From the
Centre for Biomolecular Sciences, School of Biology, University of St.
Andrews, North Haugh, St. Andrews, Fife KY169AL, United Kingdom,
Glaxo SmithKline, Medicines Research Centre,
Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom, and
GlaxoSmithKline, Research Triangle Park, North
Carolina 27709
Received for publication, December 29, 2002 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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In vitro ubiquitylation of p27Kip1 in cell extracts requires a continuously active NEDD8 conjugation system, thus suggesting the existence of isopeptidases that are capable of hydrolyzing NEDD8 from Cul-1 (15). It is therefore likely that the extent of NEDD8 modification is controlled by a dynamic equilibrium between NEDD8 modification, mediated by APP-BP1/Uba3 and Ubc12, and NEDD8 removal catalyzed by NEDD8-specific proteases. A NEDD8-specific protease activity has been reported to be associated with the COP9 signalsome, and while a metalloprotease motif in Jab1/Csn5 is required for this activity the isolated protein did not display NEDD8 protease activity (16, 17). NEDD8, like all ubiquitin-like proteins, is synthesized as an inactive precursor and has to be processed by a NEDD8-specific protease to expose the diglycine motif at the COOH terminus that is required for conjugation. Here we describe NEDP1, a highly conserved, NEDD8-specific protease that precisely cleaves NEDD8 after the diglycine motif to generate mature NEDD8. NEDP1 appears to be specific for NEDD8 as neither ubiquitin nor SUMO bearing COOH-terminal extensions are utilized as substrates. Mutagenesis and inhibitor studies indicate that the protease uses cysteine as the active site nucleophile. NEDP1 is capable of removing NEDD8 from Cullins both in vitro and in vivo. It is likely that NEDP1 will have an important role in establishing the extent of NEDD modification of Cullins in vivo.
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EXPERIMENTAL PROCEDURES |
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Plasmid ConstructionsPlasmid pGST-NEDD8-Myc-His was a kind gift from H. Yasuda (Tokyo University of Pharmacy and Life Science, Hachioji, Japan), pcDNA3-Cullin-2 was from D. Girdwood (St. Andrews), and myc-Cullin 4A was from P. Zhou (Cornell University, New York). His-NEDD8 in pcDNA3 was obtained from D. Xirodimas (University of Dundee, Dundee, UK). A bacterial expression construct for the His6-tagged human NEDD8 gene was generated by PCR amplification using the 5' primer (5-TTGGGATCCATGCTAATTAAAGTG-3) and 3' primer (5'-GCAAAGCTTTCACTGCCTAAGACCACCTCCTCC-3'), which contain recognition sites for BamHI and HindIII, respectively. The PCR product was digested with BamHI and HindIII and ligated into pHISTEV vector (a gift of H. Liu, University of St. Andrews). The insert was verified by restriction endonuclease digestion and automated DNA sequence analysis. A blast search of protein data bases using 181 amino acids of the protease domain of the yeast Ulp1 (18) identified a number of Ulp-like proteins. Hidden Markov model searches (using the HIMMER suite) of a translated EST assembly data base with hidden Markov models generated from protein multiple sequence alignments of the Ulp-like proteins identified NEDP1 (accession number AAH31411 [GenBank] ).
To confirm and identify a cDNA containing the full-length gene of NEDP1, PCR analyses of tissues of human cDNA (Clontech) were used to determine expression of NEDP1. Oligonucleotides (5'-GATCCGCCAAGCTGGCTCAATGACC-3' and 5'-GCGTGAACTGAGTTGCTCCTGCTATGG-3') were designed that flanked the region of the protease domain and cDNAs were screened for expression. A kidney cDNA library from Origene was screened to identify bacterial clones containing the cDNA. Positive clones from the library were then rescreened using a primer in the vector, pCMV6-XL4, to detect the 5' end of the gene. The clone containing the longest insert was determined by DNA sequence analysis. A stop codon in the 5'-untranslated region was used as confirmation that the cDNA was a full-length gene. Oligonucleotides (5'-GCCACCATGGACCCCGTAGTCTTG-3' and 5'-CTACTACTTTTTAGCAAGTGTGGCAATGAG-3') of the coding region including the stop codon of NEDP1 were designed for cloning NEDP1 into the pcDNA3.1/V5-His vector (Invitrogen). This construct was used for further analysis.
