Human Molecular Genetics, 2002, Vol. 11, No. 5 515-523
© 2002 Oxford University Press
Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy
1Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 208 Bluemle Life Sciences Building, 233 S. 10th Street, Philadelphia, PA 19107, USA, 2Neuroscience Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA, USA and 3Department of Radiation and Stress Cell Biology, University of Groningen, Groningen, The Netherlands
Received October 15, 2001; Revised and Accepted December 20, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal and bulbar muscular atrophy (SBMA) is one of a growing number of neurodegenerative diseases caused by a polyglutamine-encoding CAG trinucleotide repeat expansion, and is caused by an expansion within exon 1 of the androgen receptor (AR) gene. The family of polyglutamine diseases is characterized by the presence of ubiquitinated, intranuclear inclusions associated with molecular chaperones and 26S proteasome components, although the role of these inclusions in the pathogenesis of polyglutamine diseases remains unclear. The over-expression of molecular chaperones of the Hsp70 and Hsp40 families has been shown to modulate inclusion frequency and cellular toxicity. We developed a cell culture system which enables the quantitative analysis of the effects of molecular chaperones on the biochemical properties of an expanded repeat AR. Using this approach, we demonstrate that Hsp70 and its co-chaperone Hsp40 not only increase expanded repeat AR solubility, but function to enhance the degradation of expanded repeat AR through the proteasome. Furthermore, our studies indicate that these molecular chaperones significantly decrease the half-life of an expanded repeat AR. Molecular chaperone enhancement of protein degradation points to the modulation of molecular chaperones as a potential therapeutic target for polyglutamine diseases.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS) and polyglutamine diseases [spinobulbar muscular atrophy (SBMA), Huntington’s disease (HD), spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7 and 17 and dentatorubropallidoluysian atrophy (DRPLA)] are characterized by the presence of neuronal inclusions (1–5). Such inclusions represent the abnormal accumulation of mutant or otherwise misfolded proteins. In polyglutamine diseases, which are caused by an expansion of a polyglutamine-encoding CAG repeat in the associated gene, neuronal intranuclear inclusions (NIIs) are found in the distinct neuronal populations that are dysfunctional and destined to degenerate, despite widespread expression of the mutant protein. In SBMA, caused by a CAG expansion in exon 1 of the androgen receptor (AR) gene (6), lower motor neurons are primarily affected, resulting in progressive bulbar and proximal limb muscle weakness and atrophy (7,8). Molecular chaperones and components of the ubiquitin-dependent proteasome degradation pathway are typically found in the neuronal inclusions associated with many neurodegenerative diseases (9,10). These findings implicate the pathways of protein repair and degradation in at least some aspects of the pathogenesis of neurodegenerative diseases.
Despite the presence of inclusions in the susceptible neuronal populations, the role of NIIs as a primary cause of pathogenesis in polyglutamine diseases remains controversial (11–14). The finding that ubiquitin and components of the multicatalytic 26S proteasome are associated with nuclear inclusions implies that the mutant protein is targeted for degradation (15–20). However, the presence of NIIs suggests that polyglutamine-containing proteins are not adequately cleared by the ubiquitin-dependent proteasome pathway (UPP), and accumulate as a result. Furthermore, a role for the UPP in aggregation is suggested by the finding that the inhibition of ubiquitination results in a decrease in inclusion frequency, even while increasing cellular toxicity (12,21).
Molecular chaperones, which function in protein repair (22,23) as well as in ubiquitin-dependent proteasomal degradation (24–28), play a role in polyglutamine diseases. Chaperones of the heat shock protein 70 (Hsp70) and Hsp40 families are sequestered into polyglutamine-containing inclusions (15,18,20,29,30–32). The interaction of heat shock proteins and proteasome components with ubiquitin-positive inclusions may represent an unsuccessful cellular attempt to refold or degrade expanded polyglutamine repeat proteins, suggesting that impairment of the UPP is an underlying cause of polyglutamine pathogenesis. Alternatively, the presence of protein repair and degradation machinery in NIIs may be indicative of a functional cellular process to solubilize and degrade the accumulated mutant protein. The elimination of NIIs subsequent to suspension of huntingtin expression in a conditional HD mouse model (33) supports the idea that inclusions may not simply represent a static accumulation of mutant protein.
