correspondence to: Akira Hattori, Ph.D. Department of System Chemotherapy and Molecular Sciences Graduate School of Pharmaceutical Sciences, Kyoto University Sakyo, Kyoto 606-8501, Japan Tel.: +81-75-753-9267; Fax: +81-75-753-4591 E-mail: [email protected]
Hideaki Kakeya, Ph.D. Department of System Chemotherapy and Molecular Sciences Graduate School of Pharmaceutical Sciences, Kyoto University
© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 1
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Jinhua Piao, Aika Tashiro, Minako Nishikawa, Yutaka Aoki, Eiko Moriyoshi, Akira
Sakyo, Kyoto 606-8501, Japan Tel.: +81-75-753-4524; Fax: +81-75-753-4591 E-mail: [email protected]
Running title : Characterization of recombinant human USP47
repeat-containing
protein;
DUB,
de-ubiquitinating
enzyme;
GST,
glutathione-s-transferase; GrB, granzyme B; NEM, N-ethylmaleimide; Ub, ubiquitn; UCH, ubiquitin C-terminal hydrolases; USP, ubiquitin-specific protease.
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Abbreviations : AMC, aminoacyl-4-metheylcoumaryl-7-amide; β-TrCP, β-transducin
Summary In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and
with a molecular mass of approximately 146 kDa on SDS-PAGE. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. A assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N-ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.
Key words: Ubiquitin, De-ubiquitinating enzyme, Ubiquitin-specific protease, C19 cysteine peptidase family
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purified to homogeneity. The purified protein was shown to be a monomeric protein
It is widely recognized that the ubiquitin (Ub) conjugation system is involved in a variety of cellular processes such as proteasome-dependent degradation of unnecessary proteins, membrane trafficking, transcription and DNA repair (1, 2). Ub is a 76-amino acid-long polypeptide, the primary structure of which is highly conserved in eukaryotes. Ub has a four-stranded mixed parallel/antiparallel β-sheet
structure is termed “the β-grasp fold” and shared among Ub-like molecules, although no significant similarity among their primary sequences is found. The C-terminal Gly residue of Ub is attached to the ε-amino group of a Lys residue of its target protein via a cascade of three separate biochemical reactions catalyzed by a Ub-activating enzyme (E1 enzyme), Ub-conjugating enzymes (E2 enzyme), and Ub ligases (E3 enzyme) (6-10). Subsequently, ubiquitinylation recurs on an internal Lys residue(s) (e.g., Lys48 and Lys63) of the Ub attached to the target protein through the Ub conjugation system, which, in turn, forms polyUb chains. A Lys48-linked polyUb chain acts as a signal for degradation by the proteasome (2, 11). A Lys63-linked polyUb chain serves as a scaffold activating signal transduction cascades involved in several cellular events, such as the interleukin-1β and tumor necrosis factor signaling pathways (2, 12, 13). Growing evidence has recently demonstrated that Lys11-linked polyUb chains have a non-degradative role in, for example, an NF-κB-activating scaffold in tumor necrosis factor signaling as well as a degradative role in cell cycle progression and cellular protein quality control mediated by endoplasmic reticulum associated protein degradation (12, 14, 15). Moreover, recent studies have uncovered that polyUb chains generated through the α-amino group of the N-terminal Met residue of Ub, referred to as linear polyUb
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with two α-helices located on its concave side (3-5). This characteristic tertiary
chains, are also involved in the interleukin-1β and tumor necrosis factor-induced activation of NF-κB (15, 16). Ub modification is a reversible process balanced by the action of de-ubiquitinating enzymes (DUBs) (17-19). DUBs specifically liberate Ub from substrates conjugated with mono Ub and/or a Lys48- or Lys63-linked polyUb chain.
precursor genes, i.e., the human Ub B gene (NM_018955.2), the human Ub C gene (NM_021009) and the human ribosomal protein S27a gene (NM_002954). Over 90 DUB genes are encoded in the human genome.