Site-directed mutagenesis using the QuikChange system (Stratagene) was used to alter the active site cysteine residue in NEDP1. The template DNA contained the full-length gene of NEDP1 under control of the cytomegalovirus promoter. Mutagenic oligonucleotides (5'-CCAACAAAACAGCTATGACGCTGGGATGTACGTGATATG-3' and 5'-CATATCCGTACATCCCAGCGTCATAGCTGTTTTGTTGG-3') were used to alter the active cysteine (TGT) to an alanine (GCT). The complete coding region of NEDP1 was sequenced to verify that the cysteine had been altered to alanine and the sequence was otherwise unaltered.
pGEX-4T3-NEDP1 plasmid for the expression of NEDP1 was constructed by PCR amplification of NEDP1 cDNA with the primers 5'-GCAGAATTCCGACCCCCTAGTCTTGAGTTAC-3' and 5'-CCAGCTCGAGCTACTTTTTAGCAAGTGTGGC-3'. The PCR product was restricted with EcoRI and XhoI before insertion into a similarly restricted pGEX-4T3. pGEX-4T3-NEDP1mut(C163A) was constructed as above by amplifying NEDP1(C163A) cDNA.
pGEX-2T-Ub-H-PK for the expression of GST-Ub-His-PK was constructed by PCR amplification of pGST-UbGG with the primers 5'-GCGGGATCCCAGATCTTCGTGAAGACCCTG-3' and 5'-GCGGAATTCCACCACCTCTCAGACGCAGGAC-3'. The PCR product was restricted with BamHI and EcoRI prior to insertion into a similarly restricted pGEX-2T-H-PK vector, provided by R. E. Randall, St. Andrews.
Expression and Purification of Recombinant
ProteinsC52A-SUMO-1, GST-NEDP1, and GST-SENP2 were expressed in,
and purified from, Escherichia coli B834 as described previously
(20). Proteins were eluted
from glutathione-agarose with buffer containing 10 mM glutathione
and stored at 70 °C. A Drosophila ubiquitin
carboxyl-terminal hydrolase was expressed in E. coli as a GST fusion
protein (GST-UCH) and purified as described
(21). GST-NEDD8-Myc-His and
GST-Ub-H-PK were expressed in E. coli BL21(DE3) as described
previously (22). The
His6-NEDD8 was expressed in E. coli strain BL21(DE3) at 37
°C. At an A600 of 0.50.6,
isopropyl-1-thio--D-galactopyranoside was added to a final
concentration of 0.4 mM and the cultures were incubated for 4 h at
37 °C. Cells from a 1-liter culture were collected by centrifugation,
resuspended in 30 ml of lysis buffer (phosphate buffer saline containing in
addition, 0.5 M NaCl, 1 mM EDTA, and 2 mM
bezamidine), and disrupted by sonication. His6 NEDD8 inclusion
bodies were collected by centrifugation (20,000 x g for 30
min), and the insoluble material was washed three times with 30 ml of buffer
containing 1 M urea, 0.1 M
NaH2PO4, 50 mM Tris-HCl, pH 8.0. Purified
inclusion bodies containing His6-NEDD8 were solubilized in 20 ml of
8 M urea, 0.1 M NaH2PO4, 50
mM Tris-HCl, pH 8.0, and held at room temperature for 30 min. Any
remaining particulate material was removed by centrifugation (20,000 x
g, 30 min) and the supernatant was bound to 10 ml of Ni-NTA resin.
The resin was washed successively with 50 ml of 8 M urea, 0.1
M NaH2PO4, 50 mM Tris-HCl, pH 8.0,
and 50 ml of 8 M urea, 0.1 M
NaH2PO4, 50 mM Tris-HCl, pH 6.3.
His6-NEDD8 was eluted with 8 M urea, 0.1 M
NaH2PO4, 50 mM Tris-HCl, pH 4.5. The eluted
His6-NEDD8 was refolded by dialysis at 4 °C into 50
mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM
dithiothreitol and insoluble material was removed by centrifugation. About 40
mg of purified His6-NEDD8 was recovered per 1 liter of culture and
the purity of the protein was >95% as evaluated by SDS-PAGE and Coomassie
staining.
Mass SpectrometryFull-length His6-NEDD8 or NEDP1-processed NEDDs (20 µl, 10 pmol/µl) was desalted on-line through a XTerra MS C8 2.1 x 10-mm column, eluting with an increasing acetonitrile concentration (2% acetonitrile, 98% aqueous, 1% formic acid to 98% acetonitrile, 2% aqueous, 1% formic acid) and delivered to an electrospray ionization mass spectrometer (LCT, Micromass, Manchester, UK) that had previously been calibrated using myoglobin. An envelope of multiple charged signals was obtained and deconvoluted using MaxEnt1 software to give the molecular mass of the protein.
