A Novel Type of Deubiquitinating Enzyme*,

Paul C. Evans {ddagger} §, Trevor S. Smith {ddagger}, Meng-Jiun Lai ¶, Mark G. Williams ¶ ||, David F. Burke ¶, Karen Heyninck **, Marja M. Kreike **, Rudi Beyaert **, Tom L. Blundell ¶ and Peter J. Kilshaw {ddagger}

From the {ddagger}Molecular Immunology Programme, The Babraham Institute, Cambridge CB2 4AT, United Kingdom, the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom, the **Department of Molecular Biomedical Research, Unit of Molecular Signal Transduction in Inflammation, University of Ghent-VIB, Ghent B-9000, Belgium

Received for publication, February 21, 2003 , and in revised form, April 4, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A previous report from this laboratory described two novel proteins that have sequence similarity to A20, a negative regulator of NF-{kappa}B (Evans, P. C., Taylor, E. R., Coadwell, J., Heyninck, K., Beyaert, R., and Kilshaw, P. J. (2001) Biochem. J. 357, 617–623). One of these molecules, cellular zinc finger anti-NF-{kappa}B (Cezanne), a 100-kDa cytoplasmic protein, also suppressed NF-{kappa}B. Here we demonstrate that Cezanne is a novel deubiquitinating enzyme, distinct from the two known families of deubiquitinases, Types I and II. We show that Cezanne contains an N-terminal catalytic domain that belongs to the recently discovered ovarian tumor protein (OTU) superfamily, a group of proteins displaying structural similarity to cysteine proteases but having no previously described function. Recombinant Cezanne cleaved ubiquitin monomers from linear or branched synthetic ubiquitin chains and from ubiquitinated proteins. Mutation of a conserved cysteine residue in the catalytic site of the proteolytic domain caused Cezanne to co-precipitate polyubiquitinated cellular proteins. We also provide evidence for an additional ubiquitin binding site in the C-terminal part of the molecule. Our data provide the first demonstration of functional activity among OTU proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The activities of numerous cellular proteins are regulated by covalent attachment of ubiquitin (1). Polyubiquitin genes encode tandem repeats of several ubiquitin units which are joined by {alpha}-amino, peptide bonds. These linear polyubiquitin chains are processed to generate reactive ubiquitin monomers. Ubiquitin is covalently attached to substrate lysine residues through the concerted action of E1,1 E2, and E3 ubiquitin ligases, which together determine substrate specificity. Successive rounds of ubiquitination, where ubiquitin is itself ubiquitinated, generate an isopeptide-linked branched chain. Proteins modified with a polyubiquitin chain conjugated through lysine 48 are targeted to the proteasome, where they are degraded. Recent data suggest that alternative forms of ubiquitin modification, such as monoubiquitination or assembly of Lys63-linked chains, regulate intracellular localization of proteins and signaling activities.

Deubiquitinating enzymes are important regulators of this process (2). Molecules belonging to the ubiquitin C-terminal hydrolase family (Type I) are small (20–30 kDa) and highly conserved. They cleave {alpha}-amino linked (linear) polyubiquitin chains and are required for the generation of reactive ubiquitin monomers from polyubiquitin gene products.

The UBP family (Type II) comprises over 90 proteases that have a range of cellular functions. Molecules belonging to this family are large (60–300 kDa) and highly divergent except for two short conserved sequences that surround the catalytic cysteine and histidine residues. UBP enzymes are able to hydrolyze branched polyubiquitin chains. They play an important role in proteasome function (3, 4) and in generating free ubiquitin monomers from branched chains for recycling (5). In addition, some UBP molecules can regulate the fate of specific cellular proteins. Thus, removal of ubiquitin chains will extend the half-life of these molecules and therefore modulate their particular activities in the cell. For example, genetic experiments in Drosophila have revealed that liquid facets protein is the specific target of Fat facets, a UBP molecule involved in eye development (6). Fat facets stabilizes liquid facets through deubiquitination (7). For the great majority of UBP molecules, however, substrate specificity has not been determined. The JAMM family of metalloproteases that cleave ubiquitin or ubiquitin-like molecules has also been identified recently (810). Deubiquitinating activity by one of these enzymes, Rpn11, is essential for degradation of substrate proteins by the 26 S proteasome (9, 10).

We have recently discovered two novel proteins, Cezanne and TRABID, with sequence similarity to A20, an important negative regulator of the transcription factor NF-{kappa}B (11). Cezanne also has the capacity to suppress NF-{kappa}B. Here, we demonstrate that Cezanne is a novel type of deubiquitinating enzyme. This activity may explain its inhibitory effect on NF-{kappa}B.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian Cells—COS7 and HeLa cells were cultured using Dulbecco's modified Eagle's medium, 10% fetal-calf serum, supplemented with antibiotics.

