Human Molecular Genetics, 2002, Vol. 11, No. 22 2689-2700
© 2002 Oxford University Press
Aggregate formation inhibits proteasomal degradation of polyglutamine proteins
Microbiology and Tumor Biology Center, Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden
Received May 7, 2002; Accepted August 8, 2002
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Insoluble protein aggregates are consistently found in neurodegenerative disorders caused by expanded polyglutamine [poly(Q)] repeats. The aggregates contain various components of the ubiquitin/proteasome system, suggesting an attempt of the cell to clear the aberrant substrate. To investigate the effect of expanded poly(Q) repeats on ubiquitin/proteasome-dependent proteolysis, we targeted these proteins for proteasomal degradation by the introduction of an N-end rule degradation signal. While soluble poly(Q) proteins were degraded, they resisted proteasomal degradation once present in the aggregates. Stabilization was also observed for proteins that are co-aggregated via interaction with the expanded poly(Q) domain. Introduction of a degradation signal in ataxin-1/Q92 reduced the incidence of nuclear inclusions and the cellular toxicity, conceivably by accelerating the clearance of the soluble substrate.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Expansion of polyglutamine [poly(Q)] repeats causes a group of inheritable disorders that are characterized by progressive neuronal loss in specific regions of the central nervous system causing cognitive decline and uncontrolled locomotion (1). These presently incurable and often-lethal diseases comprise Huntington's disease, Kennedy's disease and spinocerebellar ataxias (SCA) types 1–3, 6 and 7. Animal models revealed that these diseases are mainly caused by a toxic gain of function, attributable to the expanded poly(Q) repeats (2,3). Despite intensive research, the precise molecular mechanism by which the poly(Q) repeats implement their devastating effect on neurons remains elusive (4).
The expanded poly(Q) repeats can form stable ß-sheets that mediate intermolecular contacts between repeats (5,6). Consequently, cells expressing such proteins often contain insoluble cytosolic or nuclear aggregates that are predominantly composed of tightly packed poly(Q)-containing proteins. The inclusions also contain various molecular chaperones and components of the ubiquitin/proteasome system (7,8), which are dedicated to refolding or proteolytic destruction of misfolded proteins (9,10).
Although protein aggregates are generally found in the affected neurons of many neurodegenerative disorders, it is still disputed whether they are a major determinant of cellular pathology or whether they counteract toxicity by sequestering the aberrant proteins (11–13). Recent studies providing evidence for a ‘one-hit model’ in neurodegeneration (14) have inspired Perutz and Windle (15) to postulate that the nucleation of misfolded proteins into aggregates is the single biochemical event that initiates neurodegeneration. Indeed, protein aggregates display an inherent cellular toxicity (16), which may be due to the sequestration of essential components of the ubiquitin/proteasome machinery into aggregates (7). Redistribution of the ubiquitin/proteasome system may promote the accumulation of pro-apoptotic substrates of the proteasome (17,18). In support of the alternative scenario, it should be mentioned that the aggregates found in neurodegenerative disorders share many similarities with the aggresome (19–21), a perinuclear proteolytic center that is induced by overexpression of hydrophobic misfolded proteins or by treatment with proteasome inhibitors (22,23). Hence, the formation of aggregates could be an innate protective response of cells in conditions where the amount of misfolded proteins exceeds the proteolytic capacity (4).
Independently of the pathophysiological significance of the aggregates, the question remains why the aggregation-prone poly(Q) proteins are not efficiently eliminated by proteasomal degradation before they form the insoluble protein deposits. A glycine–alanine repeat (GAr), present in the Epstein–Barr virus nuclear antigen 1, inhibits proteasomal degradation by blocking events downstream of ubiquitination (24,25). It has been proposed that, similar to the viral repeat, the poly(Q) repeat may interfere with proteasomal degradation in a length-dependent manner (26). Indeed, proteasomal degradation of mutant ataxin-1, which is responsible for SCA1, was substantially delayed in vitro by the presence of a pathologic poly(Q) repeat although ubiquitination was not affected (26). More recently, a similar inhibitory effect was reported for the expanded poly(Q) repeat in the N-terminal fragment of huntingtin (18).
We have investigated the susceptibility of poly(Q) proteins to proteasomal degradation by introducing poly(Q) repeats of various lengths into designed proteasomal substrates or by introducing a strong degradation signal in mutant ataxin-1. We show that soluble poly(Q) proteins that contain a strong degradation signal are efficiently degraded independently of the length of the repeat. However, once captured in the aggregates, both poly(Q) proteins and co-aggregating proteins are stable despite the presence of strong degradation signals. Thus, the formation of aggregates renders the toxic proteins resistant to proteasomal degradation and initiates the accumulation of poly(Q) proteins and poly(Q)-interacting proteins.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Efficient proteasomal degradation of poly(Q) proteins provided with a strong degradation signal
To evaluate the effect of poly(Q) repeats on proteasomal degradation, a poly(Q) repeat of 16 amino acids or pathologic expanded repeats of 65 or 112 residues were linked to the C terminus of the rapidly degraded N-end rule proteasomal substrate Ub–R–green fluorescent protein (GFP) or the control Ub–M–GFP, which lacks such degradation signal (27) (Fig. 1A). The ubiquitin moiety of these Ub–X–GFP fusions is cleaved by endogenous ubiquitin C-terminal hydrolases upon which the variable X amino acid is exposed as the new N terminus of the GFP moiety. Processed X–GFP that contains an N-terminal arginine residue is subject to rapid ubiquitination and proteasomal degradation, as predicted according to the N-end rule (28), while X–GFP with an N-terminal methionine is not recognized by the N-end rule pathway. A Ub–R–GFP–GAr chimera containing a 239-amino-acid long–GAr sequence, which was previously shown to protect Ub–R–GFP from proteasomal degradation (29), was used as a control.
