NEDP1, a Highly Conserved Cysteine Protease That deNEDDylates Cullins*

Heidi M. Mendoza, Lin-nan Shen, Catherine Botting, Alan Lewis {ddagger}, Jingwen Chen §, Barbara Ink {ddagger} and Ronald T. Hay 

From the Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews, Fife KY169AL, United Kingdom, {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ubiquitin-like protein NEDD8 is essential for activity of SCF-like ubiquitin ligase complexes. Here we identify and characterize NEDP1, a human NEDD8-specific protease. NEDP1 is highly conserved throughout evolution and equivalent proteins are present in yeast, plants, insects, and mammals. Bacterially expressed NEDP1 is capable of processing NEDD8 in vitro to expose the diglycine motif required for conjugation and can deconjugate NEDD8 from modified substrates. NEDP1 appears to be specific for NEDD8 as neither ubiquitin nor SUMO bearing COOH-terminal extensions are utilized as substrates. Inhibition studies and mutagenesis indicate that NEDP1 is a cysteine protease with sequence similarities to SUMO-specific proteases and the class of viral proteases typified by the adenovirus protease. In vivo NEDP1 deconjugates NEDD8 from a wide variety of substrates including the cullin component of SCF-like complexes. Thus NEDP1 is likely to play an important role in ubiquitin-mediated proteolysis by controlling the activity of SCF complexes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquitin and ubiquitin-like proteins are conjugated to acceptor lysine residues on target proteins and have diverse effects on the modified proteins. Whereas conjugation of multiple copies of ubiquitin targets proteins for degradation via the proteasome, addition of SUMO or NEDD8 can alter the function of the conjugated protein (1). Formation of the isopeptide bond between the COOH-terminal glycine of the Ulp and the {epsilon}-amino group of lysine in the modified protein is accomplished by an enzymatic cascade that typically involves three enzymes, E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ligase). In the case of NEDD8, or its yeast equivalent Rub1, the ubiquitin-like protein is activated by a heterodimeric complex of APP-BP1 and Uba3, and is conjugated to substrates by the conjugating enzyme Ubc12 (2, 3). Although an E3 ligase specific for NEDD8 has yet to be identified, the parallels with the ubiquitin and SUMO systems indicate that it is likely such an activity will exist. To date the only targets for NEDD8 modification that have been described are members of the Cullin family of proteins (28). Cullins are important components of multiple ubiquitin ligase complexes that also contain Rbx1, Skp1 (or homologue), and a substrate receptor protein that contains an F-box motif. These SCF-like complexes are responsible for the ubiquitination of proteins such as phosphorylated I{kappa}B{alpha} and hydroxylated HIF1{alpha}. Genetic experiments in yeast and plants indicate that Rub1 (NEDD8) modification is important for SCF ubiquitin ligase activity (3, 911), whereas biochemical experiments demonstrated that NEDD8 modification of Cul-1 was responsible for recruitment of the Ubc4-ubiquitin thioester to the SCF complex (12). It has been demonstrated that the Rbx1 component of SCF complexes activates Ubc12-mediated NEDD8 modification of Cdc53 and Cul-2 (5). Whereas a complete Rub1 (NEDD8) modification pathway is not required for the viability of Saccharomyces cerevisiae (3, 9) it is required for viability of Schizosaccharomyces pombe (11). In ts41 hamster cells, a temperature-sensitive mutation in APP-BP1 results in cell cycle defects and indicates that NEDD8 modification is required for entry into mitosis and inhibition of entry into S-phase (13, 14). Deletion of the Uba3 gene in mice leads to embryonic lethality and establishes an essential function of NEDD8 modification in higher eukaryotic cells (12).

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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—GST1-Ub-H-PK was detected in Western blot experiments using mouse monoclonal sv5 PK Tag 336, antibody (at a 1:1000 dilution), kindly provided by R. E. Randall (St. Andrews). GST-Nedd8-Myc-His6 was detected in Western blot experiments using mouse monoclonal anti-His antibody (at a 1:2000 dilution), obtained from Amersham Biosciences. Sheep anti-mouse horseradish peroxidase-conjugated IgG (Amersham Biosciences) was used to detect primary antibody at 1:5000 dilution. Anti-Myc 9E10 monoclonal mouse antibody was used at 1:500 in Western blot experiments to detect Cul-4A-Myc.

Plasmid Constructions—Plasmid 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 Proteins—C52A-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 0.5–0.6, isopropyl-1-thio-{beta}-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 Spectrometry—Full-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 Assay—In 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 {beta}-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 {beta}-mercaptoethanol. Reactions were incubated at 37 °C for 3 h, terminated with SDS sample buffer containing {beta}-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 Assay—NEDD8 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 {beta}-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 {beta}-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 {beta}-mercaptoethanol for 3 h at 37 °C. After termination with SDS sample buffer containing {beta}-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 {beta}-mercaptoethanol, reaction products were fractionated by gel electrophoresis in 12.5% polyacrylamide gels containing SDS, stained and destained.