Further supporting a role of molecular chaperones in the pathogenesis of polyglutamine diseases is the finding that over-expressing molecular chaperones modulates both the aggregation frequency and cellular toxicity caused by the expression of expanded polyglutamine repeat proteins. Both Hsc70 and Hsp70 (constitutive and inducible forms of Hsp70, respectively) function to decrease polyglutamine protein aggregation (31,32). In addition, inclusion formation is modulated by the Hsp40 family of co-chaperones (15,17,18,31,32), although Hsp40/HDJ-1 (HDJ-1) has been shown to be more effective than HDJ-2/HSDJ (HDJ-2) in this regard (17,32). Furthermore, HDJ-1 and Hsc70/Hsp70 ameliorate the toxicity in cell culture models of various polyglutamine diseases (17,31,32). Finally, Hsp70 suppresses the neuronal degeneration and neurological deficits, with little effect on NIIs, in Drosophila and mouse models of polyglutamine diseases (30,34).
The mechanism by which heat shock proteins decrease the aggregation and toxicity of polyglutamine-containing proteins is important for understanding both disease pathogenesis and developing therapeutic approaches directed at modulating chaperones. It remains unclear how chaperones mitigate polyglutamine-dependent toxicity; potential mechanisms include refolding denatured mutant proteins, promoting protein degradation or blocking apoptosis. In an in vitro model of truncated huntingtin aggregation, Hsp70 and HDJ-1 were shown to alter the physical state of polyglutamine-containing protein, allowing the formation of SDS-soluble, amorphous aggregates in lieu of SDS-insoluble fibrils (35). In addition, while over-expression of Hsp70 and dHdj1, the Drosophila homolog of human HDJ-1, suppressed retinal degeneration in a Drosophila model of SCA3, ataxin-3 NIIs were not significantly altered, although the level of soluble ataxin-3 was markedly increased (36). These results indicate that molecular chaperones modify the physical state of expanded polyglutamine-containing proteins from an insoluble to a soluble form, but whether molecular chaperones simply alter the physical state of expanded polyglutamine repeat proteins or further control their degradative fate (particularly in mammalian cells) remained unknown.
In the present study, we examine the ability of excess chaperones in a mammalian cell environment to promote the refolding and/or degradation of an expanded polyglutamine repeat protein. To address this question we analyzed the effects of Hsp40 and Hsp70 proteins on the metabolism of an expanded repeat, truncated androgen receptor in a neuronal cell culture model. The use of truncated AR constructs is supported by the finding of only N-terminal epitopes within NIIs of SBMA motor neurons (16), suggesting that proteolysis of full-length AR occurs during the course of the disease. This truncated AR form thus represents a potential proteolyzed species of the mutant full-length AR. Furthermore, the expression of this truncated form reproduces many aspects of the aggregation and toxicity of SBMA (20,37–39). In the current study, we expressed AR and enhanced green fluorescent protein (EGFP) from a bicistronic construct, enabling the stable EGFP to be used to normalize AR expression levels. We quantified the levels of soluble and insoluble AR protein using western blot and filter-trap assays, respectively. We show that the molecular chaperones HDJ-1, HDJ-2 and Hsp70 modulate the levels of soluble and insoluble forms of AR. In addition, Hsp70 with its co-chaperone HDJ-1 facilitate the degradation of AR through the proteasome, decreasing the half-life of the mutant AR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Sequestration of endogenous molecular chaperones with AR inclusions
We previously described a cell culture model of SBMA, which reproduces several aspects of SBMA pathophysiology (37). In Cos-7 or the mouse motor neuron–neuroblastoma (MN) hybrid cells (40) transient expression of an expanded polyglutamine repeat (65Q or 112Q) truncated androgen receptor (AR
HA) results in AR aggregation and cellular toxicity. In MN hybrid cells both cytoplasmic and nuclear inclusions are observed, while in Cos-7 cells the inclusions are cytoplasmic. The basis of this difference is unknown. The truncated AR constructs used here contain 57 amino acids N-terminal and 52 amino acids C-terminal to the polyglutamine tract (
10% of full-length AR), and a C-terminal hemagglutinin (HA) epitope tag.