They are structurally classified
into five families: Ub C-terminal hydrolases (UCHs), Ub-specific proteases (USPs), Machado-Joseph disease proteases (MJDs) otubain proteases (OTUs) and JAB1/MPN/Mov34 motif metalloenzymes (JAMMs). USPs form the C19 family within clan CA of cysteine protease in MEROPS database, which is the largest gene family comprising over 50 members (17-19) USP47 [EC 3.4.19.12] is a novel member belonging to the C19 USP family. Its biological and physiological functions as well as enzymatic properties remain unclear. For example, although all active site residues are conserved, it was shown that recombinant human USP47 did not reveal any Ub-liberating activity toward Ub-β-galactosidase, an N-terminal Ub fusion β-galactosidase (20). On the other hand, another group detected Ub aminoacyl-4-metheylcoumaryl-7-amide (AMC) hydrolytic activity in partially purified recombinant human USP47 ( USP7 > USP8 > USP47. It was,
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recombinant USP47 virus, Sf9 cells (7.5 × 107 cells) were collected and lysed in PBS
nevertheless, demonstrated that USP47 is able to liberate a Ub moiety in a dose-dependent manner (Fig. S1A). Neither the primary structure similarity within the catalytic core domain nor the domain composition of those DUBs was also found to be correlated with the hydrolyzing profile towards the fluorescent DUB substrates (Fig. S2).
fusion protein, Ub-GrB. When Ub-GrB (~48kDa) was incubated with USP47, a mature GrB (~40kDa) was efficiently generated (Fig. 3A). However, in contrast to what was observed for LRGG-AMC and Ub-AMC, the mobility shift of Ub-GrB into GrB by UCH-L3, USP7, and USP8 was less apparent than that by USP47. The Ub-liberating activity of DUB by monitoring the Ac-IETD-AMC hydrolysis activity of the DUB-producing mature GrB was also demonstrated. As shown in Fig. 3B, although Ub-GrB showed no hydrolytic activity toward Ac-IETD-AMC, removal of a Ub moiety by each DUB resulted in a remarkable increase in fluorescence intensity in a dose- and time-dependent manner, which was significantly correlated with the Ub-GrB/GrB-converting activity of each DUB (Fig. S1B). The Ub-GrB hydrolytic activity of a USP47 mutant was also investigated by the GrB reporter DUB assay (Fig. 3B). In this mutant enzyme, the potential nucleophile Cys109 was replaced with Ser (Cys109Ser USP47). As expected, the substitution severely abrogated the Ub-releasing activity of USP47. These results strongly indicated that USP47 is indeed a cysteine protease with Ub-releasing activity. The optimal pH at which human USP47 can employ Ub-AMC as a substrate was determined. Figure 4 shows that USP47 was active above pH 6.0. The activity reached a plateau at pH 7.0–10.0, suggesting that human USP47 has a broad
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Next, the DUB activity of USP47 was tested by employing the N-terminal Ub
optimal pH.
DUB Activity of a Recombinant Human USP47 toward PolyUb Substrates Next, the DUB activity of USP47 toward isopeptide-linked Ub dimers was investigated (Fig. 5). USP47 cleaved an isopeptide bond between the carboxyl group
Ub. The enzyme showed limited DUB activity toward Met1-, Lys27-, Lys29-, and Lys33-linked di-Ubs. These results suggest that USP47 has a marginal specificity toward the isopeptide linkage between two Ub molecules, indicating the involvement of the enzyme in various Ub-regulated cellular processes. The DUB activity of USP47 toward polyUb chains was also tested. As shown in Figure 6, USP47 processed Lys48- and Lys63-linked polyUb chains into a monomeric Ub in a time-dependent manner. The kinetics of the generation of a monomeric Ub was clearly shows that USP47 cleaved both Lys48- and Lys63-linked polyUb chains much more efficiently compared to USP7.
Effects of Protease Inhibitors and Ub Derivatives on USP47 Activity The effects of various known protease inhibitors on the enzymatic activity of USP47 were examined. As shown in Fig. 7A, NEM, a Michael acceptor which alkylates-reactive thiol groups of cysteine proteases, inhibited the Ub-GrB processing activity of USP47 with an IC50 value of = 921 ± 287 nM, although NEM inhibited USP7 more effectively (IC50 = 8.11 ± 2.94 nM). Even though the catalytic core structure of the USP family DUBs has been shown to have a papain-like architecture, both DUBs (i.e. USP47 and USP7) were not susceptible to E-64, which
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of Gly76 of one Ub and the ε-amino group of Lys6, Lys11, Lys48 or Lys63 of another
is an irreversible inhibitor of papain-like cysteine proteases up to 100 µM.
