From the
Derald H. Ruttenberg Cancer Center and
Department of Human Genetics, The Mount Sinai
School of Medicine, New York, New York 10029-6574,
¶Department of Biochemistry, Emory University,
Atlanta, Georgia 30322, ||Department of
Biochemistry and Molecular Biology, University of Medicine and Dentistry of
New Jersey-New Jersey Medical School, Newark, New Jersey 07103, and
**Department of Molecular, Cellular, and Developmental
Biology, Yale University, New Haven, Connecticut 06520
Received for publication, March 20, 2003 , and in revised form, May 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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Using in vitro systems, several studies have shown that Nedd8
activates the ubiquitination of IB
(8) or p27
(9), through its conjugation to
cullin 1 (CUL1). These reactions are mediated by SCF E3 Ub ligases, in which
CUL1 functions as a molecular scaffold
(1012).
Subsequently, it was observed that degradation of HIF-
by von
Hippel-Lindau tumor suppressor required Nedd8
(13). In this case, Nedd8 was
conjugated to CUL2 that assembles the von Hippel-Lindau protein E3 Ub ligase
(reviewed in Ref. 14). These
studies thus suggest a role for Nedd8 in the assembly of an active
cullin-based E3 Ub ligase.
We initially reported that conjugation of Nedd8 to CUL1 increases the
ability of ROC1-CUL1, a sub-complex within the SCF E3 Ub ligase, to assemble
polyubiquitin chains in a reaction catalyzed by the Cdc34 E2 conjugating
enzyme (15). Subsequently, we
showed that the activation function of Nedd8 in polyubiquitin chain assembly
is critically dependent on its charged surface residues, suggesting that Nedd8
mediates electrostatic interactions to facilitate the recruitment of an E2
(16). Consistent with these
observations, it was shown that immunoprecipitates containing exclusively
neddylated forms of cullin were more active in assembling polyubiquitin chains
(17,
18). Furthermore, Nedd8
conjugation was found to increase the interaction between
SCFTRCP and the Ubc4
S
Ub conjugate
(19). More recently, x-ray
crystallographic studies have predicted that the covalent attachment of Nedd8
at CUL1 lysine residue 720 (Lys-720CUL1) positions Nedd8 within
close proximity of ROC1/Rbx1
(20), a subunit whose function
is to recruit an E2
(2123).
These studies support a model suggesting that Nedd8 activates ubiquitination
by increasing the recruitment of an E2, thereby facilitating the assembly of
polyubiquitin chains. Additionally, recent studies
(24,
25) suggest a role of
neddylation in disrupting an interaction between CUL1 and an inhibitor called
p120CAND1.
Rather unexpectedly, studies from Lyapina et al. (17) and Schwechheimer et al. (26) have demonstrated a critical role for COP9 signalosome (CSN) in promoting the cleavage of Nedd8 from CUL1. CSN, an eight-subunit complex, was originally identified as a suppressor of plant photomorphogenesis (reviewed in Ref. 27). In a more recent study, Cope et al. (28) showed that the Nedd8 isopeptidase activity is dependent on the MPN/JAMM domain within the Jab1 (CSN-5) subunit of CSN, suggesting that CSN acts as a metalloprotease. Surprisingly, while characterizing proteolytic activities from HeLa extracts that deconjugated Nedd8 from CUL1, we purified a novel cysteinyl protease we called DEN1 (human deneddylase 1). DEN1 selectively binds Nedd8, efficiently processes the C terminus of Nedd8, and deconjugates hyper-neddylated CUL1. These data suggest a role for DEN1 in regulating the Nedd8 pathway.
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EXPERIMENTAL PROCEDURES |
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To identify DEN1, the peak fraction (number 21) from the Superdex 75 column was trichloroacetic acid-precipitated and size-fractionated by 420% SDS-PAGE followed by Coomassie staining. Each band was then excised and analyzed via mass spectrometry as described previously (30). One detected tryptic peptide, with the sequence QQTESLLQLLTPAYITK, was matched to AAH31411 [GenBank] .1 (see Fig. 2, AC).