Yeast Expression Vectors—Cloning was performed by recombinant PCR using Pfu turbo polymerase (Stratagene). The yeast expression vector pGBKT7 (Clontech Laboratories) was used to express fusions between the DNA-binding domain of the Gal4 transcription factor, a Myc epitope tag, and Cezanne (driven by the ADH1 promoter). The primers used to amplify regions corresponding to Cezanne residues 17–858 (5'-GCTGGAATTCACCCTGGACATGGATGCTGTTC-3' and 5'-GGGTTTGTCGACTCAGAACCTGTGCACCAGGAG-3'), residues 17–443 (5'-GCTGGAATTCACCCTGGACATGGATGCTGTTC-3' and 5'-GGGTTTGTCGACCATGTAGCTATGCAGCAGATGC-3'), or residues 444–858 (5'-GCTGGAATTCAATGTGAAGTGGATCCCACTG-3' and 5'-GGGTTTGTCGACTCAGAACCTGTGCACCAGGAG-3') incorporated SalI or EcoRI restriction sites, which facilitated directional cloning of cDNA in frame with GAL4 DNA-binding domain.

Bacterial Expression Vectors—pDEST15 (Invitrogen) was used for expression of glutathione S-transferase (GST)-Cezanne fusion proteins in Escherichia coli in response to isopropyl-{beta}-D-thiogalactosidase (IPTG). PCR primers amplified cDNA corresponding to residues 126–455 (sense, 5'-CACCGAGAACCTGTACTTCCAGGGTCGGTCCCATGTCTCCTCCAA-3'; antisense, 5'-TTACTAGAACTCCTCTGCATCAGAGGACAGTGGGATCCACTT-3') or residues 182–455 (sense, 5'-CACCGAGAACCTGTACTTCCAGGGTGCAGGGCGTTTGAACTGG-3'; antisense as above). Sense primers incorporated nucleotides encoding the tobacco etch virus protease recognition site and nucleotides required for topoisomerase cloning. Antisense primers incorporated stop codons. Amplified sequences were cloned initially into pENTR/D-TOPO vectors (Invitrogen) and then into pDEST15, using the GATEWAY system (Invitrogen).

Mammalian Expression Vectors—pHM6 was used to express molecules tagged at the N terminus with hemagglutinin (HA) in mammalian cells (driven by a cytomegalovirus immediate early promoter). Construction of pHM6-Cezanne 1–858 and pHM6-Cezanne 1–443 has been described previously (11). Expression of FLAG-tagged ubiquitin driven by the cytomegalovirus immediate early promoter was achieved using pcDNA3.1-FLAG-ubiquitin, kindly supplied by Dr. I. Dikic (Ludwig Institute for Cancer Research, Uppsala, Sweden).

Mutagenesis—QuikChange technology (Stratagene) was used to change two nucleotides at codon 209 (Cys to Ala) in pGBKT7-Cezanne 17–858, pHM6-Cezanne 1–858, and pHM6-Cezanne 1–443 using complementary primers (sense, 5'-CTACTGGAGATGGGAACGCCCTCCTGCATGCAGCCTC-3'). This method was also used to introduce a point mutation at codon 209 (Cys to Ser) in pHM6-Cezanne 1–858 (sense, 5'-GGAGATGGGAACAGCCTCCTGCATGCAG-3').

QuikChange PCR using this primer set was unsuccessful when pG-BKT7-Cezanne 17–443 was used as template. An alternative strategy was employed, which involved amplification of two overlapping products (using primers 5'-GCTGGAATTCACCCTGGACATGGATGCTGTTC-3' and 5'-CTGAATCAAGTAGGCTGTCTCCTGCAGTCC-3' or 5'-GGACTGCAGGAGACAGCCTACTTGATTCAG-3' and 5'-GGGTTTGTCGACTCAGAACCTGTGCACCAGGAG-3', respectively). The two products were then denatured and annealed, and 3' ends were extended using polymerase. cDNA corresponding to residues 17–443 was then amplified by PCR and inserted into pGBKT7, following restriction with EcoRI and SalI. DNA sequencing was used to ensure that each construct was inserted in frame with N-terminal residues and to verify changes at codon 209.

Yeast Two-hybrid System—The yeast two-hybrid system was performed according to the manufacturer's recommendations (Clontech). In the first instance, it was ensured that co-transformation of yeast strain YRG-2 with various pGBKT7-Cezanne constructs (17–858, 17–443, 444–858, 17–443 C209S, 17–858 C209A, or 17–443 C209A) plus empty pGAD424 would not generate colony growth on minimal medium lacking Trp, Leu, and His in the presence of 5 mM 3-amino-1,2,4-triazole (–Trp, –Leu, –His, +3AT). The cDNA library used was derived from whole mouse embryos and cloned into pGAD424 in frame with the activation domain of GAL4 (AD) using methods described previously (12). This library was screened using pGBKT7-Cezanne 17–858, and clones containing putative interacting proteins were identified by growth on minimal medium lacking essential amino acids (–Trp, –Leu, –His, +3AT) and by expression of {beta}-galactosidase. pGAD424 plasmid DNA was harvested from positive clones and amplified using Escherichia coli as host. Each plasmid was then used to retransform yeast together with either pGBKT7-Cezanne 17–858 or empty pGBKT7. Colony growth on minimal medium lacking Trp and Leu was assessed to ensure that the transformation efficiency using either construct was identical. Clones that reacted exclusively with pGBKT7-Cezanne 17–858 were sequenced.

