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Genes to Cells (2008) 13, 827-838. doi:10.1111/j.1365-2443.2008.01214.x
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p97 homologs from Caenorhabditis elegans, CDC-48.1 and CDC-48.2, suppress the aggregate formation of huntingtin exon1 containing expanded polyQ repeat

 Shingo Nishikori1, Kunitoshi Yamanaka1, Toshihiko Sakurai2, Masatoshi Esaki1 and Teru Ogura1,*

1 Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
2 Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8552, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Polyglutamine (polyQ)-expanded proteins are associated with cytotoxicity in some neurodegenerative disorders such as Huntington's disease. We have reported that the aggregation of the polyQ-expanded protein is partially suppressed by co-expression of either of two homologs of an AAA chaperone p97, CDC-48.1 or CDC-48.2, in Caenorhabditis elegans, but how p97 regulates the aggregation of polyQ-expanded proteins remains unclear. Here we present direct evidence that CDC-48.1 and CDC-48.2 suppress the aggregation of a huntingtin (Htt) exon1 fragment containing an expanded polyQ repeat in vitro. CDC-48.1 and CDC-48.2 bound the Htt exon1 fragment directly, and suppressed the formation of SDS-insoluble aggregates of Htt fragments containing 53 glutamine residues (HttQ53) independently of nucleotides. CDC-48.1 and CDC-48.2 also modulated the oligomeric states of HttQ53 during the aggregate formation. In the absence of CDC-48.1 and CDC-48.2, HttQ53 formed 70–150 kDa oligomers, whereas 300–500 kDa oligomers as well as 70–150 kDa oligomers accumulated in the presence of CDC-48.1 and CDC-48.2. Taken together, these results suggest that p97 plays a protective role in neurodegenerative disorders by directly suppressing the protein aggregation as a molecular chaperone.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Mutational expansion of CAG repeats encoding polyglutamine (polyQ) tracts is responsible for at least nine different neurodegenerative diseases, including Huntington's disease (Cummings & Zoghbi 2000). PolyQ-expanded proteins have been shown to aggregate both in vitro (Scherzinger et al. 1997, 1999) and in vivo (Davies et al. 1997; DiFiglia et al. 1997). A characteristic feature of polyQ diseases is the presence of intranuclear and cytoplasmic inclusions in affected neurons. The neuronal inclusions in Huntington's disease have fibrillar morphology and contain aggregated amino-terminal fragments of huntingtin (Htt) (DiFiglia et al. 1997). Similar inclusions containing aggregated polyQ fragments are also detected in other polyQ diseases (Paulson et al. 1997; Skinner et al. 1997). Thus, there is a close relationship between these neurodegenerative disorders and the formation of fibrillar aggregates (Rochet & Lansbury 2000). Recently, it has been reported that some chaperones, such as members of the Hsp70 family or chaperonin TRiC/CCT, modulate the polyQ aggregation and suppress its toxicity (Muchowski et al. 2000; Wacker et al. 2004; Behrends et al. 2006; Kitamura et al. 2006; Tam et al. 2006).

p97 (also called VCP in mammals and Cdc48p in yeast) is a member of the AAA (ATPases associated with diverse cellular activities) protein family. p97 is an essential and abundant protein involved in numerous diverse cellular activities including homotypic fusion of endoplasmic reticulum (ER) and Golgi membranes (Latterich et al. 1995; Rabouille et al. 1995; Patel et al. 1998; Roy et al. 2000), ER-associated protein degradation (ERAD) (Braun et al. 2002; Jarosch et al. 2002; Rabinovich et al. 2002; Ye et al. 2003), nuclear envelope reassembly (Hetzer et al. 2001), transcription activation (Wang et al. 2004) and meiotic progression (Sasagawa et al. 2007). Underlying function of p97 seems to be chaperone activities. During the ATPase cycle, p97 undergoes conformational changes and modifies the structure of the bound substrates. Chaperone activity of p97 is required to disassemble the SNARE complexes in membrane fusion processes, and to retrotranslocate unfolded proteins through the ER membrane in ERAD. Recently it has been reported that p97 suppresses the aggregation of unfolded rhodanese and luciferase in vitro, indicating that p97 interact with unfolded proteins in general (Thoms 2002; Song et al. 2007).

