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Address correspondence to Nico P. Dantuma, Microbiology and Tumor Biology Center, Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden. Tel.: 46-8-728-7147. Fax: 46-8-331-399. E-mail: nico.dantuma{at}mtc.ki.se
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Abstract |
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Key Words: proteasome; neurodegeneration; aggregate; tauopathies; polyglutamine disorders
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Introduction |
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The demonstration that components of the ubiquitin/proteasome system often are involved in neurodegeneration prompted us to examine whether a general impairment of the proteolytic machinery may contribute to the pathology. Recently, an aberrant form of ubiquitin was found in affected neurons of patients with different tauopathies such as sporadic and familial Alzheimer's disease, Down syndrome (van Leeuwen et al., 1998), progressive supranuclear palsy (Fergusson et al., 2000), Pick's disease, frontotemporal dementia, argyrophilic grain disease, and the polyglutamine disorder Huntington's disease (unpublished data), but not in synucleinopathies, such as Lewy body disease and multisystem atrophy (van Leeuwen et al., 1998). Ubiquitin is generated from precursor proteins consisting of tandem ubiquitin moieties that are cleaved into monomeric ubiquitin by ubiquitin C-terminal hydrolases (Wilkinson, 2000). Due to a mechanism known as molecular misreading (van Leeuwen et al., 2000), a dinucleotide deletion can occur within the mRNA encoding the ubiquitin B precursor resulting in a +1 frame shift close to the C terminus of the first ubiquitin moiety (van Leeuwen et al., 1998). Translation of the shifted open reading frame results in the product UBB+1 that comprises the first ubiquitin moiety with a 19amino acid extension. Because the cleavage site of the ubiquitin C-terminal hydrolase is absent in UBB+1, the extension is not removed. The aberrant C terminus prevents the activation and conjugation of UBB+1, but due to the unaffected lysine residues, the mutant ubiquitin may serve as a scaffold for ligation of wild-type ubiquitin molecules (van Leeuwen et al., 2000). Synthetically ubiquitinated UBB+1 was shown to inhibit proteasomal degradation in vitro, and therefore it was hypothesized that its expression in neurons may disturb ubiquitin-dependent proteolysis (Lam et al., 2000). Using two different green fluorescent protein (GFP)*-based reporters that allow monitoring of ubiquitin-/proteasome-dependent proteolysis in living cells (Dantuma et al., 2000b), we show that UBB+1 acts as a strong inhibitor of the proteasome in vivo and induces a general accumulation of ubiquitinated substrates and cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) substrate and accordingly ubiquitinated at both Lys29 and Lys48 residues of its ubiquitin moiety. The inhibitory capacity relies on its recognition as a UFD substrate, as substitutions of either lysine residue releases the blockade while the inhibitory activity is further activated by enhancement of the UFD signal.
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Results |
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Western blot analysis of UBB+1-transfected HeLa and SH-SY5Y cells revealed the presence of unmodified UBB+1, as well as three slower migrating bands (Fig. 1 E; unpublished data). This pattern corresponds to that found in earlier studies in which the bands were identified as conjugates of UBB+1 with one, two, or three ubiquitin moieties (Lam et al., 2000; de Vrij et al., 2001).
Expression of UBB+1 induces accumulation of polyubiquitinated proteins and cell cycle arrest
In subsequent experiments we analyzed the ubiquitination status of accumulating proteasome substrates in UBB+1-expressing cells. UbG76V-GFP HeLa cells were transiently transfected with UBB+1 and then sorted by flow cytometry based on GFP fluorescence intensity. Western blots of lysates from GFP-positive and -negative cells probed with an anti-ubiquitin antibody demonstrated that elevated GFP levels correlated with a general accumulation of polyubiquitinated proteins (Fig. 2 A), corresponding to an approximately twofold increase in the intensity of the smear of polyubiquitin adducts (Fig. 2 B). Thus, UBB+1 is likely to affect an event downstream of polyubiquitination.