In Vitro NEDD8 Deconjugation AssayIn vitro transcription,
translation, and conjugation of Cul-2 was performed using 1 µg of plasmid
DNA and a rabbit reticulocyte lysate-coupled transcription/translation system
(Promega) in the presence of 3 µg of GST-NEDD8-GG for 2 h at 30 °C.
[35S]Methionine (Amersham Biosciences) was used in the reactions to
generate radiolabeled protein. Conjugation was terminated by the addition of
iodoacetamide to 10 mM and incubated at 20 °C for 30 min.
Iodoacetamide was quenched by the addition of -mercaptoethanol to 15
mM and incubated at 20 °C for a further 15 min.
Deconjugation of [35S]methionine-labeled Cul-2-NEDD8 was
performed in 10 µl containing 3 µlof 35S-labeled conjugated
substrate, 2 µg of GST-NEDP1 in 50 mM Tris, pH 7.5, 2
mM MgCl2, and 5 mM -mercaptoethanol.
Reactions were incubated at 37 °C for 3 h, terminated with SDS sample
buffer containing
-mercaptoethanol, and reaction products were
fractionated by electrophoresis in polyacrylamide gels (8%) containing SDS,
stained, destained, and dried prior to analysis by phosphorimaging.
In Vitro NEDD8 Processing AssayNEDD8 processing was
performed in 20 µl containing 6 µg of GST-NEDD8-Myc-His6, 50
mM Tris, pH 7.5, 2 mM MgCl2, 5 mM
-mercaptoethanol and between 166 and 0.07 ng of GST-NEDP1. Reactions
were incubated at 37 °C for 3 h. After termination with SDS sample buffer
containing
-mercaptoethanol, reaction products were fractionated by gel
electrophoresis in 12.5% polyacrylamide gels containing SDS, stained, and
destained. Dried gels were analyzed by phosphorimaging.
To determine the specificity of NEDP1 processing, NEDD8, SUMO, and Ub
processing assays were performed in 20 µl containing 2 µg of substrate
(GST-NEDD8-Myc-His6, GST-UB-H-P, or SUMO-1) and 0.5 µg of
GST-NEDP1 or GST-NEDP1mut. All processing assays were performed in buffer
containing 50 mM Tris, pH 7.5, 2 mM MgCl2,
and 5 mM -mercaptoethanol for 3 h at 37 °C. After
termination with SDS sample buffer containing
-mercaptoethanol, reaction
products were fractionated by gel electrophoresis in 12.5% polyacrylamide gels
containing SDS. NEDD8 and ubiquitin-processing assays were analyzed by Western
blotting using either anti-His or anti-PK SV5 antibody, respectively.
SUMO-processing assays were analyzed by Coomassie Blue staining.
Protease inhibition assays were performed in 10 µl containing 1 µgof
substrate (GST-NEDD8-Myc-His6) and 50 ng of GST-NEDP1. Assays were
performed in 50 mM Tris, pH 7.5 containing either
N-ethylmaleimide (2.5 and 5 mM) or EDTA (10, 30, and 50
mM) as indicated. Protease was preincubated with either inhibitor
or buffer for 5 min prior to the addition of substrate and then further
incubated at 37 °C for 3 h. After termination with SDS sample buffer
containing -mercaptoethanol, reaction products were fractionated by gel
electrophoresis in 12.5% polyacrylamide gels containing SDS, stained and
destained.
Cell Culture and TransfectionsCOS7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For analysis of NEDP1 deconjugation activity in vivo, 25-cm2 flasks of subconfluent cells were cotransfected with the expression constructs for NEDP1 and His-NEDD8 (10.6 µg of total plasmid DNA) as indicated in the figures. Lysates were prepared as described previously (19) and analyzed by Western blotting with the anti-His antibody.