In Vitro Peptidase and Isopeptidase Assays—In vitro hydrolysis assays were performed by incubating potential proteases with substrate in reaction buffer (50 mM Hepes (pH 7.8), 0.5 mM EDTA, 0.01% Brij, 3 mM dithiothreitol; 40-µl total volume) at 37 °C for varying lengths of time.

For peptidase assays, a fluorescent substrate was used (Arg-Leu-Arg-Gly-Gly-AMC (Bachem)) at a final concentration of 10 µM. Hydrolysis of the peptide bond between Gly and AMC was assessed over time using a fluorimeter (Cytofluor II; PerSeptive Biosystems). Test material was prepared as follows: E. coli (Rosetta, Novagen) transformed with pDEST (15)126–455 or pDEST (15)182–455 were cultured in the presence of 0.4 mM IPTG for 6 h to induce expression of GST fusion proteins (or remained untreated as a control). Bacteria from each 50-ml culture were harvested and lysed in 1 ml of BugBuster (Novagen), 0.04% {beta}-mercaptoethanol, 25 units of benzonase (Novagen), 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 mM EDTA and clarified by centrifugation (20,000 x g for 10 min at 4 °C). Hydrolysis reactions were performed using 5 µl of lysate, which gave a final concentration of each GST fusion protein of 15 nM (estimated by Coomassie staining of lysates alongside standards of known concentration, following gel electrophoresis).

For isopeptidase assays, a mixed population of branched polyubiquitin chains polymerized through Lys48 (Affiniti Research Products) was used as substrate at a final concentration of 1 µM. Preparation of test material was carried out as follows. (i) GST-Cezanne 126–455, GST-Cezanne 182–455, and a GST-tagged irrelevant molecule (Vav2; kindly supplied by Dr. M. Turner, Babraham Institute) were harvested from E. coli as described earlier. They were either tested in crude form (denoted as lysate) or purified using a glutathione column (Amersham Biosciences). Purified material was eluted from the column using 20 mM glutathione, 0.1 M Tris (pH 8), NaCl, 1 mM dithiothreitol. Material was then used either directly in isopeptidase assays (denoted as purified) or used after dialysis against PBS (denoted as purified/dialyzed). Analysis of the purified material by gel electrophoresis/silver staining identified only trace amounts of impurities (<1%), which were present at identical levels in each preparation (data not shown). All forms of the GST fusion protein or isopeptidase T (Affiniti Research Products), which served as a positive control, were tested at a final concentration of 15 nM. In some experiments, the GST tag was removed from GST-Cezanne 126–455 protein by digestion with tobacco etch virus protease (Invitrogen). Material was then loaded onto a glutathione column to remove undigested fusion protein and released GST, before final purification of Cezanne 126–455 by gel filtration on a Superose75 column (Amersham Biosciences). (ii) Alternatively, HA-tagged molecules were tested after immunoprecipitation from COS7 cells. Cultures in 25-cm2 flasks were transfected with various pHM6 constructs using DEAE-dextran, according to standard protocol. After 40 h, cells were washed using PBS before application of lysis buffer (20 mM Tris (pH 7.5), 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2) plus reversible protease inhibitors (10 µM EDTA, 10 µM leupeptin, 1 µM pepstatin, and 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride). Lysates were cleared by centrifugation and precleared using 150 µl of Sepharose 4B. HA-tagged molecules were then immunoprecipitated using 12 µl of anti-HA antibody bound covalently to matrix (Roche Applied Science). Beads were then washed twice using lysis buffer containing 150 mM NaCl; once using lysis buffer containing 500 mM NaCl; and then three times using reaction buffer (see above) before 50% were used in hydrolysis reactions. For inhibition of deubiquitinating activity, Cezanne was preincubated in the presence of 2 mM ubiquitin aldehyde (Affiniti Research Products) for 30 min at 37 °C. Following hydrolysis, ubiquitin was revealed by Western blotting using anti-ubiquitin antibody (1:1000; Zymed Laboratories Inc.), horseradish peroxidase-conjugated secondary antibody (Sigma), and chemiluminescent detection (Pierce).