|
In a first set of experiments, HeLa cells were transiently transfected with the Ub–M–GFP and Ub–R–GFP chimeras, with or without the addition of the proteasome inhibitors lactacystin or carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3-VS) (30) (Fig. 1B and data not shown). High expression levels were obtained with Ub–M–GFP, which were not enhanced by administration of inhibitors, while the Ub–R–GFP gave very low steady-state expression levels and strong accumulation in the presence of inhibitors, confirming that this reporter is indeed targeted for proteasomal degradation (Fig. 1B). Introduction of the GAr abrogated proteasomal degradation of the Ub–R–GFP reporter resulting in expression levels that were comparable to Ub–M–GFP. In contrast, insertion of the short or expanded poly(Q) repeats gave expression levels very similar to those observed with unmodified Ub–R–GFP, demonstrating that the poly(Q) repeat fusions are still subject to proteasomal degradation. To quantify the effect of the repeats, flow cytometric analysis was performed of cells transfected with the different fusions, and the ratio of the percentages of fluorescent cells in the absence and presence of Z-L3-VS was determined. This demonstrated that the GAr stabilized the reporter while insertion of the poly(Q) repeats resulted in a minor increase in the stability of the Ub–R–GFP (Fig. 1C and D). Notably, the minor but significant effect observed with the poly(Q) repeats was independent of the length of the repeat. Taken together, these data show that proteins containing poly(Q) repeats can be efficiently degraded by the proteasome once provided with a functional degradation signal.
Aggregated poly(Q) proteins resist proteasomal degradation
We then asked whether poly(Q) proteins containing an N-end rule degradation signal differ in their ability to form aggregates as compared with their Ub–M–GFP counterparts. Expression of a GFP reporter with a poly(Q) stretch of 16 residues gave a homogenous fluorescence staining (data not shown). However, a substantial fraction of the cells transfected with Ub–M–GFP–Q112 and Ub–R–GFP–Q112 developed strongly fluorescent aggregates in the cytosol and nucleus (Fig. 2A). The GFP fluorescence appeared in these cells as multiple small dots (Fig. 2A: left panel) or single bright fluorescent aggregates (Fig. 2A: right panel). There were no apparent differences in the morphology or intracellular localization of aggregates induced by Ub–M–GFP–Q112 or Ub–R–GFP–Q112.
|
To investigate the stability of the aggregates, we monitored the number of GFP fluorescent cells, distinguishing between cells with homogenous and punctuate fluorescence. In line with the anticipated rapid turnover of the Ub–R–GFP reporter, transfection with Ub–R–GFP–Q16 yielded a much lower number of cells with detectable GFP fluorescence as compared with Ub–M–GFP–Q16. The expression of these reporters reached a maximum after 2–3 days of transfection, and then gradually declined to background levels. This kinetics of expression was observed for cells that displayed a homogenous staining with the Ub–M–GFP–Q112 and Ub–R–GFP–Q112 constructs. In contrast, after an initial increase during the first 2 days, the number of cells with GFP aggregates remained constant for up to 9 days post transfection when the experiment was terminated (Fig. 2B). Viability assays showed that a large fraction of the aggregate-bearing cells were dead 6 days post transfection, while the fluorescent aggregates remained (see below). Importantly, there was no difference in the stability of aggregates composed of the Ub–M–GFP–Q112 or the N-end rule substrate Ub–R–GFP–Q112.
Stabilization of co-aggregated proteins
Natural proteins bearing poly(Q) repeats of a non-pathologic length co-aggregate with expanded poly(Q) proteins (31), a process that can be mimicked experimentally with GFP variants carrying short poly(Q) repeats (21). We used this characteristic to test whether the inhibitory effect of the poly(Q) repeats depends on their presence in the aggregates.
In order to investigate whether our artificial reporters co-aggregate in the nuclear inclusions (NIs) formed by a natural poly(Q) protein, HeLa cells were co-transfected with the mutant ataxin-1/Q92, together with Ub–M–GFP, Ub–M–GFP–Q16 or Ub–M–GFP–Q112. Reporters containing short or long poly(Q) repeats co-localized in the NIs, while the poly(Q)-less reporter was not found in the inclusions (Fig. 3A). The GFP reporter containing a Q112 repeat also formed large cytosolic aggregates devoid of ataxin-1/Q92 (Fig. 3A).
|
Next, we analyzed whether the localization of the Q16-containing proteins in the NIs affected their proteasomal degradation. GFP fluorescence was measured in cells transfected with ataxin-1/Q92 together with Ub–R–GFP or Ub–R–GFP–Q16. GFP-containing NIs were observed in cells co-transfected with ataxin-1 and Ub–R–GFP–Q16 after 2 days, while the NIs of Ub–R–GFP-transfected cells did not emit detectable GFP fluorescence (Fig. 3B). Counting of the cells containing fluorescent NIs 4 days post transfection revealed that the presence of the short poly(Q) repeat stabilized the Ub–R–GFP reporter, resulting in a
10-fold increase in the number of GFP-fluorescent cells (Fig. 3C). Similar data were found with UbG76V-GFP (Fig. 3B and C), which, like Ub–R–GFP, is rapidly degraded by the proteasome but is recognized as a ubiquitin fusion degradation substrate (27). Importantly, the observation that two repeat-less reporters were efficiently degraded in cells with NIs demonstrates that the stabilizing effect is not due to general impairment of the ubiquitin/proteasome system but is restricted to the aggregated proteins. Thus, proteins recruited to the NIs become resistant to proteasomal degradation independently of the repeat length or the presence of strong degradation signals.