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UBB+1 is a UFD substrate
To test whether physiological ubiquitination is required for the inhibitory activity of UBB+1 in vivo, we generated the mutant UBB+1/K48R in which the common ubiquitin conjugation site Lys48 was substituted with Arg. Surprisingly, UBB+1/K48R was still subject to ubiquitination in SH-SY5Y and HeLa cells (Fig. 3 A; unpublished data), suggesting that an alternate ubiquitination site may be used. Targeting of substrates for proteasomal degradation may also occur via the less common ubiquitination site Lys29. To date, this site has only been described for UFD substrates in yeast in which both Lys29 and Lys48 of the N-terminal ubiquitin moiety are targets for polyubiquitination (Johnson et al., 1995; Koegl et al., 1999). Therefore, we compared UBB+1 mutants carrying Lys29Arg and Lys48
Arg substitutions. Indeed, both UBB+1/K29R and UBB+1/K48R were equally efficiently ubiquitinated, whereas ubiquitin conjugation was virtually abrogated in the double mutant UBB+1/K29,48R (Fig. 3 A). Furthermore, substitution of either lysine residue was sufficient to induce a significant increase in the steady state levels of the mutant protein. The effect was most dramatic with the UBB+1/K29R mutant (Fig. 3 A), suggesting that this ubiquitination site may preferentially target UBB+1 for proteasomal degradation. Surprisingly, we observed consistently higher levels of UBB+1/K29R as compared with UBB+1/K29,48R in both HeLa and neuroblastoma cells. Although we did not fully understand this observation, subsequent analysis confirmed that this is not due to proteasomal degradation of the double mutant (Fig. 3 C; unpublished data).
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Ubiquitination as a UFD substrate is required for a full inhibitory activity
Next, we tested whether ubiquitination at specific sites is required for the inhibitory activity of UBB+1. UBB+1 mutants lacking the Lys29, Lys48, or both ubiquitination sites were transiently transfected in SH-SY5Y cells expressing the GFP reporters and the activity of the ubiquitin/proteasome system was monitored by measuring GFP accumulation. Mutation of both Lys29 and Lys48 abrogated the accumulation of both GFP reporters in the neuroblastoma cells confirming that ubiquitination is critical for the inhibitory effect (Fig. 4, A and B). Surprisingly, substitutions of single lysine residues had different effects on the degradation of UFD and N-end rule substrates. The single lysine mutants UBB+1/K29R and UBB+1/K48R were still able to inhibit the degradation of UbG76V-GFP, although the inhibitory effect was strongly compromised. In contrast, substitution of either lysine residue was sufficient to fully abrogate the effect of UBB+1 on accumulation of the Ub-R-GFP reporter, demonstrating that both ubiquitination sites are required to block the degradation of N-end rule substrates. Thus, efficient inhibition of the ubiquitin/proteasome system can only be accomplished by UBB+1 containing both ubiquitination sites.
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No impaired proteasomal degradation in response to overexpression of other substrates
A possible explanation for the inhibitory activity of UBB+1 is that overexpression of proteasome substrates will saturate the system and competitively affect degradation of the Ub-R-GFP and UbG76V-GFP substrates. To address this issue, we designed substrates whose expression was driven by the CMV promotor similar to the UBB+1 constructs. These substrates were FLAGUb-R-nfGFP and FLAGUbG76V-nfGFP, which are based on a nonfluorescent variant of GFP (nfGFP), and FLAGp53. UbG76V-GFP HeLa cells expressing the substrate were identified by the FLAG tag present on each of the substrates. Microscopic and flow cytometric analysis demonstrated that only UBB+1 was able to block degradation of the GFP substrate, whereas none of the other three substrates had an effect on UbG76V-GFP levels (Fig. 7). It is noteworthy that even the nonfluorescent variant of the UbG76V-GFP substrate itself did not induce accumulation. Hence, the inhibitory effect of UBB+1 is not simply due to saturating the ubiquitin/proteasome system by overexpression of a substrate.