Assay of Deubiquitination in Cultured Cells—HeLa cells cultured in 60-mm dishes were co-transfected with 0.5 µg of pcDNA3.1-FLAG-ubiquitin plus 2.5 µg of various pHM6-Cezanne constructs (or with empty pHM6) using Genejuice, following the manufacturer's recommendations (Novagen). A relatively low amount of FLAG-ubiquitin expression vector was used to ensure that proteins modified with FLAG-ubiquitin were derived from cells that also expressed the HA-tagged molecule. After 48 h, cells were treated with 20 µM MG132 for 1 h (or remained untreated). Cell lysates were then analyzed by Western blotting using anti-FLAG (1:500; Sigma) or anti-HA (1:1000; Roche Applied Science) primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and chemiluminescent detection. To control for the total amounts of protein loaded, blots were stripped and reprobed using anti-{alpha}-tubulin (1:1000; Sigma).

Co-immunoprecipitation of Ubiquitinated Cellular Proteins—COS7 cells cultured in 75-cm2 flasks were transfected with a pHM6 expression vector containing a HA-tagged version of Cezanne (as indicated) or with empty pHM6 (vector), using DEAE dextran. After 24 h, cells were treated with 20 µM MG132 for 1 h (or remained untreated, as indicated) before the addition of lysis buffer (see earlier) plus irreversible protease inhibitors (Roche Applied Sciences). Lysates were cleared by centrifugation and precleared twice using 50 µl of Sepharose 4B. HA-tagged molecules were immunoprecipitated using 30 µl of anti-HA matrix (Roche Applied Science). Beads were then washed four times using lysis buffer containing 150 mM NaCl. The levels of immunoprecipitated Cezanne protein were revealed by silver staining following gel electrophoresis, according to the manufacturer's recommendations (Bio-Rad). Co-immunoprecipitating material was detected by Western blotting using anti-ubiquitin antibody (Zymed Laboratories Inc.), horseradish peroxidase-conjugated secondary antibody (Sigma), and chemiluminescent detection (Pierce).

Sequence Structure Homology Recognition—Sequence data base searching was carried out using Psi-BLAST (available on the World Wide Web at www.ncbi.nlm.nih.gov) with the NCBI nonredundant data base, and multiple sequence alignments were generated using ClustalW (available on the World Wide Web at www.ebi.ac.uk/clustalw/). Secondary structure predictions were obtained from the programs PHD (available on the World Wide Web at cubic.bioc.columbia.edu/predictprotein/) and PSIPRED (available on the World Wide Web at bioinf.cs.ucl.ac.uk/psipred/). Structure-based sequence alignments were generated using COMPARER (13) with the resultant output formatted with JOY (14). Multiple sequence alignment of A20 family and ovarian tumor protein (OTU) superfamily sequences together with the protease structures was then created using FUGUE (15). Comparative models were constructed using Modeler (16) for Cezanne and other family members on the basis of known structures of homologous enzymes and their precursors. These were used to optimize the alignment and increase confidence about the location of the N and C termini of the protease region of Cezanne.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cezanne Cleaves Polyubiquitin Gene Products—Yeast two-hybrid screening of a murine whole embryo cDNA library with full-length Cezanne showed that 29 clones of 93 analyzed contained polyubiquitin genes. Start points of polyubiquitin cDNA varied between clones and were in frame with the GAL4 AD. Neither polyubiquitin nor Cezanne bound components of the GAL4 transcription factor, thus demonstrating that the interaction between Cezanne and polyubiquitin was genuine (data not shown).

Data base searches have previously identified weak sequence similarity between the N-terminal region of Cezanne and molecules related to Drosophila OTU (17) (see Supplementary Figure). OTU is the founder member of a superfamily of over 80 proteins predicted to be cysteine proteases. They contain conserved cysteine and histidine residues in a putative catalytic site. For Cezanne, the residues are Cys209 and His373 (Fig. 1). A proteolytic function for OTU-like molecules has not been previously demonstrated.



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FIG. 1.
Domain composition of Cezanne. Amino acid sequences were analyzed using BLAST (available on the World Wide Web at www.ncbi.nlm.nih.gov), PFAM (www.sanger.ac.uk), and ProSite (ca.expasy.org) programs. The position of recognized domains is represented together with the amino acid number. The region of A20 homology, core catalytic domain, nuclear localization sequence (NLS), and zinc (Zn) finger are depicted.

 

The yeast two-hybrid system was used to investigate whether the putative catalytic cysteine of the proposed protease domain (see Fig. 1) may effect the interaction with polyubiquitin that we observed in our initial screen. The ability of this region of Cezanne (residues 17–443) to interact with polyubiquitin was enhanced considerably by mutating cysteine 209 to serine (Fig. 2, A and B) or to alanine (Fig. 2C). This result is consistent with the interpretation that the mutation prevented proteolytic activity and trapped the substrate, thus enhancing activation of yeast two-hybrid reporter genes. Interestingly, the full-length version of wild-type Cezanne (residues 17–858) yielded a higher colony count when co-transformed with polyubiquitin than the putative protease domain alone (residues 17–443) (Fig. 2B).