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DEN1 Cloning, Expression, and Antibody Preparation
DEN1 was cloned by PCR from a human fetal brain cDNA library (a gift from
A. Chan, The Mount Sinai School of Medicine) using the sense
5'-caccctggttccgcgtggatccatggaccccgtagtcttg-3' primer (the
bold sequences encode a thrombin protease cleavage site) and the antisense
5'-ctactttttagcaagtgtgg-3' primer designed based on the data base
sequence for AAH31411
[GenBank]
.1. The PCR fragment was inserted into the Invitrogen
Gateway entry vector pENTR/D-TOPO and verified by sequencing.
Recombinant DEN1 was expressed as a glutathione S-transferase
(GST) fusion protein in BL21 (DE3) cells using the Gateway destination vector,
pDEST 17 (Invitrogen). Cells were grown and induced, and extracts were
prepared as described previously for GST-UBC12
(15), except that 0.8
mM isopropyl-1-thio--D-galactopyranoside was used
for induction, and leupeptin, as well as antipain, were omitted from the lysis
buffer. Glutathione-Sepharose purification was carried out as described
previously for GST-UBC12 (15),
except with buffers that lacked protease inhibitors.
To prepare GST-free DEN1, GST-DEN1 was incubated with biotinylated thrombin (1 unit/mg of protein; Novagen) at room temperature overnight. The thrombin and cleaved GST were then removed by passing the reaction mixture first through streptavidin-Sepharose and then glutathione-Sepharose beads (Amersham Biosciences). The cleaved DEN1 was further purified by fast protein liquid chromatography on the Superdex 75 gel filtration column. Approximately 37.5 mg of pure DEN1 was obtained per liter of culture.
Anti-DEN1 polyclonal antibody was prepared using purified, GST-free DEN1 as
antigen (Covance). To affinity purify the anti-DEN1-specific antibody, serum
(10 ml) was incubated with Affi-15 beads (0.5 ml; Bio-Rad) that had been
cross-linked with purified GST-DEN1 (4 mg of protein per ml of beads).
After extensive washing, bound antibody was eluted with 0.1 M
glycine, pH 2.5, immediately followed by neutralization with Tris-HCl, pH
8.3.
Substrate Preparation
PK-Nedd8 The pET3a PK-Nedd8 expression plasmid was
generated by inserting a DNA linker containing the cAMP-dependent kinase motif
(LRRASV) sequence and flanking NdeI restriction site compatible ends
(sense 5'-tatgcttagacgagcttctgtgcc-3', antisense
5'-tagggcacagaagctcgtctaagca-3') into the pET3a Nedd8 plasmid (a
kind gift from C. Pickart, The Johns Hopkins University) at the NdeI
site. PK-Nedd8 expression and purification were carried out as described for
the wild type Nedd8 (15),
except PK-Nedd8 was found in the soluble fraction. The extracts containing
PK-Nedd8 were first passed through Q-Sepharose (Amersham Biosciences).
PK-Nedd8 bound to SP-Sepharose (Amersham Biosciences) was eluted with a
50500 mM NaCl gradient. The peak fraction, judged by
Coomassie staining following SDS-PAGE, was used for this study.
pro-Nedd8 The coding sequence for pro-Nedd8 was PCR-amplified from a human liver B-cell cDNA library (Stratagene). Primers contained an NdeI site at the initiator Met and a KpnI site after the stop codon. The PCR product was rescued into the TA vector (Invitrogen). The insert was excised by digestion with NdeI and KpnI and ligated into a similarly digested pRSET vector (Invitrogen). The protein was expressed in BL21 (DE3) cells and purified using the same protocol as published for ubiquitin. The protein was refolded before use by denaturation with 8 M urea followed by dialysis into 50 mM Tris, pH 7.6 (31).