Accelerated proteasomal degradation reduces aggregate formation
In order to allow accurate estimation of the incidence of aggregate formation, we modified the GFP reporters by tagging the ubiquitin moiety with an eight-amino-acid FLAG epitope, resulting in FLAGUb–X–GFP (Fig. 4A). Upon cleavage of the reporter, the FLAGUb moiety is stable, allowing identification of all transfected cells regardless of their levels of the GFP reporter. We shall refer to this method, which is a modification of the ubiquitin/protein/reference technique developed by Varshavsky and co-workers (32), as the ubiquitin/fluorescence/ reference (UFR) technique.
|
Staining of HeLa cells transfected with FLAGUb–X–GFP constructs demonstrated that all the cells transfected with the stable FLAGUb–M–GFP that expressed the FLAG tag also emitted GFP fluorescence, while many of the cells transfected with destabilized FLAGUb–R–GFP were specifically stained with the anti-FLAG antibody but had undetectable levels of GFP (not shown). Quantitative analysis with the UFR technique confirmed that there were more GFP-positive cells in the FLAGUb–M–GFP–Q112-transfected cells compared with the FLAGUb–R–GFP–Q112-transfected cells (Fig. 4B). More importantly, a 4-fold lower incidence of cells with aggregates was observed in cells transfected with FLAGUb–R–GFP–Q112 compared with its counterpart lacking the N-end rule degradation signal (Fig. 4C and D). We conclude that introduction of a degradation signal into the GFP–Q112 protein reduced the incidence of aggregate formation.
Effect of degradation signal on the toxicity of poly(Q) proteins
Next, we asked whether, under our experimental conditions, cellular toxicity correlated with the presence of aggregates. Nuclear condensation and fragmentation, which are indicative of apoptosis, were evaluated in cells expressing FLAGUb–M–GFP–Q112 or FLAGUb–R–GFP–Q112. Transfected cells without GFP aggregates displayed normal nuclear morphology, while a large fraction of the cells with aggregates contained condensed or fragmented nuclei (Fig. 5A and B). This was observed for both FLAGUb–M–GFP–Q112 and FLAGUb–R–GFP–Q112.
|
We also studied the kinetics of cell death of FLAGUb–M–GFP–Q112- and FLAGUb–R–GFP–Q112-expressing cells. Transiently transfected cells were stained with a fluorescent dye that selectively stains dead cells based on membrane permeability. The kinetics of cell death was similar for cells containing both types of aggregates (Fig. 5C). However, the total number of dead cells was lower in the FLAGUb–R–GFP–Q112-transfected cells as compared with FLAGUb–M–GFP–Q112 (Fig. 5D), which reflected the
4-fold lower incidence of aggregate formation with FLAGUb–R–GFP–Q112 (Fig. 4C). We conclude that the reduced formation of aggregates accomplished by the insertion of a degradation signal is accompanied by a decrease in the cellular toxicity of the poly(Q) proteins. Yet, the correlation between aggregate formation and cell death does not imply that the aggregates cause the toxic effect, since they may equally well be a consequence of a protective reaction in cells that express high concentrations of the toxic poly(Q) proteins.
Destabilizing ataxin-1
The GFP reporters described in this work are in many respects different from the natural proteins associated with poly(Q) disorders. For example, they harbour C-terminal repeats, while in natural poly(Q) proteins the repeats are often positioned close to the N terminus. Moreover, the GFP reporters cause aggregates in the cytosol and nucleus, while natural poly(Q) proteins mainly cause NIs. We therefore chose to test some of the key findings obtained with the GFP constructs with human mutant ataxin-l/Q92. To this end, we constructed FLAGUb–M–ataxin-1/Q92 and FLAGUb–R–ataxin-1/Q92 chimeras. Expression of these modified ataxin-1/Q92 constructs in HeLa cells resulted in the formation of the characteristic NIs (Fig. 6A). Immunostaining of NIs for ataxin-1 and a proteasome core subunit revealed that the proteasomes accumulated in NIs induced by both the FLAGUb–M–ataxin-1/Q92 and the N-end rule substrate FLAGUb–R–ataxin-1/Q92 (Fig. 6A). Introduction of the N-end rule degradation signal resulted in reduced ataxin-1/Q92 level, indicating proteasomal degradation. Using the UFR technique, we observed that 75% of the cells transfected with FLAGUb–R–ataxin-1/Q92 expressed undetectable levels of ataxin-1, in contrast to FLAGUb–M–ataxin-1/Q92-transfected cells, where virtually all FLAG-positive cells co-stained for ataxin-1/Q92 (Fig. 6B). As with the GFP reporters, significantly fewer FLAGUb–R–ataxin-1/Q92-expressing cells developed NIs compared with cells expressing FLAGUb–M–ataxin-1/Q92 (Fig. 6C). Introduction of the degradation signal resulted in an 70% inhibition of the number of cells with NIs. Thus, the introduction of a degradation signal in a natural protein with expanded poly(Q) repeats can reduce the formation of aggregated protein deposits.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
A distinct feature of many neurodegenerative disorders is the presence of intracellular insoluble protein aggregates (4). The striking similarity of these structures suggests that diverse neurological disorders may have a common pathogenesis related to abnormal protein conformations (33). An important question is why these misfolded proteins escape the cellular quality control that normally eliminates aberrant and potentially hazardous proteins (4). In the present study, we have approached this question using designed and natural poly(Q) proteins.