We used this model system to examine the interaction of endogenous molecular chaperones with the AR aggregates. MN hybrid cells expressing the pathogenic ARHA construct containing 112Q (AR112HA) showed co-localization of endogenous HDJ-2 and Hsc70 with cytoplasmic and nuclear AR inclusions (Fig. 1). Staining for HDJ-1 revealed HDJ-1-positive aggregate structures in a small percentage of cells (<1%) (data not shown). The chaperones Hsp25, Hsp70 and Hsp90 were not sequestered by AR112HA aggregates in our cell culture system. Cells expressing the non-pathogenic AR16HA showed diffuse chaperone staining (data not shown).
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Suppression of AR inclusion formation by Hsp40 and Hsp70 chaperones
To determine if molecular chaperones could modulate the frequency of aggregation in our model system, we co-expressed HDJ-1, HDJ-2 and Hsp70 with AR112
HA, and determined the percentage of AR-expressing cells that contained inclusions. As shown in Table 1, molecular chaperones significantly decreased the aggregation frequency in our cell culture model. HDJ-1 expression suppressed inclusion formation of AR112HA on average by 54%, while Hsp70 expression showed a more modest effect (26% decrease). The co-expression of HDJ-1 or HDJ-2 with Hsp70 further suppressed polyglutamine aggregation; HDJ-1 was modestly more effective than HDJ-2. Notably, HDJ-2 alone did not alter AR aggregation. These data are consistent with other reports of chaperone modulation of aggregation (15,17,18,31,32), and indicate functional effects of these chaperones in this cell culture system.
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Modulation of AR solubility by Hsp40 and Hsp70 chaperones
To understand the mechanism by which the chaperones suppress aggregation, we sought to determine if these chaperones alter the solubility of AR protein. In order to ascertain the effect of chaperone over-expression on the solubility of expanded repeat AR, AR
HA protein expression levels in our transient transfection system were controlled through the use of AR–IRES–EGFP constructs allowing ARHA and EGFP proteins to be expressed from one bicistronic mRNA. This expression cassette ensures that the two proteins are translated at comparable levels; the stable EGFP protein is used as a measure of total AR protein translated, enabling AR protein levels to be normalized to EGFP protein levels. The normal repeat AR–IRES–EGFP construct contains 20 CAG repeats (AR20), whereas the expanded repeat constructs contain 66 CAGs (AR66) and 112 CAGs (AR112). Western blotting was used to quantify SDS-soluble AR, whereas a filter trap assay using cellulose acetate membrane (35,41) was used to measure SDS-insoluble AR. To demonstrate the specificity of each technique, AR20 and AR66 cell lysates were separated into SDS-soluble and insoluble fractions by centrifugation, and then total cell lysate, supernatant and pellet fractions were analyzed. Soluble AR protein was detected in total cell lysate and supernatant fractions by western blot analysis (Fig. 2A). Note the insoluble AR66 protein detected in the stacking gel portion of the western blot. Since this protein cannot be adequately quantified by western blotting, we used a filter trap assay for quantitative analysis of the insoluble form of the mutant protein. Only insoluble AR66 protein was retained on the cellulose acetate membrane (Fig. 2B, left), while the nitrocellulose membrane captures both forms of the protein (Fig. 2B, right). As expected, the normal repeat AR20 protein was not retained on the cellulose acetate membrane.