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FIG. 2.
Cezanne binds and cleaves the polyubiquitin gene product. A–C, yeast strain YRG-2 was co-transformed with a pGBKT7 construct containing full-length, truncated, or mutated versions of Cezanne (residues indicated) plus pGAD424-polyubiquitin B. {beta}-Galactosidase reporter gene expression was measured when yeast colony growth on each plate was at the maximal rate (days 4–6), as assessed by daily monitoring of colony numbers. Yeast colonies expressing reporter genes were identified (A–C) and counted (B and C). Transformation efficiency was identical within a single experiment but varied between experiments. The results are representative of three closely similar experiments. D, E. coli were transformed with an IPTG-inducible expression vector containing two lengths of sequence encompassing the putative catalytic domain of Cezanne (residues 126–455 or 182–455, as indicated) and then cultured in the presence or absence of IPTG for 6 h. E. coli lysates were tested in vitro against Arg-Leu-Arg-Gly-Gly-AMC (a fluorescent analogue representing the polyubiquitin cleavage site), and release of AMC was measured using a fluorimeter. Lysates from transformed E. coli that were not treated with IPTG did not yield fluorescence (data not shown).

 

A separate yeast two-hybrid screening experiment was performed using the C-terminal half of Cezanne as bait (residues 444–858). A total of 90 clones of 92 analyzed contained polyubiquitin genes, which were in frame with the GAL4 AD (data not shown). Analysis of the amino acid sequence of Cezanne 444–858 revealed several structural features (see Fig. 1), but sequence similarity to known ubiquitin-binding domains was not identified. We conclude, therefore, that Cezanne 444–858 may contain a novel type of binding element for ubiquitin. Further experiments demonstrated that the C-terminal region generated relatively few colonies compared with the inactive protease domain (residues 17–443, C209S) suggesting that, in relative terms, it may have a lower affinity for ubiquitin (Fig. 2B).

The greater number of colonies seen with the full-length version compared with the N-terminal region in isolation (residues 17–443) could be explained by a synergistic relationship between binding sites in the C-terminal and N-terminal halves. Alternatively, the C-terminal region may inhibit proteolytic activity, and this would have the effect of increasing colony number. When mutated versions of full-length and truncated sequences were compared, colony counts were closely similar (Fig. 2C). This suggests that inactivation of the catalytic cysteine was sufficient to allow maximal binding to polyubiquitin and full activation of yeast two-hybrid reporter genes.

Although UCH and UBP molecules have different substrate preferences, both types target the peptide bond at Gly76, which links ubiquitin units in tandem. Both have the ability to cleave short model substrates (18). Therefore, to assess directly whether Cezanne has proteolytic activity, we tested its ability to cleave a fluorophore from a short peptide corresponding to the C terminus of ubiquitin (Fig. 2D). Lysates from E. coli expressing a version of the putative protease domain (residues 126–455), cleaved the fluorophore AMC from Arg-Leu-Arg-Gly-Gly-AMC. A shorter version of the catalytic domain, Cezanne 182–455, had little or no activity. Cezanne 126–455 corresponds to the region that displays similarity to the tumor necrosis factor receptor-associated factor-binding domain of A20 (19), whereas Cezanne 182–455 corresponds to the predicted core catalytic domain that displays structural similarity to papain (Fig. 1 and Supplementary Figure). Although it is plausible that residues 126–181 are critical for substrate binding or regulation of hydrolysis, it is equally possible that the conformation of the active site of Cezanne 182–455 may have been compromised by aberrant folding. The lack of reactivity of endogenous E. coli proteins toward this substrate was not surprising, because prokaryotes lack ubiquitin hydrolase genes.

Cezanne Hydrolyzes Branched Polyubiquitin Chains—The potential ability of Cezanne to cleave branched polyubiquitin chains was examined (Fig. 3A). Hydrolysis experiments in vitro demonstrated that a GST-Cezanne 126–455 fusion protein cleaved {epsilon}-amino-linked polyubiquitin chains into monomers (lanes 4, 7, and 10). This activity was present in crude E. coli lysates containing Cezanne 126–455 (lane 4) and was elevated in purified material (lanes 7 and 10). The deubiquitinating activity observed in this experiment must be attributed to Cezanne sequences, because it was not observed using an irrelevant GST fusion protein either purified (lanes 5 and 8)orin the presence of endogenous E. coli proteins in the crude lysate (lane 2). The shortened version of the protease domain (Cezanne 182–455) had little isopeptidase activity against branched polyubiquitin chains (lanes 3, 6, and 9). Cezanne's activity was suppressed by ubiquitin aldehyde, a highly specific inhibitor of deubiquitinating enzymes (Fig. 3B).