It has been reported that p97 co-localizes with polyQ aggregates in cultured cells and with intraneuronal inclusions in several neurodegenerative diseases (Hirabayashi et al. 2001; Mizuno et al. 2003). In addition, the Drosophila p97 homolog was identified as a genetic modifier of polyQ-induced eye degeneration (Higashiyama et al. 2002). We have also reported that co-expression of either of p97 homologs from Caenorhabditis elegans with a polyQ-expanded protein partially suppressed the aggregation of the polyQ-expanded protein in C. elegans (Yamanaka et al. 2004). These results suggest that p97 plays an important role in polyQ-associated disorders. However, how p97 affects the fibrillar formation of disease-causative proteins such as polyQ-expanded proteins remains largely unknown, although p97 has been shown to suppress the aggregation of unfolded model substrates such as rhodanese and luciferase.

Here, we conducted a series of in vitro experiments to explore the suppression mechanism of the aggregate formation of Htt fragments containing polyQ repeats (Qn = 20 or 53) by p97 homologs from C. elegans, CDC-48.1 and CDC-48.2. Both CDC-48.1 and CDC-48.2 bound Htt fragments directly, and retarded the aggregate formation of HttQ53. Remarkably, CDC-48.1 and CDC-48.2 modified the oligomers of Htt fragments with expanded polyQ to higher molecular mass (300–500 kDa). These results indicate that p97 targets directly to polyQ-expanded proteins and suppresses the aggregate formation as a molecular chaperone.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Both CDC-48.1 and CDC-48.2 form a hexamer and display similar ATPase activities

CDC-48.1 and CDC-48.2 (hereafter collectively CDC-48s) were expressed in Sf9 cells using vaculovirus expression system as N-terminally His-tagged proteins. Most of CDC-48s were found in soluble fraction after ultracentrifugation (data not shown). After purification by Ni-affinity chromatography, CDC-48s were greater than 95% pure as judged by SDS-PAGE analysis (Fig. 1A). Typically, 1 mg of purified proteins was obtained from 100 mL culture of infected Sf9 cells. AAA proteins are generally expected to form hexamers, and the hexamer formation is critical for their functions (Karata et al. 1999). Mammalian p97 has been shown to be a ring-shaped hexameric structure (Rouiller et al. 2002; DeLaBarre & Brunger 2003). In order to investigate whether purified CDC-48s form a hexamer, oligomeric states of CDC-48s were analyzed by size exclusion column chromatography. The molecular masses of CDC-48s were estimated to be ca. 600 kDa (Fig. 1B). Because the deduced molecular masses of CDC-48s’ monomers were 89 kDa, it is most likely that both CDC-48s exist as a hexamer. Furthermore, in order to investigate whether CDC-48s require nucleotides to form a hexamer, we constructed nucleotide binding-defective mutants of CDC-48s. It is well known that mutations in Walker A motif of AAA proteins abolish the ATP binding. Therefore, we changed conserved lysine residues in Walker A motifs of D1 and D2 domains to alanine residues (K257A/K530A for CDC-48.1 and K256A/K529A for CDC-48.2), and analyzed the oligomeric states of these mutants. The molecular masses of these mutants were similar to that of wild-type CDC-48s (Fig. 1B). These results indicate that CDC-48s form a hexamer independently of nucleotides.


Figure 1
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  		Figure 1  Purification and characterization of C. elegans CDC-48s. (A) CDC-48s were purified as described in Experimental procedures. Purified CDC-48s were analyzed by SDS-PAGE and visualized with CBB staining. (B) The oligomeric states of wild-type CDC-48s and nucleotide binding-defective mutants were analyzed by Superose 6 10/300GL gel filtration column chromatography. All proteins form the fully assembled hexamers. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa) and BSA (67 kDa) are used as molecular weight standard. (C) ATPase activities of CDC-48s and their variants were measured at 30 °C. The released inorganic phosphate was quantified by malachite green assay (Chan et al. 1986).