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Using this approach, we analyzed the ability of the chaperones HDJ-1, HDJ-2 and Hsp70 to alter the physical state of expanded repeat AR66 and AR112 proteins and of normal repeat AR20 protein. Data representing insoluble AR protein captured on cellulose acetate membrane (filter trap assay) (Fig. 3A) and soluble protein analyzed by western blots probed with AR(N20) and anti-EGFP antibodies (Fig. 3B) for AR20 and AR66 total cell lysates are shown. The expression of HDJ-1 or Hsp70 resulted in a significant decrease in insoluble AR66 protein at 24 and 48 h post-transfection (Fig. 3A and C, bottom). The effect of HDJ-2 was generally less effective than either HDJ-1 or Hsp70, showing a significant decrease only at 24 h. Co-expressing HDJ-2 with Hsp70 resulted in only a moderate decrease in insoluble AR66. However, the co-expression of HDJ-1 with Hsp70 nearly eliminated insoluble AR66 at all time points. Similar molecular chaperone effects on AR solubility were also obtained with a more highly expanded repeat AR construct, containing 112Q. The expression of an irrelevant protein, ß-galactosidase, did not enhance the solubility of AR66 (data not shown).
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The decrease in SDS-insoluble AR66 protein was generally not accompanied by a concomitant increase in SDS-soluble AR66 protein. This was particularly evident at 24 and 48 h, at which time soluble AR66 in the presence of chaperones was similar to the level of soluble protein in the absence of exogenous chaperones (Fig. 3B and C, middle). These results suggest that the molecular chaperones, Hsp70 and Hsp40, do not simply solubilize expanded AR but may enhance its degradation as well. Of note was our observation that normal repeat AR20 was somewhat decreased by the expression of chaperones, although this decrease was not statistically significant (Fig. 3C, top).
Molecular chaperones enhance expanded polyglutamine repeat AR solubility in the absence of proteasomal degradation
Since HDJ-1 and Hsp70 produced the largest decrease in insoluble AR protein, we further analyzed the effects of these chaperones on the biochemical properties of expanded repeat AR. To determine if these chaperones promote the degradation of AR through the proteasome, we inhibited proteasomal degradation with the irreversible inhibitor, lactacystin, and examined the levels of soluble and insoluble AR66. In the absence of exogenous chaperones, the addition of lactacystin exacerbated the insolubility of expanded repeat AR (Fig. 4A and C), with a more modest effect on the soluble form of AR (Fig. 4B and C). This is consistent with previous studies reporting an increase in the aggregation of polyglutamine-containing proteins in the presence of proteasome inhibitors (21,29). While proteasome inhibition caused the further accumulation of insoluble AR66, co-expression of HDJ-1 and Hsp70 largely blocked this effect. However, unlike our results in the absence of lactacystin, HDJ-1 and Hsp70 greatly increased the level of soluble AR protein upon proteasome inhibition. Despite the strong effect of Hsp70 and HDJ-1 co-expression on reducing insoluble AR, these chaperones were less effective in the presence of lactacystin than in its absence; this may be due to the saturation of chaperoning capacity in the face of accumulating AR protein and other chaperone substrates. Our findings indicate that Hsp70 and its co-chaperone HDJ-1 promote the degradation of AR through the UPP, but when proteasomal degradation is compromised these chaperones still function to alter the physical state of expanded repeat AR, from an insoluble to a soluble form.
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Decreased half-life of expanded polyglutamine repeat AR in the presence of HDJ-1 and Hsp70
To demonstrate directly that chaperones promote the degradation of expanded repeat AR, the half-life of AR66 was measured in the presence and absence of exogenous HDJ-1 and Hsp70 (Fig. 5). Consistent with our previous results (Fig. 3), the expression of chaperones nearly eliminated the accumulation of insoluble AR66 protein (Fig. 5B and D). In the absence of exogenous chaperones, insoluble AR66 accumulated over time, preventing a meaningful half-life determination (Fig. 5C). Therefore, a comparison of only soluble AR66 in the presence and absence of chaperones was made (Fig. 5E). The half-life of the soluble form of AR66 was reduced by 40% (P < 0.01), from 1.28 h ± 0.22 in the absence of chaperones, to 0.76 h ± 0.12 in the presence of HDJ-1 and Hsp70, indicating that HDJ-1 and Hsp70 indeed enhance the rate of AR66 degradation (Fig. 5H). Furthermore, a comparison of the rates of degradation of total AR66 protein reveals that HDJ-1 and Hsp70 dramatically enhance the degradation of expanded repeat AR (Fig. 5F).