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FIG. 3.
Cezanne cleaves free, isopeptide-linked polyubiquitin chains. A, two versions of the catalytic domain of Cezanne (182–455 or 126–455) or an irrelevant molecule (control) were expressed in E. coli as GST-tagged proteins. Crude E. coli lysate, purified protein, or purified/dialyzed protein were tested (see "Experimental Procedures"). The relative amount of protein in stocks of purified or purified/dialyzed material was assessed by Coomassie staining (lower panel). Test samples containing equivalent quantities of GST-tagged protein were incubated at 37 °C with an excess of branched polyubiquitin chains (Ub). Purified isopeptidase-T served as a positive control (Iso.T), whereas polyubiquitin chains incubated alone were a negative control (No enzyme). Following hydrolysis, ubiquitin was revealed by Western blotting using anti-ubiquitin antibody. Cleavage of polyubiquitin into the monomeric form was observed for Cezanne 126–455. These data are representative of six closely similar experiments. B, equivalent quantities of purified Cezanne 126–455 or isopeptidase T (Iso.T) were preincubated in the presence or absence of ubiquitin-aldehyde as indicated. An excess of branched polyubiquitin chains (Ub) was then added before further incubation for 30 min. Polyubiquitin chains incubated alone were a negative control (no enzyme). Following hydrolysis, ubiquitin was revealed by Western blotting using anti-ubiquitin antibody. Note that monomeric ubiquitin migrated out of this gel.

 

The C-terminal Half of Cezanne Regulates Deubiquitination of Branched Polyubiquitin Chains—Hydrolysis experiments were performed to examine the influence of the C-terminal half of Cezanne on deubiquitination of branched polyubiquitin chains and to examine our prediction that Cys209 is a catalytic residue.

We compared full-length Cezanne and the isolated protease domain alone for the ability to cleave a synthetic substrate of branched polyubiquitin chains using HA-tagged versions of Cezanne immunoprecipitated from transfected COS7 cells. The full-length molecule was considerably more effective than the protease domain alone (Fig. 4, compare lanes 1 and 3). The mutation C209S abolished activity in the full-length molecule, confirming that Cys209, which is highly conserved between members of the OTU superfamily, is a catalytic residue (compare lanes 1 and 2). We observed very little activity using isolated protease domain immunoprecipitated from COS7 cells compared with that seen using preparations purified from E. coli (Fig. 3). We attribute this difference to the much greater concentration of protease domain used in the latter experiments.



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FIG. 4.
The C-terminal half of Cezanne regulates polyubiquitin hydrolysis. Various HA-tagged versions of Cezanne were immunoprecipitated from transfected COS7 cells and tested in hydrolysis reactions using an excess of branched polyubiquitin chains (Ub). Precipitated material from cells transfected with empty expression vector served as a negative control (vector). Purified isopeptidase-T was a positive control (Iso.T), whereas polyubiquitin chains incubated alone were a second negative control (No enzyme). Following hydrolysis, ubiquitin was revealed by Western blotting using anti-ubiquitin antibody (lower panel). It is possible that the substrate contained trace amounts of a circularized form of polyubiquitin (*), which is known to be resistant to cleavage by isopeptidase T. The efficiency of each precipitation was assessed by Western blotting using anti-HA antibody (upper panel).

 

Cezanne Deubiquitinates Cellular Proteins in Cultured Cells—To investigate the effect of Cezanne on ubiquitinated cellular proteins, we co-transfected HeLa cells with expression vectors containing HA-tagged versions of Cezanne and FLAG-tagged ubiquitin (at a ratio of 5:1; Fig. 5A). After 48 h, cells were either treated with a proteasome inhibitor (MG132) for 1 h or remained untreated. Levels of ubiquitinated cellular proteins in experimental and control cultures were then compared by Western blotting for FLAG.



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FIG. 5.
Cezanne deubiquitinates cellular proteins in cultured cells. A, HeLa cells were co-transfected with pcDNA3.1-FLAG-ubiquitin (0.5 µg) plus a pHM6 construct (2.5 µg) containing an HA-tagged version of Cezanne (residues as indicated) or empty pHM6 (No Cezanne). Untransfected cells served as a control (Untransfected). After 48h, cells were treated with 20 µM MG132 for 1 h or remained untreated, as indicated. Cell lysates were tested by Western blotting using anti-FLAG to detect residual ubiquitinated proteins or with anti-HA antibodies to detect Cezanne. Blots were stripped and reprobed using anti-{alpha}-tubulin to assess loading. Two independent experiments are shown (lanes 1–10 and lanes 11–16, respectively). B, COS7 cells were transfected with a pHM6 construct containing an HA-tagged version of Cezanne (residues as indicated) or with empty pHM6 (vector). After 24 h, cells were treated with 20 µM MG132 for 1 h or remained untreated, as indicated. HA-tagged molecules were immunoprecipitated from COS7 cell lysates using anti-HA antibody coupled to beads. Co-immunoprecipitating polyubiquitinated proteins were detected by Western blotting using anti-ubiquitin antibody. Levels of immunoprecipitated Cezanne were assessed by silver staining. The position of Cezanne is indicated (*).