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Discussion |
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Detailed analysis of the requirements for the inhibitory effect of UBB+1 revealed some unexpected characteristics. It was acknowledged earlier that UBB+1, even though it cannot be conjugated to substrates (van Leeuwen et al., 1998), can serve as a recipient for polyubiquitination.Therefore, it was postulated that polyubiquitinated UBB+1, similar to free polyubiquitin trees (Piotrowski et al., 1997), can block proteolysis of proteasome substrates (Lam et al., 2000). Indeed, we confirm that ubiquitination of UBB+1 is required for its inhibitory activity in vivo. However, several lines of evidence argue that ubiquitinated UBB+1 does not simply act as a free polyubiquitin tree but is instead an aberrant UFD substrate. First, we show that UBB+1 is ubiquitinated both at Lys29 and Lys48, a pattern that is unique for UFD substrates (Johnson et al., 1995; Koegl et al., 1999). Second, UBB+1 is structurally similar to a UFD substrate, as it has an N-terminal uncleavable ubiquitin moiety linked to a C-terminal extension. Third, our data clearly demonstrate that UBB+1 is degraded by the proteasome in a large number of the transfected cells.
Even though the related UFD reporter UbG76V-GFP seems to be susceptible to inhibition by UBB+1 with a single ubiquitination site to some extent, blockage of degradation of the Ub-R-GFP reporter required the Lys29 as well as Lys48 residues. One possible explanation is that the pool of inhibitory UBB+1 consists of molecules bearing two ubiquitin trees. Binding of both trees to acceptor sites in the proteasome may be required to achieve interactions sufficiently tight to prevent access to other polyubiquitinated substrates. It is noteworthy that in the crystal structure of ubiquitin the Lys29 and Lys48 residues are localized on opposite faces of the molecule and would structurally allow double ubiquitin trees (Cook et al., 1994). It is also possible that the two sites act cooperatively in optimizing ubiquitination, as suggested by the recent finding that in yeast the polyubiquitination factor E4/UFD2 requires Lys48 in a UFD signal in order to accommodate efficient polyubiquitination at Lys29 (Koegl et al., 1999). Interestingly, a recombination event in the gene encoding the murine homologue of E4/UFD2 may underlie the delayed Wallerian nerve degeneration observed in a mouse strain (Conforti et al., 2000). The experiments with the UbG76V-GFP substrate strongly support the model based on a tight interaction between UBB+1 bearing two ubiquitin trees and the proteasome, as both lysine residues can independently target this model UFD substrate to the proteasome, suggesting that Lys29 and Lys48 can each bear a functional ubiquitin tree. Several studies suggest that the rate of polyubiquitination determines the duration of the interaction between a substrate and the proteasome (Lam et al., 1997; Thrower et al., 2000), and it is likely that regardless of whether these two lysine residues are required for the formation of double ubiquitin trees or more efficient polyubiquitination at Lys29, the outcome is a polyubiquitinated UBB+1 that cannot be rapidly released from the proteasome. The combination of a tightly bound but poorly degradable proteasome substrate may clog the system by obstructing access to other substrates, especially when the UBB+1 feed in large amounts to the proteasome. It is tempting to speculate that in its short C-terminal extension may lie the reason for the inhibitory activity of UBB+1, either because it is too short to allow efficient tethering of the recruited UBB+1 into the cavity of the proteasome as has been proposed for another UFD substrate with a short extension (Johnson et al., 1992), or due to the presence of specific residues that stabilize the structure and hamper unfolding (Lee et al., 2001). Notably, during the revision of this manuscript, it was reported that introduction of stable structures within a proteasome substrate can turn an otherwise normal substrate into a potent inhibitor (Navon and Goldberg, 2001). An alternative possibility is that UBB+1 interferes more dramatically with degradation of the UbG76V-GFP substrate because these proteins are both UFD substrates and may well be targets for the same ubiquitin ligase. Accordingly, the stabilized UBB+1 may competitively inhibit the ubiquitination of UbG76V-GFP.