Purification of His6-tagged NEDD8 Cul-4A
conjugatesForty-eight hours after transfection COS7 cells were
lysed in 5 ml of 6 M guanidinium HCl, 0.1 M
Na2HPO4/NaH2PO4, 0.01 M
Tris-HCl, pH 8.0, plus 5 mM imidazole and 10 mM
-mercaptoethanol per 25-cm2 flask. After sonication, to
reduce viscosity, the lysates were mixed with 50 µl of
Ni2+-NTA-agarose beads prewashed with lysis buffer and
incubated for 2 h at room temperature. The beads were successively washed with
the following: 6 M guanidinium HCl, 0.1 M
Na2HPO4/NaH2PO4, 0.01 M
Tris-HCl, pH 8.0, plus 10 mM
-mercaptoethanol; 8 M
urea, 0.1 M
Na2HPO4/NaH2PO4, 0.01 M
Tris-HCl, pH 8.0, 10 mM
-mercaptoethanol; 8 M
urea, 0.1 M
Na2HPO4/NaH2PO4, 0.01 M
Tris-HCl, pH 6.3, 10 mM
-mercaptoethanol (buffer A) plus 0.2%
Triton X-100; buffer A and then buffer A plus 0.1% Triton X-100. After the
last wash with buffer A the beads were eluted with 200 mM imidazole
in 5% SDS, 0.15 M Tris-HCl, pH 6.7, 30% glycerol, 0.72 M
-mercaptoethanol. The eluates were subjected to SDS-PAGE (10%) and the
proteins were transferred to a polyvinylidene difluoride membrane (Sigma).
Western blotting was performed with a monoclonal antibody against the Myc
tag.
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RESULTS |
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The NEDP1 coding region of 636 nucleotides is composed of only 1
exon (data not shown) containing the complete coding region of the gene.
Recognizable within the NEDP1 coding sequence is a 212-amino acid domain that
is also present in the family of SUMO proteases. This domain contains the
putative catalytic triad of histidine, aspartate, and cysteine along with an
invariant glutamine residue. NEDP1 has 20% identity to the yeast and
human Ulps (Fig. 1A)
but differs from other members of the yeast and human family of Ulp proteases
in that the protein consists of just the protease domain with only short
NH2 and COOH-terminal extensions.
Interrogation of sequence data bases with the putative protease domain of NEDP1 revealed a highly conserved family of proteins that are present in all eukaryotes from S. pombe to Homo sapiens (Fig. 1C). PCR analysis using primers from the region encoding the protease domain of NEDP1 on a panel of cDNAs from different tissues indicated that the NEDP1 gene was widely expressed (Fig. 2). This data was also confirmed by TaqMan analysis of cDNAs (data not shown). To determine the biochemical activity of NEDP1, the complete coding region was expressed as a fusion with glutathione S-transferase in bacteria. GST-NEDP1 was isolated by affinity chromatography and the purified proteins were analyzed by electrophoresis in a polyacrylamide gel containing SDS. Coomassie Blue staining revealed that the purified GST-NEDP1 was essentially homogenous (Fig. 3A). Although NEDP1 was expected to be a SUMO-specific protease there was no evidence of activity against full-length SUMO-1, SUMO-2, or SUMO-3.
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Processing of GST-NEDD8 but Not Ub or SUMO by NEDP1Like SUMO, ubiquitin and NEDD8 are synthesized as inactive precursors that need to be precisely cleaved by proteases at a COOH-terminal diglycine motif, prior to conjugation to their substrates. Recombinant GST-NEDD8-Myc-His, GST-Ub-PK, and full-length SUMO-1 were expressed and purified from bacteria to provide model precursor substrates for NEDP1. To establish the specificity of NEDP1, 2 µg of SUMO-1, GST-Ub-H-P, or GST-NEDD8-Myc-His were incubated with 0.5 µg of GST-NEDP1 or a catalytically inactive form of the enzyme, GST-NEDP1mut. NEDP1 was unable to process either SUMO-1 or Ub, but efficiently processed NEDD8 (Fig. 3B). To confirm that NEDP1 cleaved NEDD8 precisely after the second glycine in the GG motif a His6 version of NEDD8 was processed with NEDP1 and products of the cleavage reaction were analyzed by electrospray ionization mass spectrometry (Fig. 4A). The molecular mass of the processed NEDD8 corresponds precisely to cleavage after the second Gly in the diglycine motif. Thus NEDP1 is a NEDD8-processing enzyme. To determine the efficiency of NEDP1 processing GST-NEDD8-Myc-His was incubated with a range of concentrations of purified GST-NEDP1 and the reaction products were analyzed by SDS-PAGE followed by Coomassie Blue staining (Fig. 4B). In the presence of 6 µg of GST-NEDD8-Myc-His, 2 ng of NEDP1 is capable of processing greater then 50% of the substrate in 3 h at 37 °C.