 

Full-length Cezanne prevented the buildup of ubiquitinated cellular proteins in response to MG132, whereas a catalytically inactive version had no effect (Fig. 5A, compare lanes 1 and 3 or lanes 13 and 15). This implies that Cezanne has the capacity to deubiquitinate at least part of the cellular pool of ubiquitinated proteins. Expression of the catalytic domain alone had only a marginal effect on ubiquitination levels (compare lanes 1 and 5). Thus, the C-terminal part of Cezanne is required for efficient hydrolysis of both free and conjugated forms of branched ubiquitin chains (Figs. 4 and 5).

We next examined the capacity of the mutated full-length molecule to bind to polyubiquitinated proteins in cultured cells (Fig. 5B). Ubiquitinated cellular proteins could be co-immunoprecipitated with a catalytically inactive version of full-length Cezanne (lane 6) but not with the wild-type molecule (lane 5). It is likely that the catalytically inactive version of Cezanne functions as a substrate-trapping mutant, whereas the active version would not be expected to retain polyubiquitin after hydrolysis.

Cezanne Is a Novel Type of Deubiquitinating Enzyme—Data base searching and sequence analysis was carried out to examine the relationship between members of the A20 group, the OTU-superfamily, Type I and Type II deubiquitinating enzymes, and other cysteine proteases (Fig. 6 and Supplementary Figure). Secondary structure predictions for the A20 family show close agreement to the actual structure of papain-like cysteine proteases, despite low sequence identity between these groups (Supplementary Figure). Multiple sequence alignment of the putative protease regions of the A20 family, however, revealed that Cezanne shares 39 and 25% sequence identity with A20 and TRABID, respectively. The similarity is particularly evident in the areas containing putative catalytic residues (Cys and His regions) (Fig. 5). Makarova et al. (17) suggest that the catalytic site of OTU proteins may comprise a triad of conserved Cys, His, and Asp residues (209, 373, and 206; Cezanne numbering). It is notable that TRABID does not contain the conserved Asp. We emphasize that, with the exception of Cezanne, empirical evidence for proteolytic activity is lacking among other A20 and OTU family members. The Cys and His regions of the A20 family display a significant level of sequence identity with OTU-like molecules but not to the catalytic regions of Type I or Type II deubiquitinating enzymes, apart from conservation of active site cysteine and histidine residues. We conclude that Cezanne is a novel type of deubiquitinating enzyme.



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FIG. 6.
Cys and His regions of Cezanne are distinct from catalytic regions of UCH and UBP molecules. Three multiple sequence alignments were made of putative catalytic regions of representative members of the OTU superfamily (this includes the A20 group), the UCH family, or the UBP family. Accession codes are given, and residue numbers are indicated. All sequences are human except for AAD31534 [GenBank] (mouse UCH37). Hin-1 was compiled using several GenBankTM entries. Text is highlighted for conserved putative catalytic residues (white on black background), identical residues (dark gray background), and residues with similar properties (light gray background).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have demonstrated that Cezanne has deubiquitinating activity against both linear and branched forms of polyubiquitin. We compared the activity of Cezanne with isopeptidase T, a well characterized UBP molecule that is known to hydrolyze the branched form of polyubiquitin chains in preference to linear forms (5). Although we have not yet analyzed the kinetics of Cezanne deubiquitination in detail, we noted from several experiments that equimolar quantities of Cezanne and isopeptidase T hydrolyzed free, branched polyubiquitin chains at similar rates. The activities of both molecules were suppressed by ubiquitin aldehyde, a highly specific inhibitor of deubiquitinating enzymes.

The catalytic domain of Cezanne is present in the N-terminal half. Yeast two-hybrid experiments provided preliminary evidence for an additional ubiquitin binding site in the C-terminal part of the molecule. Full-length Cezanne hydrolyzed free and conjugated forms of branched polyubiquitin chains more efficiently than the catalytic domain alone. These observations are consistent with the idea that a C-terminal ubiquitin binding element may contribute to the avidity of Cezanne for the branched form of polyubiquitin. Alternatively, this motif may be required for optimal orientation of polyubiquitin chains with the catalytic site. Other proteases are known to contain substrate-binding sites outside the catalytic region, otherwise known as exosites. Interestingly, prothrombinase contains an exosite that is responsible for the initial interaction of this protease with its substrate (20). The anchored substrate is then cleaved at two spatially separated sites. By analogy, it is conceivable that binding of the C terminus of Cezanne to a branched polyubiquitin chain may enable cleavage of multiple ubiquitin units in processive fashion.