Cell Culture and Transfections—COS7 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 conjugates—Forty-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 {beta}-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 {beta}-mercaptoethanol; 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris-HCl, pH 8.0, 10 mM {beta}-mercaptoethanol; 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris-HCl, pH 6.3, 10 mM {beta}-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 {beta}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of cDNA Encoding NEDP1—A hidden Markov model search using protein multiple sequence alignments of Ulp-like proteins identified a human gene, NEDP1, containing a putative protease domain with homology to both human and yeast Ulps (Fig. 1A). The longest open reading frame identified was confirmed by cloning of a cDNA with an upstream stop codon. The longest clone was found to contain the open reading frame as shown in Fig. 1B.



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FIG. 1.
Isolation of cDNAs encoding NEDP1. A, primary sequence alignments of H. sapiens NEDP1, SENP1, SENP2, and SUSP1, and S. cerevisiae Ulp1 and Ulp2. B, nucleotide sequence of the cDNA and deduced amino acid sequence of the NEDP1 protein. The likely active site residues of the catalytic triad (H, D, and C) are boxed. C, conservation of NEDP1 between Mus musculus, Drosophila melanogaster, Arabidopsis thaliana, and S. pombe.

 

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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FIG. 2.
Expression of NEDP1 mRNA. cDNAs prepared from a range of human tissues (Clontech) were assessed for NEDP1 expression by PCR. Products were analyzed by agarose gel electrophoresis and stained with ethidium bromide.

 


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FIG. 3.
NEDP1 is a NEDD8-specific protease. A, Coomassiestained SDS-polyacrylamide gel containing 3 µg of recombinant purified NEDP1 and NEDP1mut as indicated. B, 2 µg of GST-Ub-H-PK, GST-Nedd8-Myc-His6, or SUMO-11–101 were incubated with 0.5 µg of GST-NEDP1, GST-NEDP1mut, ubiquitin COOH-terminal hydrolase (UCH), or SUMO-specific protease (SSP) as indicated. All reaction products were fractionated by 12.5% polyacrylamide gels containing SDS. NEDD8 and ubiquitin-processing assays were further subjected to Western blot using either anti-His antibody or anti-PK SV5 antibody as detailed under "Experimental Procedures." SUMO processing was analyzed by Coomassie Blue staining.

 

Processing of GST-NEDD8 but Not Ub or SUMO by NEDP1—Like 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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FIG. 4.
Processing of NEDD8 by NEDP1 cysteine protease. A, His6-NEDD8 and His6-NEDD8 processed with NEDP1 were analyzed by electrospray ionization mass spectrometry. Experimentally derived molecular masses are indicated. Calculated molecular mass for unprocessed His6-NEDD8 is 12611.33 whereas that of His6-NEDD8 terminating after the GG motif is 12099.75. B, Coomassie-stained SDS-polyacrylamide gel showing 2 µg of full-length recombinant GST-NEDD8-Myc-His6 incubated with varying concentrations of GST-NEDP1 (166, 55, 18, 6, 2, 0.6, 0.2, and 0.07 ng). The reaction products were further subjected to Western blot using anti-His antibody. GST-NEDD8-Myc-His6 and GST-NEDD8 are indicated. C, Coomassie-stained SDS-polyacrylamide gel showing 1 µg of full-length recombinant GST-NEDD8-Myc-His6 incubated with 50 ng of GST-NEDP1 in the presence of N-ethylmaleimide (NEM), EDTA, or buffer. GST-NEDD8-Myc-His6 and GST-NEDD8 are indicated.

 

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 Vitro—To 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 {beta}-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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FIG. 5.
NEDP1 deconjugates NEDD8 from modified cullins. A, GST-NEDP1 catalyzed deconjugation of Cul-2-GST-NEDD8. GST-NEDD8-modified 35S-labeled Cul-2 was incubated with 2 µg of GST-NEDP1 for 3 h at 37 °C. Reaction products were analyzed by SDS-PAGE and radioactive species were detected by phosphorimaging. The positions of Cul-2-GST-NEDD8 and unmodified Cul-2 are indicated. B, COS7 cells were transfected with either empty pcDNA3 (–) or plasmids expressing Cul-4A-Myc, His-NEDD8, NEDP1, or NEDP1mut as indicated. His-NEDD8 conjugates were purified on Ni2+-agarose and conjugates were fractionated by gel electrophoresis in SDS-PAGE gels (8% polyacrylamide). Anti-Myc Western blots were performed as detailed under "Experimental Procedures." A nonspecific (NS) band is indicated along with the molecular weight markers.