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We next compared the half-life of soluble AR66 to that of the normal repeat AR20 (Fig. 5G). AR20 has a half-life of 1.05 h ± 0.22, which is not statistically different than the half-life determined for soluble AR66 (1.28 h ± 0.22) in this cell culture system (Fig. 5H). While the half-lives of soluble AR66 and AR20 are similar, the overall half-lives are clearly different due to the presence of accumulating insoluble AR66. Since the loss of soluble expanded repeat AR measured in these experiments occurs via two routes, degradation and aggregation, the finding of similar half-lives for soluble AR66 and AR20 suggests that the rates of aggregation and degradation may be coupled, and the processes themselves may be linked.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The finding that molecular chaperones suppress the aggregation and toxicity associated with polyglutamine disease models (15,17,18,30–32,42) formed the basis for our current studies to understand the underlying biochemical mechanism of these effects. In order to examine the effect of chaperones on the solubility of expanded repeat AR, we developed an assay in which EGFP was used to control for the level of translated AR protein. Our data reveal that the combined expression of HDJ-1 and Hsp70 alters the solubility and enhances the proteasome-mediated degradation of expanded repeat AR. These data are consistent with the in vitro findings that Hsp70 and Hsp40/HDJ-1 alter the biochemical properties of expanded repeat huntingtin protein (35), and with the in vivo finding that molecular chaperones synergistically enhance the solubility of ataxin-3 (36). However, the current study extends these ideas in a mammalian system and furthers our understanding of the role of molecular chaperones in polyglutamine disease, revealing that molecular chaperones not only alter the solubility of a polyglutamine-containing protein, but promote its degradation through the 26S proteasome.
Understanding the mechanism by which molecular chaperones act upon polyglutamine-containing proteins to suppress aggregation and toxicity has important therapeutic implications. Although the role of NIIs in pathogenesis remains controversial and the pathogenic form of expanded polyglutamine proteins is unknown, reducing the level of the mutant proteins in polyglutamine diseases is likely to be beneficial. Yamamoto et al. (33) showed that abolishing mutant protein expression in a conditional HD mouse model arrested disease progression, and furthermore resulted in the clearance of inclusions and the partial reversal of the neurological phenotype. An alternate approach for diminishing mutant protein levels is to increase protein degradation. The current finding of chaperone-mediated enhancement of polyglutamine protein degradation in a mammalian system reinforces the idea of chaperones as a therapeutic target to clear polyglutamine proteins. However, it remains to be determined if HDJ-1 and Hsp70 co-expression at the organismal level will have similar effects on mutant protein turnover. In addition, such animal studies would examine the effect of chaperone over-expression on cellular toxicity, which could not be adequately evaluated in the current cell culture system. Furthermore, it is important to determine whether HDJ-1 and Hsp70 over-expression would adversely impact normal endogenous protein turnover and function. The recent finding that Hsp70 alone mitigated the neurologic symptoms and neuronal degeneration of SCA1 transgenic mice, albeit without affecting NII formation, supports an approach that manipulates molecular chaperones (34). While Hsp70 expression alone may suppress the pathological phenotype, our results suggest that the co-expression of HDJ-1 with Hsp70 should further enhance polyglutamine protein solubility and degradation.