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The sequence of NEDP1 suggests that it is a cysteine protease with the active site cysteine located at residue 163. To address this point a version of NEDP1 was created in which cysteine 163 was changed to alanine (NEDP1 mut). This protein was expressed in bacteria and purified to homogeneity (Fig. 3A). GST-NEDP1mut, in which putative active site cysteine 163 is mutated to an alanine, was unable to process NEDD8 (Fig. 3B). To verify that NEDP1 is a cysteine protease, processing assays were set up in the presence of N-ethylmaleimide or EDTA. Although GST-NEDP1 processing activity was inhibited by 2.5 mM N-ethylmaleimide, addition of EDTA up to 50 mM had no effect on processing in this assay (Fig. 4C). Together with the lack of activity displayed by the C163A mutant these data indicate the NEDP1 is a NEDD8-specific cysteine protease.
NEDP1 Deconjugates NEDD8 from Cul-2 in VitroTo determine
whether NEDP1 is capable of acting as an isopeptidase in the presence of
unrelated proteins an in vitro deconjugation assay was designed.
Previously it was shown that Cullin-4A could be conjugated to GST-NEDD8 during
a transcription/translation reaction in rabbit reticulocyte lysates
(2). Cul-2 was therefore
labeled with [35S]methionine and conjugated to GST-NEDD8 during an
in vitro transcription-translation reaction in rabbit reticulocyte
lysates. Conjugation was terminated by incubation with iodoacetamide that also
served to inhibit any endogenous NEDD8 proteases. After quenching of the
iodoacetamide with -mercaptoethanol the reaction products were used as
substrates for NEDP1. Although Cul-2 has one major translated product there
are two lower molecular weight species that may represent internal
initiations. Full-length Cul-2 as well as incomplete translations were
utilized by the conjugation machinery in the lysates for conjugation to
GST-NEDD8. Incubation of the modified products with NEDP1 resulted in
conversion of the modified to the unmodified form of Cul-2
(Fig. 5A). Thus NEDP1
displays NEDP1 isopeptidase activity on a natural substrate in the presence of
a large excess of unrelated proteins (from rabbit reticulocyte lysate
extract).
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NEDP1 Deconjugates NEDD8 from Modified Cul-4 in VivoTo determine whether NEDP1 was active against a specific cullin family member in vivo, the cDNA encoding NEDP1 was transfected into COS7 cells along with expression plasmids for His-NEDD8 and Cul-4A-Myc. Cells were lysed under denaturing conditions and the His-NEDD8 conjugates were purified from cell lysates using Ni2+-NTA-agarose beads. Bound proteins were separated by SDS-PAGE and subjected to Western blotting with an anti-Myc monoclonal antibody. Transfection of Cul-4A-myc, His-NEDD8, and empty expression vector allowed the purification of a His-NEDD8-modified Cul-4A-myc. Transfection of these constructs in the presence of NEDP1 resulted in the absence of NEDD8-modified Cul-4A. Transfection of the catalytically inactive NEDP1mut did not affect the modification state of Cul-4A. Western blotting of the unfractionated extract with anti-Myc antibody revealed that CUL-4A expression was not affected by NEDP1 or C163A NEDP1 (Fig. 5B). Thus NEDP1 is capable of acting as a NEDD8-specific cysteine protease that can deNEDDylate cullins in vivo.
Substrate Specificity of NEDP1To determine whether NEDP1 displays a preference for particular NEDD8-conjugated substrates in vivo, a NEDP1 expression vector was transfected into COS7 cells along with the expression plasmid for His-NEDD8. As a control His-NEDD8 was transfected with empty expression vector or a vector encoding C163A NEDP1 where the cysteine residue predicted to supply the active site nucleophile was changed to alanine. 24 h post-transfection NEDD8-modified conjugates were identified by Western blotting with an anti-His antibody. Transfection of His-NEDD8 leads to the appearance of high molecular weight conjugates that disappear when NEDP1 is co-transfected. Co-transfection of catalytically inactive C163A NEDP1 does not alter the pattern of NEDD8-modified conjugates (Fig. 6). Therefore NEDP1 is active as a NEDD8 protease in vivo and is capable of deconjugating NEDD8 from all modified proteins detected in vivo.
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DISCUSSION |
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences
Research Council, GlaxoSmithKline, Canadian Institutes of Health Research, and
the Wellcome Trust. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 44-1334-463396; Fax: 44-1334-462595; E-mail: rth{at}st-and.ac.uk.
1 The abbreviations used are: GST, glutathione S-transferase;
Ni-NTA, nickel-nitrilotriacetic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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