SCFHA-Fbx22To prepare SCFHA-Fbx22, insect High Five cells (10 flasks; 150 mm2) were infected with baculoviruses expressing CUL1 (Y. Xiong, University of North Carolina at Chapel Hill), HA-tagged Fbx22 (GenBankTM accession number AY005144 [GenBank] ), His-tagged Skp1 (M. Pagano, New York University Medical School), and His-tagged ROC1. Cells were harvested, and Ni-NTA purification was carried out based on previously published procedures (32). The resulting SCFHA-Fbx22 was further purified through Q-Sepharose and Superdex 200 chromatography.
In Vitro Nedd8 ConjugationNedd8 was initially
phosphorylated in a reaction mixture (200 µl) that contained PK-Nedd8 (15
µg), 40 mM Tris-HCl, pH 7.4, 12 mM
MgCl2,2mM NaF, 50 mM NaCl, 25
µM ATP, 50 µCi of [-32P]ATP, 0.1 mg/ml
bovine serum albumin, and 20 units of cAMP kinase (Sigma). The reaction was
incubated at 37 °C for 30 min. To conjugate 32P-Nedd8 to CUL1,
a second reaction mixture was added to include 4 mM ATP,
APP-BP1/Uba3 (20 ng), 10 µg of Ubc12, and 12 pmol of
[HA-ROC1]-CUL1324776 (see
Fig. 5C, lane
1) (15) or 6 pmol of
SCFHA-Fbx22 (see Fig.
5C, lane 2). The reaction was further incubated
at 37 °C for 60 min. To purify the 32P-Nedd8-CUL1 conjugates,
the reaction mixtures were adsorbed to anti-HA antibody-linked agarose beads
(Sigma), and the bound protein complex was eluted by HA-peptide (2 mg/ml) in
Buffer A plus 1 mM DTT and 0.05 M NaCl.
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To prepare hyper-neddylated CUL1, purified SCFHA-Fbx22 (30 µg), immobilized on Ni-NTA (200 µl), was incubated in a solution (1.5 ml) containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM ATP, 0.6 mM DTT, APP-BP1/Uba3 (750 ng), GST-Ubc12 (30 µg), and Nedd8 (60 µg). After incubation at 37 °C for 60 min, the beads were washed, and the modified complexes were then eluted with 60 mM imidazole in Buffer A plus 1 mM DTT and 50 mM NaCl followed by dialysis and concentration. This procedure yielded hyper-neddylated SCFHA-Fbx22 (9.8 µg) (shown in Fig. 6B).
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Preparation of SUMO-1
Full-length cDNA coding for SUMO-1 was initially sub-cloned into pCRII
(Invitrogen) by PCR using two primers,
5'-GGATCCACCATGTCTGACCAGGAGGC-3' and
5'-GAATATCTAAACTGTTGAATGACCCCC-3'. The
BamHI-SpeI fragment containing SUMO-1 coding sequence was
then excised and inserted into pRSET-A (Invitrogen) that had been treated
sequentially with EcoRI, T4 DNA polymerase, and BamHI. The
positive pRSET-SUMO-1 clone, confirmed by DNA sequencing, was digested with
BamHI and subsequently ligated with a synthetic linker that encodes
for RRASV, the phosphorylation site by the cAMP-dependent kinase. The
synthetic linker was generated by annealing two primers,
5'-GATCTGGTACCCGTCGTGCATCTGTTA-3' and
5'-GATCTAACAGATGCACGACGGGTACCA-3'. SUMO-1 was expressed in BL21
(DE3) and purified using Ni-NTA as per the manufacturer's instructions.
Nedd8 Protease Assays
32P-Nedd8 Cleavage AssayThe reaction (10 µl)
contained 40 mM Tris-HCl, pH 7.4, 1 mM DTT, 0.1 mg/ml
bovine serum albumin, 2 mM NaF, 10 nM okadaic Acid,
purified 32P-Nedd8-CUL1 conjugates in amounts as indicated, and
DEN1 or CSN. The reaction was incubated at 37 °C for times as specified.