In
addition, leupeptin, which competitively inhibits serine and cysteine proteases with trypsin-like activity, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, showed no inhibitory activity against either of the DUBs up to 1 mM. Moreover, the metal chelator EDTA had a negligible effect on the DUB activities of
revealed only a marginal inhibition of USP7 (~40% inhibition at 1 mM) (data not shown). The effects of various divalent cations on the DUB activity of USP47 were also examined (Fig. S3). Of the metal ions tested when the Ub-releasing activity of USP47 was monitored using the Ub-GrB reporter assay, only Zn2+ inhibited USP47 activity at 1 mM. The inhibitory effect of Zn2+, which is possibly attributed to the formation of a Zn2+-thiolate complex with the catalytic triad, was observed in a dose-dependent manner (IC50 = 46.6 ± 1.1 µM) and reached a maximum at 100 µM. The susceptibility of USP47 to Ub derivative inhibitors was further examined. As shown in Fig. 7B, USP7 was much more susceptible to Ub aldehyde (IC50 = 69.2 ± 34.4 nM), a competitive DUB inhibitor, than USP47 (IC50 = 249 ± 31 nM). An irreversible Ub derivative, Ub vinylsulfone, showed only marginal inhibition of the USP7 DUB activity and was barely effective for USP47 in the concentration range tested. These differences in the susceptibility toward Ub-based inhibitors between USP47 and USP7 correlate well with the differences in hydrolytic activity toward the mono Ub derivative substrate Ub-AMC. These results also revealed that the inhibitor profile of USP47 is different from that of USP7 as in the case of their substrate specificities.
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USP7 and USP47 up to 1 mM, while another metal chelator, 1,10-phenanthroline,
In conclusion, we report the detailed characterization of the physicochemical and enzymatic properties of recombinant human USP47 for the first time, providing valuable insights into our understanding of the patho-physiological function of the enzyme. Our in vitro investigation demonstrated that USP47 capably releases a Ub moiety from Lys48-linked polyUb chains. It is well known that the covalent
degradation (2, 11). DUBs that process Lys48-linked Ub chains are shown to be involved in the stabilization of a variety of proteins such as the stabilization of p53 and MDM2 by USP7 (24, 25). Recent reports proposed that USP47 degrades Lys48-linked polyUb chains formed by the CHIP Ub E3 ligase on nascent DNA polymerase β and the katanin p60 subunit in the cytoplasm (23, 26). Very recently, the involvement of USP47 in E-cadherin homeostasis was also reported (27). In brief, a portion of USP47 forms a complex with a minus end-directed kinesin motor KIFC3, and is recruited to the cell adhesion junctions. USP47 removes Lys48-linked polyUb chains conjugated on the cytoplasmic region of E-cadherin by E3 ligase Hakai, which protects E-cadherin from polyUb chain-induced cleavage at a cytoplasmic juxtamembrane region followed by its internalization and lysosomal degradation.
Taking these findings together, it is plausible that USP47 plays a role in the stabilization of DNA polymerase β, the katanin p60 subunit and E-cadherin via its DUB activity. We also showed that USP47 efficiently liberates a Ub moiety from Lys63-linked polyUb chains. This implies that the enzyme has physiological functions other than a role in protein degradation. It is therefore important to further investigate the physiological functions of USP47 by exploring, for example, its substrates with Lys63-linked polyUb chains. On the other hand, Peschiaroli et al.
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attachment of a Lys48-linked polyUb chain to proteins results in their proteasomal
identified USP47 as an associated protein with β-TrCP, an F-box protein comprising the SCF E3 Ub ligase complex that catalyzes the formation of Lys48-linked polyUb chains on its substrates such as Cdc25A, β-catenin, NFκB, and IκB (22). Although they showed that the N-terminal domain of USP47 directly and specifically binds to the WD-repeat region of β-TrCP which is important for the interaction with the
in the removal of Ub from β-TrCP substrates. Therefore, it is interesting to investigate non-catalytic functions of USP47 in the regulation of cell proliferation and survival through the interaction with β-TrCP. USP47 shows the highest sequence similarity with USP7 among DUBs. They also share a common structural feature in that; they have an N-terminal papain-like catalytic core and a non-catalytic long C-terminal region containing multiple Ub-like domains (Fig.S2A). Recently, in an effort to explore the roles of the C-terminal Ub-like domains of USP7, it was revealed that two Ub-like domains located at the C-terminal tail are necessary for the rearrangement of its catalytic triad, which, in turn, promotes the affinity of the enzyme for Ub and increases its activity (28). In addition to the investigation of USP47 as a novel DUB capable of releasing a Ub moiety from both Lys48- and Lys63-linked polyUb chains, a difference in the enzymatic properties of USP47 versus USP7 was revealed in this
in vitro study. Despite the significant homology in DUB domains of USP47 and USP7 (Fig.S2B), we found a higher susceptibility of USP7 for Ub derivatives with a small C-terminal chemical adduct (i.e. Ub-AMC, Ub vinylsulfone and Ub aldehyde) than USP47. Because a deletion of the C-terminal region of USP7 led to a marked decrease in the affinity to Ub-AMC (28), it is plausible that this region can induce a conformational
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β-TrCP substrate, no evidence has been provided indicating that USP47 is involved
change in the S1 pocket required for the accommodation of those mono Ub-like molecules. Results obtained in this study also strongly suggest that USP47 may play
distinct roles in Ub-mediated cellular processes via DUB activity. It is, therefore, important to understand the catalytic mechanisms of USP47 and clarify the differences in the regulatory mechanism between USP47 and USP7, leading to a
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better comprehension of their functional differences.