A major role of the ubiquitin/proteasome system is the destruction of misfolded and potentially hazardous proteins. Yet, insertion of an expanded poly(Q) repeat domain in the control Ub–M–GFP reporter did not induce proteasomal degradation, suggesting that just the presence of the toxic domain is not sufficient for recognition by the ubiquitin/proteasome system. This is in line with the observation that huntingtin proteins with normal and expanded poly(Q) repeats have very similar half-lives (34). The presence of proteasomes in NIs composed of poly(Q) proteins suggests, however, that the aberrant proteins are at some point targeted for degradation (7). An attractive hypothesis is that mobilization of the proteasomes against the poly(Q) proteins may be initiated in the process of aggregate formation. Indeed, it was recently proposed that aggregation and targeting for degradation of the androgen receptor with expanded poly(Q) repeat are linked events (35). Moreover, earlier work indicates that the misfolding of aggregation-prone proteins is a consequence of their aggregation (36). In the present study, we have shown that, once captured in the aggregates, the poly(Q) proteins become extremely stable, regardless of the presence of a strong N-end rule degradation signal. Hence, it seems that the poly(Q) repeat proteins are only recognized as misfolded substrates that are deemed for proteasomal degradation once they reach the aggregates, but paradoxically it is the aggregation event that renders the proteins resistant to proteasomal degradation.
Recent in vitro studies demonstrate that stable domains in designed substrates resist unfolding by the 19S subunit of the proteasome, which abrogates their degradation (37,38). Conceivably, while the isolated expanded poly(Q)-containing proteins may not form stable structures, aggregated poly(Q) proteins may challenge the unfolding attempts of the proteasome. It is noteworthy that the expanded poly(Q) repeat and the viral GAr appear to exploit different mechanisms for their inhibitory activity on proteasomal degradation. The functional and microscopic analysis of chimeras carrying the GAr suggests that they do not form large aggregates (25,29,39). Moreover, structural analysis of an I
B–GAr chimera revealed that this repeat forms a flexible domain (40). An intriguing possibility is that the viral repeat may adapt a tight conformation upon binding to a yet-unidentified partner; the stable complex may be capable of frustrating the unfolding attempt of the proteasome.
We have observed that the presence of poly(Q) aggregates correlates with enhanced apoptosis. This distinct correlation support the view that the aggregates are the major cause of cellular toxicity. However, this is not conclusive evidence, since the presence of aggregates in the dying cells may also reflect an attempt to restrict toxicity in cells that are exposed to high levels of the soluble toxic protein. Notably, Watase and co-workers (41) presented data suggesting that the soluble mutant ataxin-1 is the main toxic determinant in an animal model of SCA1.
Several proteins harbouring short poly(Q) repeats or glutamine-rich domains are captured in the NIs formed by the expanded repeat. Among these are important regulators such as the CREB-binding protein (31) and the TATA-binding protein (42), which have been proposed as mediators of the neuronal toxicity observed in aggregate-containing cells (43). The present study predicts that the co-aggregated proteins are also stabilized as a consequence of their redistribution. The extent to which the anticipated rise in steady-state levels may contribute to the cellular pathology remains to be resolved.
The process of poly(Q) aggregate formation shares many similarities to the orchestrated sequence of events that lead to the formation of aggresomes in cells that overexpress hydrophobic proteasome substrates (20,21). However, it is unclear whether the formation of aggregates contributes to the clearance or inactivation of the toxic proteins. While our data do not exclude the possibility that aggregate formation may be initiated or accelerated by the same mechanism that directs the generation of aggresomes, they strongly suggest that the formation of these aggresome-like structures do not accomplish destruction of the poly(Q) protein but rather induces further accumulation of the toxic poly(Q) and co-aggregated proteins.
The co-localization of proteasomes within the aggregates in the absence of degradation supports the idea that the redistribution of the ubiquitin/proteasome system may indirectly hinder the proteolysis of other substrates (7,17,18). However, the proteolytic capacity of the ubiquitin/proteasome system largely exceeds the level required for household activities, since as much as 80% of the enzymatic activity of the proteasome can be blocked without affecting cell viability (17,27). Interestingly, we observed that aggregate formation did not cause accumulation of the proteasome substrates Ub–R–GFP or UbG76V–GFP. Different results were reported in a recent study using a similar GFP-based proteasome substrate carrying the CL1 degradation signal, from which it was concluded that aggregates cause a general impairment of the ubiquitin/proteasome system (17). The discrepancy may be explained by experimental procedure, since we transiently expressed the GFP substrates, while Bence and co-workers (17) used a stably expressing cell line. However, studies performed with a stable transfectant carrying the UbG76V–GFP reporter did not reveal accumulation of the GFP substrate in aggregate-containing cells (L.G.G.C. Verhoef, K. Lindsten and N.P. Dantuma, unpublished observations). While a cell-type-specific effect cannot be excluded, a provocative explanation may be that the aggregates block the degradation of some but not all substrates. Further studies are required to resolve this important issue.