The reaction products were then separated by 420% SDS-PAGE and
visualized by autoradiography.
Nedd8 Hydrolase AssayThe reaction (20 µl) contained 40 mM Tris-HCl, pH 7.4, 1 mM DTT, 100 nM pro-Nedd8, and DEN1 or CSN in amounts as indicated. The reaction was incubated at 37 °C for times as specified. The reaction products were then separated by 16% SDS-PAGE and visualized by silver staining.
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RESULTS |
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To determine its molecular identity, the proteins contained in the DEN1
activity peak fraction, number 21, were separated by SDS-PAGE, and
individually excised polypeptides were analyzed by mass spectrometry. The
results showed that the protein band of 24 kDa contained a mixture of
tryptic peptides that corresponded to two proteins: thioredoxin peroxidase 1
and a previously uncharacterized protein, AAH31411
[GenBank]
.1/SENP8 (see
Fig. 1B, lane
3 and Fig. 2,
AC). Intriguingly, the predicted
AAH31411
[GenBank]
.1 open reading frame encodes for a protein that shares a strong
homology with the Ulp1 SUMO-1 family of isopeptidases
(33). Subsequent immunoblot
analysis, using polyclonal antibodies raised against the recombinant
AAH31411
[GenBank]
.1 protein, detected a polypeptide of 24 kDa that peaked coincident
with the DEN1 cleavage activity (Fig.
1C, lanes 35). These results, together
with the subsequent demonstration of Nedd8 protease activities by the
recombinant AAH31411
[GenBank]
.1 protein (see below), unequivocally identify that DEN1
is encoded by AAH31411
[GenBank]
.1.
As revealed by sequence analysis, DEN1 contains a His/Asp/Cys catalytic
triad, the signature motif for a large family of cysteinyl protease
(Fig. 2D)
(34). Further, homologues of
DEN1 are found in mouse, Drosophila, and Arabidopsis, the
latter two of which exhibit 35% sequence identity and
58% overall
amino acid conservation with the human counterpart. Taken together, these data
suggest that DEN1 is an evolutionarily conserved cysteinyl protease.
DEN1 Specifically Binds Nedd8 and Efficiently Processes the C Terminus of Nedd8 We first examined whether DEN1 interacted with Nedd8. For this purpose, DEN1 was expressed and purified as a GST fusion protein from bacteria. GST-DEN1 was then incubated with 32P-Nedd8 followed by addition of glutathione-Sepharose beads to sequester the fusion protein (Fig. 3A, bottom panel). After stringent washing, bound 32P-Nedd8 was released from the beads, electrophoresed through SDS-PAGE, and visualized by autoradiography. The results showed that GST-DEN1, but not GST, exhibited a remarkable affinity for Nedd8 (Fig. 3A, upper panel). Using this assay, we compared Nedd8 with Ub or SUMO-1 for their ability to interact with GST-DEN1 (Fig. 3B). The results, quantitated by phosphorimaging analysis, indicated that whereas GST-DEN1 bound greater than 24% of the Nedd8 input, it retained less than 1% of Ub or SUMO-1. Thus, DEN1 binds Nedd8 selectively.
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Next, we investigated the ability of DEN1 to process the C terminus of Nedd8. Nedd8 is synthesized as a precursor protein, -G75G76GGLRQ, which is then converted into the matured form, -G75G76, by a C-terminal hydrolytic activity. Only the mature Nedd8 is functional for being conjugated to cullin molecules, which leads to the production of active E3 ligases. To measure the Nedd8 C-terminal hydrolytic activity, the purified, bacterially expressed DEN1 (Fig. 4C, lane 1) was incubated with a Nedd8 precursor protein (pro-Nedd8) that contained the GGLRQ sequence. As revealed by silver staining/SDS-PAGE analysis, DEN1 catalyzed the conversion of a substantial portion of the substrate to the mature Nedd8 within 1 min of incubation (Fig. 4A, compare lanes 1 and 2). Notably, this reaction occurred at an enzyme/substrate ratio of 1:50. Further, significant hydrolytic activity was observed at an enzyme/substrate ratio of 1:500 (Fig. 4B, lane 3). From these results we conclude that DEN1 hydrolyzes C-terminal derivatives of Nedd8 catalytically.