Both the combination of chaperones expressed and the level of chaperone expression achieved are likely to determine the extent to which chaperones affect polyglutamine solubility and degradation. In the SCA1 transgenic mouse study described above, which showed mitigation of the disease phenotype, a dose-dependent effect was observed within the range of Hsp70 expression achieved, suggesting that further amelioration of the disease process, including a detectable change in protein solubility, might occur with increased expression of exogenous Hsp70 (34). Given that Hsp70 proteins act in conjunction with their Hsp40 co-chaperones, the chaperoning capacity of Hsp70 could also be greatly enhanced by co-expressing an Hsp40 family member. A comparison of two Hsp40 proteins revealed that HDJ-1 was more effective than HDJ-2 in the current study of AR solubility and in other studies of polyglutamine protein aggregation and cellular toxicity (17,32). While we saw a substantial difference in the level of co-chaperone expression achieved above endogenous levels in our study (HDJ-1 was greater than HDJ-2, data not shown), a functional difference between the two Hsp40 proteins may also be a contributing factor. Indeed, when expressed at equivalent levels, HDJ-1 was found to be more effective than HDJ-2 in alleviating polyglutamine toxicity (36).
The variation of endogenous chaperone levels and stress response capacity among different cell types may contribute to the neuronal susceptibility in polyglutamine repeat diseases. While Hsc70 and its co-chaperones were found to co-localize with inclusions in our neuronal cell culture model, we found no evidence for the induction of a stress response in these cells. Inducible Hsp70 was not detected in AR-expressing MN hybrid cells up to 72 h post-transfection by immunofluorescence or western blotting techniques (data not shown). In addition, the levels of the other chaperones tested, HDJ-1, HDJ-2 and Hsc70, were not increased in the presence of pathogenic forms of ARHA, as determined by western blotting (data not shown). This lack of chaperone upregulation indicates that a stress response is not induced in this cell culture model. This finding differs from that of other polyglutamine disease cell culture models in which the expression of expanded polyglutamine-containing proteins elicited a stress response as determined by detection of inducible Hsp70 (15,17,18,31). However, there is little data currently available that indicate a stress response occurs in patients. While Hsp70 was observed in a small subset of NII-positive neurons in a SCA3 brain (17), Hsp70 was not detected in the brain of a SCA1 patient (15). Therefore, a weak or absent neuronal stress response may contribute to polyglutamine disease pathogenesis, and further indicate the importance of chaperone manipulation in modulating disease progression.
Aside from their role in protein refolding, molecular chaperones and their co-chaperones are involved in ubiquitin-dependent degradation via the proteasome, although their role in the degradation pathway is not well understood (10,43). Hsc70 is necessary for the ubiquitin-dependent degradation of certain protein substrates, specifically for the ubiquitin-conjugation reaction (26). The yeast Hsp40 homolog, Ydj1, is necessary for the ubiquitin-dependent degradation of abnormal and certain short-lived proteins (24,25). Further supporting molecular chaperone involvement in protein degradation are the findings that the co-chaperone CHIP plays regulatory roles in protein folding and degradation. CHIP, a tetratricopeptide repeat-containing protein, interacts with Hsc70/Hsp70, inhibiting its ATPase activity (27), as well as with Hsp90, participating in the ubiquitination of protein substrates and their transfer to the proteasome (28). The role of molecular chaperones in protein degradation continues to be investigated, and such knowledge will likely uncover other potential co-factors whose modulation may further enhance the clearing of mutant polyglutamine-containing proteins through the proteasome.
In the current study we also compared the half-lives of the soluble forms of expanded and normal repeat AR, and found them to be quite similar (AR66: 1.28 h versus AR20: 1.05 h). Given the very long half-life of the insoluble AR66 (Fig. 5F), the overall half-lives of the normal and expanded repeat AR proteins are clearly different. It is notable that the half-lives of the soluble forms of these proteins are similar, since the normal repeat AR is lost solely through its degradation, while the expanded repeat AR is lost through both degradation and aggregation. These data indicate that the rate of AR66 aggregation and degradation is equivalent to the rate of AR20 degradation, and suggest that the process of AR66 aggregation may be linked to its degradation. This idea is consistent with the finding that inhibition of ubiquitination in models of polyglutamine disease inhibits aggregation (12,21). Further evaluation of the early steps in ubiquitin-dependent degradation should clarify this point. In addition, such a link between aggregation and degradation of polyglutamine-containing proteins likely exists in the formation of aggresomes, in which the cytoplasmic accumulation of misfolded proteins and the concentration of proteasomes at the centrosome is an active, microtubule transport-dependent process (44–46).