In contrast to the aggregates, soluble poly(Q) proteins are efficiently destroyed by the proteasome once provided with a strong degradation signal. Importantly, introduction of a degradation signal dramatically reduced the incidence of cells with aggregates or NIs, as well as the toxicity of poly(Q) protein. Thus, the induction of accelerated degradation could be an interesting therapeutic approach for neurodegenerative disorders associated with poly(Q) proteins. Recently, strategies have been developed to redirect the cellular ubiquitination machinery to target proteins of interest. One method is based on the expression of a chimeric ubiquitin ligase adaptor harboring binding motifs for the target proteins (44), while the second approach utilizes small peptides that form a trimeric complex with the target protein and the ubiquitin ligase (45). We believe that, even though the nature of these compounds will demand state-of-the-art gene therapeutic approaches, the lack of treatments and the fact that genetic tests can identify individuals at risk long before the onset of the disease justify further attempts to develop similarly designed ubiquitin ligase adaptors for poly(Q) proteins.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Construction of plasmids
All open reading frames were expressed from a cytomegalovirus (CMV) promoter in the mammalian expression vectors pBK–CMV (Stratagene, La Jolla, CA) or EGFP–N1 (Clontech, Palo Alto, CA). The poly(Q)-encoding repeats were PCR-amplified from the androgen receptor containing repeats of 16, 65 and 112 CAG (a gift from Dr Christine Bailey, University of Pennsylvania School of Medicine, Philadelphia, PA) and cloned in frame with Ub–X–GFP (27), generating Ub–X–GFP–Q16, Ub–X–GFP–Q65 and Ub–X–GFP–Q112. The tagged GFP–reporter constructs were generated by cloning of a double-stranded DNA oligonucleotide encoding a FLAG epitope in the 5' NheI and EcoRI restriction sites in the Ub–X–GFP constructs. The Ub–X–ataxin-1/92Q constructs were generated by cloning in the pBK–CMV backbone FLAGUb–X in frame with the ataxin-1/92Q, excised with HindIII from pXHC-92Q (a gift from Dr Huda Zoghbi, Baylor College of Medicine, Houston, TX).
Transfections and tissue culture
The human cervical epithelial carcinoma line HeLa was cultured in Iscove's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Grand Island, NY), 10 U/ml penicillin and 10 µg/ml streptomycin. Cells were transiently transfected with Lipofectamine (Life Technologies) and analyzed at the indicated time points. For treatment with proteasome inhibitor, cells were incubated for 16 h with medium containing 10 µM lactacystin (Affiniti Research Products Ltd, Devon, UK) or Z-L3-VS (a gift from Dr Hidde Ploegh, Harvard Medical School, Boston, MA).
Western blot analysis
Total cell lysates were fractionated by 10% SDS–PAGE and transferred to Protan BA 85 nitrocellulose filters (Schleicher and Schuell). The filters were blocked in phosphate-buffered saline (PBS) supplemented with 5% skim milk and 0.1% Tween-20 and incubated with a rabbit polyclonal anti-GFP antibody (Molecular Probes Europe, Leiden, The Netherlands). After subsequent washings and incubation with peroxidase-conjugated goat anti-rabbit serum, the blots were developed by enhanced chemiluminiscence (Amersham-Pharmacia Biotech, Uppsala, Sweden).
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 a rabbit polyclonal anti-ataxin-1 antibody (a gift from Dr Huda Zoghbi), a mouse anti-FLAG monoclonal antibody (M5, Sigma, St Louis, MO) or a mouse monoclonal antibody directed against the HC3 
-subunits of the proteasome (Affiniti Research Products Ltd, Devon, UK). After subsequent washing steps, cells were incubated with the Alexa Fluor 594 or FITC-labelled secondary antibodies (Molecular Probes and DAKO). All antibodies were diluted in 50 mM Tris pH 7.4, 0.9% NaCl, 0.25% gelatine and 0.5% Triton-X100. Cells were routinely counterstained with Hoechst 33258 (Molecular Probes). Fluorescence was analyzed using a LEITZ-BMRB fluorescence microscope (Leica, Heidelberg, Germany) and images were captured with a cooled CCD camera (Hamamatsu, Osaka, Japan). Analysis of aggregate stability was performed by counting per field the number of cells with homogenous and punctuate GFP staining at the indicated time points, using an inverted fluorescence microscope (DMIL, Leica). For identification of viable cells, cells were stained with an amine-reactive membrane-impermeable red fluorescent dye at the indicated time points (LIVE/DEAD Cell Viability Kit No. 3, Molecular Probes) and analyzed with flow cytometry and fluorescence microscopy. For quantitative analysis with the fluorescence microscope, 100–400 GFP-positive cells were scored for the presence of punctuate or homogenous staining and cell viability. Flow cytometry was performed with a FACSort flow cytometer (Beckton Dickinson, Mountain View, CA), and data were analyzed with CellQuest software.