Previous studies (17, 26) have demonstrated that CSN contains cullin-Nedd8 isopeptidase activity. For comparison studies, we affinity purified the CSN complex from 293 cells that had been engineered to constitutively express FLAG-CSN-2 and CSN-3-V5 (Fig. 4C, lane 2). However, no C-terminal hydrolytic activity was observed with CSN (Fig. 4B, lanes 68).
The observed maximal yield of cleavage was 50%
(Fig. 4A), which was
not increased by addition of excess enzyme (data not shown). Similar results
were obtained with UCH-L3 (data not shown; see Ref.
35). This is possibly because
of incomplete refolding of the substrate that was isolated from inclusion body
in bacteria following overexpression (see "Experimental
Procedures").
DEN1 Deconjugates Hyper-neddylated Forms of CUL1We analyzed the deconjugation activity of DEN1 by incubating the recombinant enzyme with the ROC1-CUL1324776-[32P-Nedd8] substrate, which contained predominantly the CUL1324776-Nedd81 conjugate (Fig. 5A, upper panel, lane 1). As shown, DEN1 almost completely converted the substrate to the monomeric 32P-Nedd8 (Fig. 5A, upper panel, compare lanes 1 and 4). Although this deconjugation reaction was both dose- and time-dependent (Fig. 5A, upper panel), it required addition of the enzyme in substantial molar excess relative to the input substrate.
To test whether the observed inefficient cleavage by DEN1 was because of the use of the CUL1 C-terminal fragment, 32P-Nedd8 was conjugated to the full-length CUL1 within the SCFHA-Fbx22 complex (Fig. 5C, lane 2), yielding predominantly the CUL1-Nedd81 conjugate (Fig. 5A, bottom panel, lane 5). When this substrate was used in a cleavage reaction, it was completely processed, resulting in accumulation of free 32P-Nedd8 (Fig. 5A, bottom panel, lane 8). Additionally, at similar concentrations, UCH-L3 did not cleave CUL1-Nedd81 (data not shown), despite its ability to hydrolyze the C-terminal derivatives of Nedd8 (see Ref. 35 and accompanying manuscript (41)) (data not shown). However, this reaction still required excess DEN1 (Fig. 5A, bottom panel, lanes 68). As CUL1-Nedd81 represents the conjugate formed between Lys-720CUL1 and Nedd8, these results suggest that under the conditions used, DEN1 did not cleave the Lys-720CUL1-Nedd8 isopeptide bond catalytically.
In contrast, the purified CSN complex catalyzed the deconjugation of Nedd8 from either CUL1324776 (Fig. 5B, upper panel) or full-length CUL1 (Fig. 5B, bottom panel), with an enzyme/substrate ratio as low as 1:500. These studies demonstrate the remarkable efficiency with which CSN cleaves the Lys-720CUL1-Nedd8 conjugate.
As shown above, the HeLa DEN1, when present in nanomolar quantity, was able to cleave CUL1324776-Nedd82 (Fig. 1A), suggesting that this protease may preferentially process Nedd8 conjugates that are not linked through Lys-720CUL1. To investigate this possibility, we prepared a substrate containing hyper-neddylated forms of CUL1. Immunoblot (Fig. 6A, lane 1) and silver staining (Fig. 6B) analysis revealed that hyperneddylated CUL1 contained predominantly CUL1-Nedd81 and CUL1-Nedd82, as well as low levels of CUL1-Nedd83 and the unmodified form. Remarkably, at an enzyme/substrate ratio of 1:10, DEN1 converted a majority of the substrate into the mono-neddylated form (Fig. 6A, compare lanes 1 and 2). Increasing the enzyme/substrate ratio to near stoichiometric level led to a further accumulation of the CUL1-Nedd81 conjugate without significantly increasing the levels of the unmodified CUL1 (Fig. 6A, lane 3). Eventually, when excess DEN1 was added, the mono-neddylated CUL1 was converted into the unconjugated form (Fig. 6A, lanes 4 and 5). These results established that at low concentrations, DEN1 efficiently processed hyper-neddylated CUL1 to yield the mono-neddylated form. When present at high levels, it cleaved the mono-neddylated conjugate to generate free CUL1, similar to that was observed with the substrate containing predominantly CUL1-Nedd81 (Fig. 5A).