Acknowledgements This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Ministry of Health, Labor and Welfare of Japan (MHLW). Downloaded from http://jb.oxfordjournals.org/ at University of California, San Diego on September 2, 2015
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NF-kappaB activation. EMBO Rep. 10, 10 706-713
Oncogene 26, 26 7262-7266 25. Sheng, Y., Saridakis, V., Sarkari, F., Duan, S., Wu, T., Arrowsmith, C.H., and Frappier, L. (2006) Molecular recognition of p53 and MDM2 by USP7/HAUSP .
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and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate
Figure Legends Figure Figure 1.
Purification and physicochemical characterization of a recombinant
human USP47 expressed in Sf-9 cells. A., SDS-PAGE of purified recombinant human USP47.
Recombinant USP47 (1 µg) was run on an 8% SDS-PAGE gel, and
stained with Coomassie brilliant blue. B., Gel filtration analysis of recombinant
G3000SW; 7.8 × 300 mm; Tosoh, Tokyo) was carried out with a mobile phase of 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM Na2SO4 at a flow rate of 0.5 mL/min.
The elution profiles were monitored by ultraviolet absorbance at 280 nm.
The molecular size markers (GE Healthcare) used were thyroglogulin (669 kDa, 1), catalase (235 kDa, 2), aldolase (158 kDa, 3), ovalbumin (69 kDa, 4) and chymotrypsin (25 kDa, 5).
Figure 2.
DUB activity of recombinant human USP47 toward synthetic
fluorogenic substrates. Recombinant DUBs (10 µg/mL) were incubated with 50 µM LRGG-AMC (A) or 2 µM Ub-AMC (B) at 37 °C for 1 h. Each reaction was conducted at 37 °C in 50 mM Tris-HCl pH 8.0, 10 mM DTT, and 100 mM NaCl. The results represent the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments.
Figure 3.
DUB activity of recombinant human USP47 toward Ub-GrB.
Recombinant DUB (10 µg/mL) was incubated with Ub-GrB (10 µg/mL) at 37 °C for 1 h in 50 mM Tris-HCl pH 8.0, 10 mM DTT and 100 mM NaCl. A., Following the reaction, samples were separated by SDS-PAGE and mobility shift of GrB was visualized with Lumitein. The relative amounts of mature GrB to the input Ub-GrB
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USP47. Size exclusion chromatography of recombinant USP47 (1 µg) (TSKgel
were quantified by scanning densitometry using a Typhoon 9410 scanner (Ex, 488 nm; Em, 610 nm). B., Following DUB treatment, GrB activity was measured using Ac-IETD-AMC (50 µM, 37 °C, 30 min) as a GrB substrate. The results represent the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments.
The pH profile of recombinant human USP47.
Each reaction was
conducted at 37 °C for 1 h. Ub-AMC (1 µM) was incubated with USP47 (20 µg/mL) in various buffers. Each point represents the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments.
Figure 5.
Cleavage of di-Ub by recombinant USP47.
Recombinant USP47
(15 µg/mL) was incubated with di-Ub (5 µg/mL) in 50 mM Tris-HCl pH 8.0, 0.05% CHAPS, and 10 mM DTT at 37 °C. Following incubation, samples were separated by SDS-PAGE on a 16% tricine-glycine gel, and were visualized with Lumitein. The gel image was obtained by scanning using a Typhoon 9410 scanner (Ex, 488 nm; Em, 610 nm). Similar results were obtained in three separate experiments.
Figure 6.