|
ACKNOWLEDGEMENTS |
|---|
We thank Marianne Jellne for excellent technical assistance, Drs Huda Zoghbi and Paul Taylor for helpful suggestion and reagents, and Drs Christine Bailey and Hidde Ploegh for the generous gifts of the reagents. This work was supported by grants awarded by the Swedish Research Council (N.P.D.), the Swedish Cancer Society (N.P.D. and M.G.M.) the Swedish Foundation of Strategy Research (M.G.M.), the Swedish Alzheimer Foundation (N.P.D.) and the Karolinska Institute. N.P.D. is supported by a fellowship awarded by the Swedish Research Council. L.G.G.C.V. was supported by a fellowship awarded by the European Commission Training and Mobility Program (ERBFMRXCT960026).
|
FOOTNOTES |
|---|
* To whom correspondence should be addressed. Tel: +46 87287147; Fax: +46 8331399; Email: nico.dantuma{at}mtc.ki.se
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Zoghbi, H.Y. and Orr, H.T. (2000) Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci., 23, 217–247.[Web of Science][Medline]
2 Heintz, N. and Zoghbi, H.Y. (2000) Insights from mouse models into the molecular basis of neurodegeneration. Annu. Rev. Physiol., 62, 779–802.[Web of Science][Medline]
3 Rubinsztein, D.C. (2002) Lessons from animal models of Huntington's disease. Trends Genet., 18, 202–209.[Web of Science][Medline]
4 Sherman, M.Y. and Goldberg, A.L. (2001) Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron, 29, 15–32.[Web of Science][Medline]
5
Perutz, M.F., Johnson, T., Suzuki, M. and Finch, J.T. (1994) Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl Acad. Sci. USA, 91, 5355–5358.
6
Bevivino, A.E. and Loll, P.J. (2001) An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel ß-fibrils. Proc. Natl Acad. Sci. USA, 98, 11955–11960.
7 Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T. and Zoghbi, H.Y. (1998) Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet., 19, 148–154.[Web of Science][Medline]
8 Alves-Rodrigues, A., Gregori, L. and Figueiredo-Pereira, M.E. (1998) Ubiquitin, cellular inclusions and their role in neurodegeneration. Trends Neurosci., 21, 516–520.[Web of Science][Medline]
9 Voges, D., Zwickl, P. and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem., 68, 1015–1068.[Web of Science][Medline]
10 Ben-Zvi, A.P. and Goloubinoff, P. (2001) Mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones. J. Struct. Biol., 135, 84–93.[Web of Science][Medline]
11 Saudou, F., Finkbeiner, S., Devys, D. and Greenberg, M.E. (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell, 95, 55–66.[Web of Science][Medline]
12 Sisodia, S.S. (1998) Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial? Cell, 95, 1–4.[Web of Science][Medline]
13 Floyd, J.A. and Hamilton, B.A. (1999) Intranuclear inclusions and the ubiquitin–proteasome pathway: digestion of a red herring? Neuron, 24, 765–766.[Web of Science][Medline]
14 Clarke, G., Collins, R.A., Leavitt, B.R., Andrews, D.F., Hayden, M.R., Lumsden, C.J. and McInnes, R.R. (2000) A one-hit model of cell death in inherited neuronal degenerations. Nature, 406, 195–199.[Medline]
15 Perutz, M.F. and Windle, A.H. (2001) Cause of neural death in neurodegenerative diseases attributable to expansion of glutamine repeats. Nature, 412, 143–144.[Medline]
16 Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M. and Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature, 416, 507–511.[Medline]
17
Bence, N.F., Sampat, R.M. and Kopito, R.R. (2001) Impairment of the ubiquitin–proteasome system by protein aggregation. Science, 292, 1552–1555.
18
Jana, N.R., Zemskov, E.A., Wang, G. and Nukina, N. (2001) Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release. Hum. Mol. Genet., 10, 1049–1059.
19
Ma, J. and Lindquist, S. (2001) Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc. Natl Acad. Sci. USA, 98, 14955–14960.
20
Waelter, S., Boeddrich, A., Lurz, R., Scherzinger, E., Lueder, G., Lehrach, H. and Wanker, E.E. (2001) Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol. Biol. Cell, 12, 1393–1407.
21
Rajan, R.S., Illing, M.E., Bence, N.F. and Kopito, R.R. (2001) Specificity in intracellular protein aggregation and inclusion body formation. Proc. Natl Acad. Sci. USA, 98, 13060–13065.
22
Garcia-Mata, R., Bebok, Z., Sorscher, E.J. and Sztul, E.S. (1999) Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. J. Cell Biol., 146, 1239–1254.
23
Johnston, J.A., Ward, C.L. and Kopito, R.R. (1998) Aggresomes: a cellular response to misfolded proteins. J. Cell Biol., 143, 1883–1898.
24
Levitskaya, J., Sharipo, A., Leonchiks, A., Ciechanover, A. and Masucci, M.G. (1997) Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly–Ala repeat domain of the Epstein–Barr virus nuclear antigen 1. Proc. Natl Acad. Sci. USA, 94, 12616–12621.