The presence of two ubiquitin binding sites could also explain our observation, using yeast two-hybrid analysis, that full-length Cezanne generated more colonies when co-transformed with polyubiquitin than the protease domain in isolation. A synergistic relationship between binding sites in the N- and C-terminal halves would have the effect of increasing the avidity of Cezanne for linear polyubiquitin chains. An alternative explanation is that the C-terminal half may inhibit hydrolysis of the linear form of polyubiquitin, which would also increase the number of colonies generated. The latter model implies that the C-terminal half may have opposing effects on the hydrolysis of different structural forms of polyubiquitin (i.e. it may inhibit cleavage of linear forms but promote cleavage of branched polyubiquitin chains). A precedent for this mode of regulation is provided by a study of a Type II deubiquitinating enzyme, UBP-t (21). A region outside the catalytic domain of this molecule was found to regulate the preference for linear or branched polyubiquitin substrates. At present, we cannot distinguish whether the C-terminal half of Cezanne regulates binding to or hydrolysis of the linear form of polyubiquitin.

Our experiments demonstrated that Cezanne can cleave branched polyubiquitin chains from a substantial proportion of cellular proteins. We cannot, however, draw firm conclusions about substrate specificity at this stage. These studies have relied on overexpression of Cezanne in cell lines, and it is quite possible that in such circumstances substrate specificity will be broadened well beyond the normal physiological range. The domain composition among deubiquitinating enzymes is diverse, and it has been proposed that regions outside the catalytic domain may target specific cellular proteins for deubiquitination. This concept has been established for several UBP molecules including HAUSP, which targets the apoptosis regulator p53 (22, 23). Substrate specificity remains poorly defined, however, for the great majority of ubiquitin hydrolases. The capacity of Cezanne to interact with other cellular proteins that could focus its activity has not yet been investigated in any depth.

Our previous study revealed that transcripts for Cezanne are expressed widely and are particularly abundant in fetal liver and kidney (11). We have also recently found that Cezanne is expressed differentially among B-cells at distinct developmental stages (reviewed in Ref. 24). Primary immature B-cells (IgM+, IgD–) contained 100-fold more Cezanne mRNA than their mature counterparts (IgM–, IgD+) as assessed by real time PCR using {beta}-actin transcripts as a standard.2 The potential role of Cezanne during B-cell development awaits further investigation. It is possible that the capacity of Cezanne to suppress NF-{kappa}B, a powerful survival factor, may influence the fate of immature B-cells.

Ubiquitin plays an essential role at several levels during NF-{kappa}B signaling, the best characterized being ubiquitination and degradation of I{kappa}B{alpha} (25). Ubiquitination also provides the signal for cleavage of NF-{kappa}B precursors into active forms (26) and activation of tumor necrosis factor receptor-associated factor- and transforming growth factor-{beta}-activated kinase (TAK1) (27, 28) signaling intermediaries. We have previously demonstrated that Cezanne has the capacity to suppress NF-{kappa}B and are currently investigating the intriguing possibility that this may be mediated through deubiquitination of key signaling intermediaries.

The amino acid sequence of the protease domain of Cezanne is similar to corresponding regions found in A20 and TRABID (11). In addition, we have recently discovered a close homologue of Cezanne, which we have named Cezanne-2 (GenBankTM accession number NM_130901 [GenBank] ). The catalytic cysteine and histidine residues of Cezanne are conserved throughout the group. Our discovery of enzymatic activity for Cezanne suggests strongly that proteolysis may be a common function throughout this family. We are currently testing the potential of other A20 family members to function as deubiquitinating enzymes.

Our study also provides the first demonstration of proteolytic activity in the OTU superfamily. It remains to be established whether other members of the OTU superfamily can also function as deubiquitinating enzymes, but it is highly likely that at least one other human OTU family member does have this activity (29). This molecule, called HSPC263, was identified recently by tandem mass spectrometry after capture using an alkylhalide-modified ubiquitin probe, which reacted with the putative active site cysteine. We propose, therefore, that Cezanne and HSPC263 should be classified as founder members of a Type III or OTU-type family of deubiquitinating enzymes.


    FOOTNOTES
 
* 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

The on-line version of this article (available at http://www.jbc.org) contains an additional figure. Back

|| Present address: Astex Technology Ltd., Cambridge, United Kingdom. Back

§ A Fellow of the National Kidney Research Fund, UK. To whom correspondence should be addressed: Molecular Immunology Programme, The Babraham Institute, Cambridge CB2 4AT, United Kingdom. Tel.: 44-1223-496633; Fax: 44-1223-496023; E-mail: paul.evans{at}bbsrc.ac.uk.

1 The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; UCH, ubiquitin C-terminal hydrolase; UBP, ubiquitin-specific processing protease; AMC, 3-amino-1,2,4-triazole; AD, Gal-4 activation domain; IPTG, isopropyl-{beta}-D-thiogalactosidase; HA, hemagglutinin; GST, glutathione S-transferase; OTU, ovarian tumor protein. Back

2 P. C. Evans, M. Turner, and P. J. Kilshaw, unpublished observations. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Alan Barrett (MEROPS group, The Sanger Institute, Cambridge) and members of the Barrett laboratory for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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