Our model deviates from an earlier presented model that proposed poor deubiquitination of UBB+1 as a possible cause for inhibition of the ubiquitin/proteasome system. Although we show that UBB+1 can indeed inhibit the proteasome in vivo, and that this inhibitory activity relies on ubiquitination of UBB+1, in accordance with the in vitro data (Lam et al., 2000), our results warrant a reevaluation of some of the observations in this earlier study. In the light of our results it is not surprising that ubiquitinated UBB+1 is less efficiently disassembled than free polyubiquitin trees by isoT, considering that this deubiquitination enzyme is highly specific for free polyubiquitin trees rather then ubiquitinated substrates (Wilkinson et al., 1995). It will be interesting to compare in a similar deubiquitination assay if UBB+1 is also more refractory to deubiquitination when compared with an authentic UFD substrate. The length dependence of the ubiquitin tree is another puzzling aspect. We confirmed that the bulk of UBB+1 in cell lysates contains one, two, or at most three conjugated ubiquitin moieties, whereas in the in vitro assay, UBB+1 with synthetically linked Lys48 tetraubiquitin was used, which fulfill much better the minimal length requirement for inhibitory polyubiquitin (Thrower et al., 2000). However, the interaction between substrates simultaneously ubiquitinated at Lys29 and Lys48 and the proteasome is not well understood, and it is possible that with these unique trees UBB+1 can interact with the proteasome while bearing only a limited number of ubiquitins.
The critical significance of the UFD nature of UBB+1 is further emphasized by the finding that introduction of multiple UFD signals had a dramatic enhancing effect on its inhibitory activity. Contrary to what we had expected on the basis of previously reported data (Stack et al., 2000), addition of one or two uncleavable ubiquitin moieties resulted in further accumulation of UBB+1 and a stronger inhibition of the ubiquitin/proteasome system. Thus, in line with the hypothesis that cells can cope only with a certain level of ubiquitinated UBB+1, when this level is increased by accelerating targeting UBB+1 starts to accumulate and further inhibits its own degradation. The inhibitory activity of UBB+1 may then establish a destructive feedback loop, which may ultimately result in overall inhibition of the ubiquitin/proteasome system.
In conclusion, we have provided evidence that UBB+1 acts as a potent inhibitor of the ubiquitin/proteasome system in neuronal cells, and we have uncovered some important features of its mechanism of action. It remains to be seen whether and under what conditions this impaired proteolysis contributes to the generation of the protein aggregates that characterize many UBB+1-associated pathologies. Finally, of paramount importance will be the identification of factors that can override the inhibitory effect of UBB+1.
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Materials and methods |
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Transfections and tissue culture
The human cervical epithelial carcinoma line HeLa and neuroblastoma cell line SH-SY5Y were cultured in Iscove's modified Eagle's medium and high-glucose Dulbecco's modified Eagle medium, respectively, supplemented with 10% fetal calf serum (Life Technologies), 10 U/ml penicillin, and 10 µg/ml streptomycin. HeLa and SH-SY5Y cells were transiently transfected with Lipofectamine (Life Technologies) and calcium phosphate method, respectively. Cells were analyzed 48 h posttransfection unless stated otherwise. Stably transfected cell lines were selected in the presence of 0.5 mg/ml geneticin (Sigma-Aldrich) and screened for GFP fluorescence upon administration of proteasome inhibitors. Where indicated transfected cells were treated the reversible proteasome inhibitor MG132 (Affinity) or the irreversible proteasome inhibitors lactacystin, epoxomicin (Affinity) or Z-L3-VS, a gift from Dr. Hidde Ploegh (Harvard Medical School, Boston, MA) (Bogyo et al., 1997)
Western blot analysis
Cell lysates were fractionated on SDS-PAGE and transferred to Protan BA 85 nitrocellulose filters (Schleicher & Schuell). The filters were blocked in PBS supplemented with 5% skim milk and 0.1% Tween-20, were and incubated with rabbit polyclonal antibody specific to UBB+1 (Ubi-3, 050897; van Leeuwen et al., 1998), ubiquitin (Dako), or GFP (Molecular Probes). After subsequent washings and incubation with peroxidase-conjugated goat antirabbit serum, the blots were developed by enhanced chemiluminiscence (ECL; Amersham Pharmacia Biotech). Quantification of Western blot bands was performed by densitometry (Molecular Dynamics).