Surprisingly, CSN did not efficiently cleave hyper-neddylated CUL1 (Fig. 6A, lanes 79). Note that in this experiment, the concentrations of CSN were identical to those used for efficient deconjugation of the mono-neddylated CUL1 (Fig. 5B, bottom panel). Thus, hyper-neddylated CUL1 appears to be a relatively poor substrate for CSN. Future studies are required to determine whether these two enzymes can function cooperatively in deconjugating hyper-neddylated CUL1. Initial attempts of depleting endogenous DEN1 by RNAi have not been successful, precluding an assessment of the role of DEN1 in regulating the neddylation of cullins in cultured mammalian cells at the present time.
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DISCUSSION |
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DEN1 belongs to the thiol protease superfamily (Fig. 2D). The catalytic action of cysteinyl proteases typically involves utilization of the side chain of cysteine for peptide bond cleavage and the histidine residue as the general base that is usually stabilized by Asp/Asn (34). Additionally, it appears that the active site of thiol Ub/Ubl deconjugating enzymes commonly contains a structurally distinct groove to accommodate the Ub/Ubl C-terminal Gly-Gly motif for cleavage (3638).
The specificity of DEN1 is likely conferred by its unique structural
organization, which allows a strong interaction with Nedd8 and permits the
recognition and accommodation of the Nedd8 C-terminal
Gly75-Gly76 motif for proteolysis. As we have shown
(Fig. 3), DEN1 specifically
recognizes Nedd8 and moreover, we demonstrate that DEN1 binds Nedd8 with a
Kd of about 180 nM
(41). The Ulp1-Smt3/SUMO
binding interface may prove instrumental for understanding the interaction
between DEN1 and Nedd8, because Smt3/SUMO exhibits a high degree of similarity
to Rub1 (Nedd8 orthologue in yeast)
(37). The Ulp1-Smt3
interaction surface encompasses the exposed sheet of Smt3/SUMO and the
entire face of the protease, comprised of six conserved Ulp1 motifs. It would
be intriguing to examine whether the seven motifs conserved among DEN1
orthologues (Fig. 2D)
mediate interaction with Nedd8.
To account for its biochemical properties revealed in this study, we suggest that the enzymatic action of DEN1 is critically dependent on its ability to bind Nedd8. In this model, DEN1 efficiently recognizes the Nedd8 precursor to promote rapid cleavage of the C-terminal sequence GGLRQ from Nedd8, as we observed (Fig. 4). However, DEN1 may not readily bind to a Nedd8 moiety that is conjugated to Lys-720CUL1, presumably because the Lys-720CUL1-linked Nedd8 is within close proximity to ROC1/Rbx1 (20), which could cause structural constrains for DEN1 recognition. This hypothesis could explain the observation that excess DEN1 was able to deconjugate the mono-neddylated CUL1 (Fig. 5A), because the elevated enzyme/substrate ratio would increase the binding of DEN1 to the Lys-720CUL1-linked Nedd8 moiety.