Our studies indicate that exploiting the role of molecular chaperones in enhancing polyglutamine degradation is an important point of intervention for polyglutamine diseases. Further studies to define the molecular regulators of polyglutamine protein solubility and degradation should provide insights into new therapeutic approaches. In addition, drug screens designed to target these processes will likely yield effective therapeutic strategies for preventing or slowing the inexorable progression of these debilitating and lethal diseases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression constructs
The IRES constructs were created by inserting the EcoRI/PvuII fragment from the previously published AR
HA constructs (37) into the EcoRI/SmaI sites of pIRES2-EGFP (Clontech). Human HDJ-2/HSDJ was a generous gift from Dr Huda Zoghbi (Baylor College of Medicine, Houston, TX). Human Hsp40/HDJ-1 and human inducible Hsp70 were published previously (23).
Cell culture
The mouse MN hybrid cell line (MN hybrid cells, clone 2F1.10.14.7) (40) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine in 5% CO2. MN hybrid cells were transfected with Lipofectamine Plus (Life Technologies Inc.), following the manufacturer’s suggested protocol, using 1 µg AR plasmid DNA per 35 mm well for endogenous chaperone studies. In co-transfection experiments a total of 1 µg of plasmid DNA was used per well, using a molar ratio of 1 AR:3 Hsp40:3 Hsp70. For proteasome inhibition studies, 50 µM synthetic lactacystin (10 mM stock in DMSO, Calbiochem) was used, and was added upon removal of transfection medium. Samples were harvested after 24 h of inhibitor treatment, and analyzed by western blot and filter trap assays.
Immunofluorescence
MN hybrid cells were plated on Permanox chamber slides coated with poly L-lysine (50 µg/ml), and transfected as described above. Approximately 72 h post-transfection, cells were stained as follows. Cells were rinsed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 2% goat serum. Cells were then incubated with primary antibody for 1 h, incubated with fluorochrome-conjugated secondary antibody for 45 min, and stained with Hoechst 33258 (5 µg/ml in PBS, Molecular Probes) for 10 min. Primary antibodies were obtained from the following sources: AR N20 used at 1:100 (Santa Cruz Biotechnologies); Hsp70 (SPA-810) used at 1:100, Hsc70 (SPA-815) used at 1:100 and Hsp40 (SPA-400) used at 1:200 (StressGen); HDJ-2/DnaJ used at 1:200 (NeoMarkers). Fluorochrome-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and used at a dilution of 1:100.
Soluble and insoluble protein analysis by western blot and filter trap assays
To test the specificity of western blot and filter trap assays for soluble and insoluble protein, respectively, a total cell lysate (in SDS lysis buffer, see below) was fractionated by centrifugation at 16 000 g. The supernatant was removed and the pellet was resuspended in an equal volume of lysis buffer. Equal volumes of total cell lysate, supernatant and pellet fractions were then analyzed by western blot and filter trap assay.
Samples for soluble and insoluble protein analysis by western and filter trap assays were analyzed and normalized for EGFP expression, as measured by EGFP fluorescence, prior to AR protein analysis. To do this, harvested cells were separated into two aliquots. The first aliquot was resuspended in sonication buffer (200 mM NaCl, 50 mM NaH2PO4, 10 mM Tris, final pH 8, 50 µg/ml PMSF and 10 µg/ml pepstatin), sonicated and analyzed for EGFP fluorescence following Clontech’s protocol for EGFP quantification using a Biotek FL600 fluorometer. Total relative fluorescence units (RFU) were obtained for each sample. Based on the EGFP quantification, the second aliquot was then lysed in SDS lysis buffer (10 mM Tris pH 8.0, 150 mM NaCl, 2% SDS, 50 µg/ml PMSF and 10 µg/ml pepstatin) in a volume that would give equivalent RFU/µl for each sample, and equal volumes were used for AR protein analysis. Soluble AR protein was analyzed by western blotting (see below), using the anti-AR antibody, AR(N20). Western blots were subsequently probed using the anti-EGFP antibody (Living Colors antibody 8372), in order to measure EGFP protein. Insoluble AR was analyzed by filter trap assay (see below), using AR(N20). Data were analyzed using a Molecular Dynamics densitometer, and quantified by ImageQuant 5.1 software. Solubility data are represented as a ratio of (either soluble or insoluble) AR(N20) optical density (OD) to (soluble) EGFP OD, in order to more precisely normalize for EGFP expression.