Pulse-chase analysis
Neuroblastoma cells, SK-N-SH, were cultured and differentiated with retinoic acid. Differentiated SK-N-SH cells were transfected with a lentiviral-based vector (Naldini et al., 1996), containing the UBB+1 open reading frame (lenti-UBB+1). 2448 h after transduction, cells were incubated in medium lacking methionine and cysteine for 1 h, and were subsequently metabolically labeled by incubating them with medium containing 100 µCi Tran35S-label for 4 h. After the labeling period, medium was replaced by Dulbecco's modified Eagle medium with 10% FCS medium. Cells were washed, chased with culture medium, and harvested at the indicated time points in 10 mM Tris, 0.15 M NaCl, 0.1% NP40, 0.1% Triton X-100, 20 mM EDTA, pH 8.0 buffer containing 0.1% SDS and protease inhibitors. UBB+1 was immunoprecipitated overnight at 4°C with anti-UBB+1 antibody Ubi-3 (1:1,000), and protein-A Sepharose beads were added to the UBB+1 infected cell lysates. Analysis and quantification of the pulse-chase experiments were performed with the usage of a phosphoimager and the software package Imagequant software.
Fluorescence microscopy and flow cytometry
For fluorescence microscopy, the cells were grown and transfected on coverslips. After rinsing in PBS and fixation in 4% paraformaldehyde, immunostaining was performed using an anti-UBB+1 rabbit polyclonal antibody or anti-FLAG mouse monoclonal antibody (M5; Sigma-Aldrich). After subsequent washing steps with PBS, cells were incubated with the secondary antibodies labeled Alexa Fluor 594 (Molecular Probes) or Texas red (Dako). All antibodies were diluted in 50 mM Tris, pH 7.4, 0.9% NaCl, 0.25% gelatine, and 0.5% Triton X-100. Cells were counterstained with Hoechst 33258 (Molecular Probes). Fluorescence was analyzed using a LEITZ-BMRB fluorescence microscope (Leica) and images were captured with a Hamamatsu cooled CCD camera. For quantitative analysis, 100 200 UBB+1 or FLAGUb-positive cells per sample were scored for GFP fluorescence. Flow cytometry was performed with a FACSort flow cytometer (Becton Dickinson) and data were analyzed with CellQuest software. For analysis of cell cycle distribution, cells were harvested 2 d post transfection and fixed in 1% paraformaldehyde. After two washings in PBS, the cells were permeabilized with 70% ethanol and then incubated with propidium iodide. Flow cytometric analysis of the stability of UbG76V-GFP mutants was performed as described before (Dantuma et al., 2000a).
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Footnotes |
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Acknowledgments |
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This work was supported by grants awarded by the Swedish Cancer Society (M.G. Masucci), the Swedish Foundation of Strategy Research (M.G. Masucci), the European Commission Training and Mobility Program (ERBFMRXCT960026; L.G.G.C. Verhoef), the Swedish Research Council (N.P. Dantuma), and a collaborative grant from the Dutch Zon-MW and the Swedish Research Council (910-32-401; E.M. Hol and N.P. Dantuma). F.M.S. de Vrij, D.F. Fischer, F.W. van Leeuwen, and E.M. Hol were supported by NWO-GPD (970-10-029), Human Frontier Science Program Organization (HFSP:RG0148/1999-B) (E.M. Hol), 5th framework EU grant (QLRT-022338), Stichting "De Drie Lichten", Hersenstichting Nederland, Jan Dekkerstichting, Dr. Ludgardine Bouwmanstichting (99-17), and Internationale Stiching Alzheimer Onderzoek.
Submitted: 9 November 2001
Revised: 14 March 2002
Accepted: 14 March 2002
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