Digestion
of
Lys48-
and
Lys63-linked
polyUb
chains
by
recombinant USP47. Lys48- or Lys63-linked polyUb chain (4 µg/mL) was incubated with USP47 (A) or USP7 (B) (40 µg/mL) in 50 mM Tris-HCl pH 8.0, 10 mM DTT, and 100 mM NaCl. Digested Ub chains were separated by SDS-PAGE, followed by Western blot analysis with anti-Ub mAb. C., Kinetic comparison of the
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Figure 4.
USP47-catalyzed (closed square) and the USP7-catalyzed (closed circle) hydrolysis of polyUb chains. The relative amount of the generated Ub monomer to that of 16 h-incubated sample (∞) was plotted at each time point.
Figure 7.
Susceptibility of recombinant USP47 toward various protease
were examined in the presence of cysteine protease inhibitors (A) or Ub derivatives (B). DUB-mediated GrB activity was measured by using Ac-IETD-AMC (50 µM, 37 °C, 30 min). Residual activity was calculated as a percentage of the activity in a vehicle-treated control. The results represent the mean of triplicate determinations with standard deviations (S.D.). Similar results were obtained in three separate experiments. Ub-VS, Ub vinylsulfone; Ub-CHO, Ub aldehyde.
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inhibitors and Ub derivatives. DUB activities of USP47 (upper) and USP7 (lower)
Manuscript Submitted to JB
Page 28 of 38
DECLARATION OF FUNDING This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Ministry of Health, Labor and Welfare of Japan (MHLW). Downloaded from http://jb.oxfordjournals.org/ at University of California, San Diego on September 2, 2015
CONFLICT OF INTEREST None declared.
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Figure 1 Piao JH et al.
A kDa 200 116 97.4 66 45
31 21
Absorbance ( 280 nm )
B (mAU) 2.0
USP47 1
23
4
5
1.5 1.0 0.5 0 0
9 10 11 12 13 14 15 16 17 18 19 20 21 22 Retention Time ( min )
Relative Fluorescent Unit (x104)
LRGG-AMC Hydrolytic Activity 4
3
2
1
0 Ub-AMC Hydrolytic Activity
A Relative Fluorescent Unit (x104)
Figure 2 Piao JH et al.
B 12
10
8
6
4
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Ub-GrB Hydrolytic Activity (%) 70
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IETD-AMC Hydrolytic Activity
Figure 3 Piao JH et al.
Ub-GrB
B
GrB
6
5
4
3
2
1
0 –
Relative Fluorescent Unit (x104)
Ub-AMC Hydrolytic Activity
Figure 4 Piao JH et al.
10 8 6 Acetate buffer
4
Phosphate buffer
2
Tris-HCl buffer Glycine-NaOH buffer
0
5
6
7
8
pH
9
10
Figure 5 Piao JH et al.
Met1 0 15 30 60 120 0
Lys6 15 30 60 120 0
Lys11 15 30 60 120 0
Lys27 15 30 60 120 (min)
dimer monomer
Lys29 0 15 30 60 120 0
Lys33
Lys48
Lys63
15 30 60 120 0 15 30 60 120 0 15 30 60 120 (min)
dimer monomer
Figure 6 Piao JH et al.
A
USP47 Lys48-linked polyUb chain 0
3
Lys63-linked polyUb chain
10 20 30 60 120 ∞ (min)
5
Ub6 Ub5 Ub4 Ub3
0
3
5 10 20 30 60 120 ∞ (min)
Ub6 Ub5 Ub4 Ub3
Ub2
Ub2
Ub1
Ub1
B
USP7 Lys48-linked polyUb chain 0
3
Lys63-linked polyUb chain
5 10 20 30 60 120 ∞ (min)
Ub6 Ub5 Ub4 Ub3
0
3
5 10 20 30 60 120 ∞ (min)
Ub6 Ub5 Ub4 Ub3
Ub2
Ub2
Ub1
Ub1
C
Lys48-linked polyUb chain
Lys63-linked polyUb chain Ub1 Generation (%)
Ub1 Generation (%)
100 80 60 40 20
100 80 60 40 20 0
0 0
20
40
60
80
100 120 (min)
0
20
40
http://mc.manuscriptcentral.com/oup/jb
60
80
100 120 (min)
Figure 7 Piao JH et al.
A
B 120
USP47
100 80 60 40 20 0 140 120
USP47
100
IETD-AMC Hydrolytic Activity (% of Control)
IETD-AMC Hydrolytic Activity (% of Control)
120
80 60 40 20
0 120
USP7
USP7
100
100 80 60 40
80 60 40
20
20
0
0
N-ethylmaleimide (μM)
E-64 (μM)
Ub-VS (μM)
Ub-CHO (μM)