 

NEDP1 Deconjugates NEDD8 from Modified Cul-4 in Vivo—To 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 NEDP1—To 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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FIG. 6.
Specificity of NEDP1 in vivo. COS7 cells were transfected with either empty pcDNA3 (–) or plasmids expressing His-NEDD8, NEDP1, or NEDP1mut as indicated. Anti-His Western blots were performed on cell lysates as detailed under "Experimental Procedures." A nonspecific (NS) band is indicated along with molecular weight markers.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Here we describe NEDP1, a NEDD8-specific protease present in human cells. NEDP1 is sensitive to the alkylating reagent N-ethylmaleimide and site-directed mutagenesis indicates that the active site nucleophile is cysteine 163. The protein is highly conserved throughout evolution and is ~40% identical over the protease domain to two predicted gene products present in S. pombe. NEDP1 has highly related homologues in most organisms for which sequence information is available, suggesting that the homologues are also NEDD8 proteases. Sequence comparisons indicate that NEDP1 is unrelated to deubiquitinating enzymes although both are part of the cysteine protease superfamily. Although NEDD8 is much more similar to ubiquitin than SUMO, NEDP1 displays sequence similarity to the SUMO-specific proteases including an arrangement of proposed catalytic residues (His-Asp-Cys) also present in yeast Ulp1 (23). This suggests that NEDP1 is part of a distinct cysteine protease subfamily that contains SUMO-specific protease along with adenoviral (24) and other viral proteases. However, the structural relatedness of NEDP1 to this class of cysteine proteases awaits the determination of its structure. Bacterially expressed and purified NEDP1 cleaves the NEDD8 precursor precisely after the diglycine motif, but is unable to utilize ubiquitin or SUMO with COOH-terminal extensions, indicating that NEDP1 is highly specific for NEDD8. This is in contrast to two previously described proteases that utilized both NEDD8 and ubiquitin as substrates (25, 26). In addition to processing NEDD8 precursor to the mature form NEDP1 is also capable of acting as a NEDD8 isopeptidase by deconjugating NEDD8 from Cul-2 in vitro. In vivo, expressed NEDP1 removes NEDD8 from Cul-4A. Whereas Cullins are the only recognized substrates for NEDD8 modification described to date it is clear that exogenous NEDD8 is conjugated to a large number of high molecular weight proteins that have apparent molecular weights that are considerably in excess of that predicted from NEDD8-modified Cullins. The identity of these NEDD8-modified proteins remains to be determined, but NEDP1 is capable of removing NEDD8 from all of the modified proteins. Thus NEDP1 appears to be highly specific for NEDD8 but is not specific with regard to the modified substrate. An important role for a deNEDDylating activity is in the COP9 signalsome-mediated removal of NEDD8 from Cullins (27). The COP9 signalsome is an 8-subunit multiprotein complex that is similar to the "lid" of the proteasome (28). Within the COP9 signalsome, the Jab1/Csn5 subunit contains a JAMM motif that is predicted to form the active site of a metalloprotease (29). Jab1/Csn5 is required for the deNEDDylation activity of the COP9 signalsome but it was reported that the deNEDDy-lation activity was sensitive to alkylating agents (16, 17) thus suggesting the involvement of a cysteine protease. The role of NEDP1 in vivo is difficult to predict as it could have a number of potentially antagonistic effects. Whereas an increase in NEDP1 activity in the cell would be predicted to result in the deNEDDylation of Cullins this effect might be counteracted by an increase in the availability of processed NEDD8, if NEDD8 processing is limiting. If the supply of processed NEDD8 is not limiting then NEDP1-mediated deNEDDylation of Cullins would be predicted to block degradation of proteins ubiquitinated by Cullin containing SCF-like ubiquitin-ligase complexes. This would result in a block to cell cycle progression. Another potential role for NEDP1 could be to mobilize NEDD8. If most NEDD8 in the cell is conjugated to protein substrates then the free pool of NEDD8 will be small and major changes in the pattern of proteins modified by NEDD8 will require the action of a NEDD8-specific protease to generate free NEDD8 for conjugation to new substrates. Given the requirement for multiple rounds of activation and inactivation of SCF complexes as cells traverse the cell cycle, it is likely that NEDP1 will have an important role in controlling NEDD8 modification in vivo.


    FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AAH31411 [GenBank] .

* 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. Back

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. Back


    ACKNOWLEDGMENTS
 
We thank R. E. Randall, H. Yasuda, H. Liu, and D. Xirodimas for the generous provision of materials and Alex Houston for DNA sequencing.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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