Although the nature of the Nedd8 linkages within hyperneddylated CUL1 remains to be determined, it can be speculated that these extensively modified forms were formed by the assembly of mono-, di-, and tri-Nedd8 chains onto Lys-720CUL1. Alternatively, they could result from the conjugation of Nedd8 moieties to Lys-720CUL1 and two other unidentified CUL1 lysine residues. Conceivably, DEN1 recognizes a Nedd8 moiety that either is located at the distal end of a Nedd8 chain or is conjugated to a non-Lys-720 CUL1 lysine residue. This could explain why DEN1 efficiently cleaved the CUL1-Nedd82 and CUL1-Nedd83 conjugates, yielding the Lys-720-neddylated CUL1 as proposed (Fig. 6A).
In essence, we postulate that the ability of a Nedd8 conjugate to be processed by DEN1 will critically depend on the availability/accessibility of the Nedd8 moiety for interactions with the protease. CSN appears to act differently. Although lacking Nedd8 C-terminal hydrolytic activity (Fig. 4B), CSN rapidly cleaves the Lys-720CUL1-Nedd8 conjugate (Fig. 5B). In light of findings that CSN is required for the de-neddylation of several cullins (17, 18), we propose that the CSN-mediated cleavage requires the interaction between the protease complex and a distinct structural motif that harbors the conserved cullin lysine residue for Nedd8 conjugation. As revealed by x-ray crystallographic studies, Lys-720CUL1 is positioned at the rim of a "cleft" formed by conserved residues from the CUL1 WH-B helix, four-helix bundle, as well as the ROC1/RBX1 RING domain (20). It can thus be suggested that this cleft structure may be conserved among cullins and recognized by CSN. The active site of CSN must then accommodate/position the conserved cullin lysine residue, such as Lys-720CUL1, as well as the Nedd8 Gly75-Gly76 motif, in a manner that is optimal for proteolytic cleavage. In support of this hypothesis, the interaction between CSN and CUL1 has been observed in transfected cells (39), as well as in vitro.2 Hyper-neddylation may weaken the CSN-CUL1 interaction, resulting in less efficient cleavage as we observed (Fig. 6A). These studies raise an intriguing question of whether CSN and DEN1 could complement to efficiently deconjugate hyper-neddylated CUL1.
Based on the results from this study, DEN1 may play a critical role in the Nedd8 regulatory pathway. Utilizing its intrinsic Nedd8 C-terminal hydrolytic activity, DEN1 could participate in the maturation of the C terminus of Nedd8, thereby generating its functional form for conjugation to cullins. Also, DEN1 may regulate the neddylation of cullins in a concentration-dependent manner. At a low concentration, this protease would remove any Nedd8 moieties that are not directly linked to the conserved cullin lysine residue, maintaining cullins in a mono-neddylated status. In this manner, DEN1 may reverse any hyper-neddylation that might be disruptive of normal regulatory interactions. At elevated concentrations, DEN1 could help complete the removal of Nedd8, yielding free cullins. These activities could be crucial in light of the observation that CSN did not efficiently process hyper-neddylated CUL1 (Fig. 6A). The existence of cullins conjugated with multiple Nedd8 moieties is suggested by the identification of CUL4A/4B-Nedd8 conjugates that migrated significantly larger than the mono-neddylated species (30). Another possible role for DEN1 is in the salvage of adventitiously trapped derivatives of Nedd8. The C-terminal hydrolytic activity of DEN1 could be used to regenerate Nedd8 trapped as catalytic intermediates by excess thiols or amines, in a manner analogous to the action of deubiquitinating enzymes (40). Additionally, as we observed that DEN1 deconjugated Nedd8 from Ubc12 in vitro (data not shown), this protease may have a role in preventing the non-productive auto-neddylation of the E2 conjugating enzyme.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 212-659-5500; Fax:
212-849-2446; E-mail:
zhen-qiang.pan{at}mssm.edu.
1 The abbreviations used are: Ub, ubiquitin; E1, ubiquitin-activating enzyme;
E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; CUL1,
cullin 1; SCF, Skp1-CUL1-F-box protein; CSN,
COP9 signalosome; DTT, dithiothreitol; GST, glutathione
S-transferase; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic
acid.
2 K. Yamoah and Z.-Q. Pan, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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