For western blotting, protein samples were electrophoresed by SDS–PAGE and transferred to Immobilon-P PVDF membrane (Millipore), using a semi-dry transfer apparatus. The membranes were subsequently blocked in 5% milk in TBS containing 0.05% Tween-20, and incubated for 1 h with appropriate primary antibodies. Primary antibodies were used at the following concentrations: AR N20 (1:1000, Santa Cruz); EGFP (1:10 000, Living Colors antibody 8372, Clontech); Hsp70 (1:1000, SPA-810, StressGen); Hsp40 (1:10 000, SPA-400, StressGen); Hdj-2/DnaJ (1:2000, NeoMarkers). HRP-conjugated secondary antibodies used were anti-rabbit (1:2500, Santa Cruz Biotechnologies), anti-rat (1:2500, Santa Cruz Biotechnologies) and sheep anti-mouse (1:1000, Amersham Pharmacia), and were detected using enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia).
The filter trap assay was performed with 0.22 µm cellulose acetate (Osmonics) or 0.45 µm nitrocellulose (Schleicher & Schuell), and four pieces of Whatman qualitative 3 filter paper to support the membrane, using a slot-blot apparatus (BioRad). The membrane was washed three times with wash buffer (0.1% SDS, 10 mM Tris pH 8.0, 150 mM NaCl). Samples for filter trap analysis were prepared in a final volume of 200 µl in lysis buffer, boiled for 3 min and then loaded and gently vacuumed. Three dilutions were typically used for each sample. Membranes were washed twice with wash buffer, and then removed from the apparatus. Slot-blots were probed as described for western blots.
Metabolic labeling and immunoprecipitation for half-life determination
Cells were transfected as described above. Approximately 42 h after transfection medium change, cells were pulsed for 35 min using 200 µCi/well of Trans 35S Label (ICN Biomedicals), containing labeled methionine and cysteine. Radioactive labeling was followed by the addition of chase media, containing five times the concentration of unlabeled methionine and cysteine. Pellets were lysed in RSB250 buffer (10 mM Tris pH 7.5, 250 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, 50 µg/ml PMSF, 10 µg/ml pepstatin). Immunoprecipitation was performed using 250 µg cell lysate, 50 µl protein A-coated metallic beads (Dynal) and 50 µl AR(N20) antibody (Santa Cruz Biotechnologies), following Dynal’s recommended procedure. Protein was eluted from beads by boiling for 3 min in 125 µl of elution buffer (50 mM Tris pH 7.5, 2% SDS, 20 µl/ml ß-mercaptoethanol). Equal volumes of eluate (50 µl) were loaded on SDS–polyacrylamide gels and slot-blots to analyze soluble and insoluble protein, respectively. A Typhoon 8600 phosphorimager (Molecular Dynamics) was used to detect radioactivity, and the signal was quantified using ImageQuant 5.1 software. The half-life of AR protein in each experiment was calculated using the equation derived from a logarithmic plot of percentage of AR protein remaining versus time (in h), using Microsoft Excel software.
ACKNOWLEDGEMENTS
We thank Dr Ramesh Raghupathi for help with statistical analysis and the Kimmel Cancer Center for the use of their phosphorimager and densitometer. This research was supported by a National Institutes of Health grant to D.E.M. (R29 NS36248).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 215 503 4907; Fax: +1 215 923 9162; Email: diane.merry@mail.tju.edu
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REFERENCES |
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