25
Sharipo, A., Imreh, M., Leonchiks, A., Imreh, S. and Masucci, M.G. (1998) A minimal glycine–alanine repeat prevents the interaction of ubiquitinated 1B- with the proteasome: a new mechanism for selective inhibition of proteolysis. Nat. Med., 4, 939–944.[Web of Science][Medline]
26 Cummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y., Ciehanover, A., Orr, H.T., Beaudet, A.L. and Zoghbi, H.Y. (1999) Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron, 24, 879–892.[Web of Science][Medline]
27 Dantuma, N.P., Lindsten, K., Glas, R., Jellne, M. and Masucci, M.G. (2000) Short-lived green fluorescent proteins for quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotechnol., 18, 538–543.[Web of Science][Medline]
28
Varshavsky, A. (1996) The N-end rule: functions, mysteries, uses. Proc. Natl Acad. Sci. USA, 93, 12142–12149.
29
Dantuma, N.P., Heessen, S., Lindsten, K., Jellne, M. and Masucci, M.G. (2000) Inhibition of proteasomal degradation by the Gly–Ala repeat of Epstein–Barr virus is influenced by the length of the repeat and the strength of the degradation signal. Proc. Natl Acad. Sci. USA, 97, 8381–8385.
30
Bogyo, M., McMaster, J.S., Gaczynska, M., Tortorella, D., Goldberg, A.L. and Ploegh, H. (1997) Covalent modification of the active site threonine of proteasomal ß subunits and the Escherichia coli homolog HslV by a new class of inhibitors. Proc. Natl Acad. Sci. USA, 94, 6629–6634.
31
Nucifora, F.C., Jr, Sasaki, M., Peters, M.F., Huang, H., Cooper, J.K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V.L. et al. (2001) Interference by huntingtin and atrophin-1 with CBP-mediated transcription leading to cellular toxicity. Science, 291, 2423–2428.
32
Levy, F., Johnsson, N., Rumenapf, T. and Varshavsky, A. (1996) Using ubiquitin to follow the metabolic fate of a protein. Proc. Natl Acad. Sci. USA, 93, 4907–4912.
33 Carrell, R.W. and Lomas, D.A. (1997) Conformational disease. Lancet, 350, 134–138.[Web of Science][Medline]
34 Persichetti, F., Carlee, L., Faber, P.W., McNeil, S.M., Ambrose, C.M., Srinidhi, J., Anderson, M., Barnes, G.T., Gusella, J.F. and MacDonald, M.E. (1996) Differential expression of normal and mutant Huntington's disease gene alleles. Neurobiol. Dis., 3, 183–190.[Web of Science][Medline]
35
Bailey, C.K., Andriola, I.F., Kampinga, H.H. and Merry, D.E. (2002) Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy. Hum. Mol. Genet., 11, 515–523.
36 Harper, J.D. and Lansbury, P.T., Jr (1997) Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem., 66, 385–407.[Web of Science][Medline]
37 Lee, C., Schwartz, M.P., Prakash, S., Iwakura, M. and Matouschek, A. (2001) ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell, 7, 627–637.[Web of Science][Medline]
38 Navon, A. and Goldberg, A.L. (2001) Proteins are unfolded on the surface of the ATPase ring before transport into the proteasome. Mol. Cell, 8, 1339–1349.[Web of Science][Medline]
39
Heessen, S., Leonchiks, A., Issaeva, N., Sharipo, A., Selivanova, G., Masucci, M.G. and Dantuma, N.P. (2002) Functional p53 chimeras containing the Epstein–Barr virus Gly–Ala repeat are protected from Mdm2- and HPV-E6-induced proteolysis. Proc. Natl Acad. Sci. USA, 99, 1532–1537.
40
Leonchiks, A., Liepinsh, E., Barishev, M., Sharipo, A., Masucci, M.G. and Otting, G. (1998) Random coil conformation of a Gly/Ala-rich insert in IB- excludes structural stabilization as the mechanism for protection against proteasomal degradation. FEBS Lett., 440, 365–369.[Web of Science][Medline]
41 Watase, K., Weeber, E.J., Xu, B., Antalffy, B., Yuva-Paylor, L., Hashimoto, K., Kano, M., Atkinson, R., Sun, Y., Armstrong, D.L. et al. (2002) A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration. Neuron, 34, 905–919.[Web of Science][Medline]
42
Perez, M.K., Paulson, H.L., Pendse, S.J., Saionz, S.J., Bonini, N.M. and Pittman, R.N. (1998) Recruitment and the role of nuclear localization in polyglutamine-mediated aggregation. J. Cell Biol., 143, 1457–1470.
43 Steffan, J.S., Bodai, L., Pallos, J., Poelman, M., McCampbell, A., Apostol, B.L., Kazantsev, A., Schmidt, E., Zhu, Y.Z., Greenwald, M. et al. (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature, 413, 739–743.[Medline]
44 Zhou, P., Bogacki, R., McReynolds, L. and Howley, P.M. (2000) Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins. Mol. Cell, 6, 751–756.[Web of Science][Medline]
45
Sakamoto, K.M., Kim, K.B., Kumagai, A., Mercurio, F., Crews, C.M. and Deshaies, R.J. (2001) Protacs: chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. USA, 98, 8554–8559.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Catalgol, I. Ziaja, N. Breusing, T. Jung, A. Hohn, B. Alpertunga, P. Schroeder, N. Chondrogianni, E. S. Gonos, I. Petropoulos, et al. The Proteasome Is an Integral Part of Solar Ultraviolet A Radiation-induced Gene Expression J. Biol. Chem., October 30, 2009; 284(44): 30076 - 30086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Raspe, J. Gillis, H. Krol, S. Krom, K. Bosch, H. van Veen, and E. Reits Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity J. Cell Sci., September 15, 2009; 122(18): 3262 - 3271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Maynard, C. Bottcher, Z. Ortega, R. Smith, B. I. Florea, M. Diaz-Hernandez, P. Brundin, H. S. Overkleeft, J.-Y. Li, J. J. Lucas, et al. Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment PNAS, August 18, 2009; 106(33): 13986 - 13991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. P. Dantuma and K. Lindsten Stressing the ubiquitin/proteasome system Cardiovasc Res, August 11, 2009; (2009) cvp255v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Salomons, V. Menendez-Benito, C. Bottcher, B. A. McCray, J. P. Taylor, and N. P. Dantuma Selective Accumulation of Aggregation-Prone Proteasome Substrates in Response to Proteotoxic Stress Mol. Cell. Biol., April 1, 2009; 29(7): 1774 - 1785. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mitra, A. S. Tsvetkov, and S. Finkbeiner Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease J. Biol. Chem., February 13, 2009; 284(7): 4398 - 4403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. G. G. C. Verhoef, C. Heinen, A. Selivanova, E. F. Halff, F. A. Salomons, and N. P. Dantuma Minimal length requirement for proteasomal degradation of ubiquitin-dependent substrates FASEB J, January 1, 2009; 23(1): 123 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang, C.-E. Wang, A. Orr, S. Tydlacka, S.-H. Li, and X.-J. Li Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice J. Cell Biol., March 24, 2008; 180(6): 1177 - 1189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sano, S. Tokudome, N. Shimizu, N. Yoshikawa, C. Ogawa, K. Shirakawa, J. Endo, T. Katayama, S. Yuasa, M. Ieda, et al. Intramolecular Control of Protein Stability, Subnuclear Compartmentalization, and Coactivator Function of Peroxisome Proliferator-activated Receptor {gamma} Coactivator 1{alpha} J. Biol. Chem., August 31, 2007; 282(35): 25970 - 25980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Kwun, S. R. da Silva, I. M. Shah, N. Blake, P. S. Moore, and Y. Chang Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing J. Virol., August 1, 2007; 81(15): 8225 - 8235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Bodner, T. F. Outeiro, S. Altmann, M. M. Maxwell, S. H. Cho, B. T. Hyman, P. J. McLean, A. B. Young, D. E. Housman, and A. G. Kazantsev From the Cover: Pharmacological promotion of inclusion formation: A therapeutic approach for Huntington's and Parkinson's diseases. PNAS, March 14, 2006; 103(11): 4246 - 4251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Bett, G. M. Goellner, B. Woodman, G. Pratt, M. Rechsteiner, and G. P. Bates Proteasome impairment does not contribute to pathogenesis in R6/2 Huntington's disease mice: exclusion of proteasome activator REG{gamma} as a therapeutic target Hum. Mol. Genet., January 1, 2006; 15(1): 33 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Menendez-Benito, L. G.G.C. Verhoef, M. G. Masucci, and N. P. Dantuma Endoplasmic reticulum stress compromises the ubiquitin-proteasome system Hum. Mol. Genet., October 1, 2005; 14(19): 2787 - 2799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Rangone, R. Pardo, E. Colin, J.-A. Girault, F. Saudou, and S. Humbert Phosphorylation of Arfaptin 2 at Ser260 by Akt Inhibits PolyQ-huntingtin-induced Toxicity by Rescuing Proteasome Impairment J. Biol. Chem., June 10, 2005; 280(23): 22021 - 22028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Bowman, S.-Y. Yoo, N. P. Dantuma, and H. Y. Zoghbi Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation Hum. Mol. Genet., March 1, 2005; 14(5): 679 - 691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. de Pril, D. F. Fischer, M. L.C. Maat-Schieman, B. Hobo, R. A.I. de Vos, E. R. Brunt, E. M. Hol, R. A.C. Roos, and F. W. van Leeuwen Accumulation of aberrant ubiquitin induces aggregate formation and cell death in polyglutamine diseases Hum. Mol. Genet., August 15, 2004; 13(16): 1803 - 1813. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Abu-Baker, C. Messaed, J. Laganiere, C. Gaspar, B. Brais, and G. A. Rouleau Involvement of the ubiquitin-proteasome pathway and molecular chaperones in oculopharyngeal muscular dystrophy Hum. Mol. Genet., October 16, 2003; 12(20): 2609 - 2623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Michalik and C. Van Broeckhoven Pathogenesis of polyglutamine disorders: aggregation revisited Hum. Mol. Genet., October 15, 2003; 12(90002): R173 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Mandrusiak, L. K. Beitel, X. Wang, T. C. Scanlon, E. Chevalier-Larsen, D. E. Merry, and M. A. Trifiro Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamine-expanded androgen receptor Hum. Mol. Genet., July 1, 2003; 12(13): 1497 - 1506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Webb, B. Ravikumar, J. Atkins, J. N. Skepper, and D. C. Rubinsztein {alpha}-Synuclein Is Degraded by Both Autophagy and the Proteasome J. Biol. Chem., June 27, 2003; 278(27): 25009 - 25013. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||