 
Next, we analyzed the ATPase activity of CDC-48s using an assay that measures the release of free inorganic phosphate when ATP is hydrolyzed to ADP (Chan et al. 1986). ATPase activities of CDC-48.1 and CDC-48.2 were 437 and 423 nmol/mg/min at 30 °C, respectively (Fig. 1C). When treated with 1 mM N-ethylmaleimide (NEM), a cysteine-sensitive alkylating agent, for 20 min on ice, CDC-48s showed dramatically reduced ATPase activity (80%–90% reduction) (Fig. 1C), which is similar to a previous report about ATPase reduction of mammalian p97 with NEM treatment (Song et al. 2003). In order to eliminate the possibility that the observed ATPase activities are due to the contamination, we measured the ATPase activities of nucleotide binding-defective mutants of CDC-48s. Both mutants displayed no detectable ATPase activity as expected (Fig. 1C). Taken these results together, we conclude that both CDC-48s indeed has basal ATPase activities. It is interesting to mention that mammalian p97 forms a hexamer independently of nucleotides (Wang et al. 2003) and has an ATPase activity of approximately 500 nmol/mg/min at 37 °C (Song et al. 2003; Ye et al. 2003). Thus, both p97 homologs from C. elegans seem to have biochemical properties similar to those of mammalian p97.

CDC-48.1 and CDC-48.2 suppress the aggregation of chemically-denatured rhodanese

Mammalian p97 and yeast Cdc48p suppress the aggregation of heat-denatured or denaturant-induced unfolded proteins in vitro (Thoms 2002; Song et al. 2007). To test if CDC-48s also suppress the protein aggregation, rhodanese from bovine liver was used as a substrate. Rhodanese is known to difficult to fold by itself, and it is often used as a model protein for aggregate formation. Rhodanese was denatured in 6 M guanidine hydrochloride, and then diluted into a buffer in the presence of either BSA, CDC-48.1 or CDC-48.2. The aggregate formation of rhodanese was monitored by turbidity at 320 nm. Addition of CDC-48s before dilution of chemically-denatured rhodanese strongly suppressed the aggregate formation of rhodanese (Fig. 2). The suppression of aggregate formation of rhodanese was independent of nucleotide. These results are consistent with the previous reports (Thoms 2002; Song et al. 2007). Thus, we conclude that C. elegans CDC-48s interact with unfolded proteins and suppress their aggregation as a molecular chaperone.


Figure 2
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  		Figure 2  Suppression of aggregate formation of chemically-denatured rhodanese by CDC-48s. Rhodanese was denatured in guanidine hydrochloride and diluted 50-fold in the buffer containing CDC-48.1 (A) and CDC-48.2 (B). Aggregation of rhodanese was then monitored photometrically. A final concentration of rhodanese was 1.2 µM, and CDC-48s and BSA were added to a final concentration of 1.2 µM as hexamer. Nucleotide was added at a final concentration of 3 mM.

 
CDC-48.1 and CDC-48.2 interact directly with Htt exon1 fragments

We have reported that CDC-48s partially suppressed the aggregate formation of polyQ in vivo (Yamanaka et al. 2004). Several studies have also suggested that p97 works as a pathological effector for polyQ diseases (Hirabayashi et al. 2001; Nollen et al. 2004). However, it is unclear whether or not p97 targets to polyQ proteins directly or not. In order to investigate the direct interaction between CDC-48s and Htt exon1 fragment, we purified proteins encoding the first exon of Htt with polyQ repeats in the normal (HttQ20) and pathogenic range (HttQ53) as fusions to glutathione-S-transferase (GST) (see Fig. 4A) (Muchowski et al. 2000), and we performed in vitro pull down assays. His-tagged CDC-48s and GST-HttQ were incubated at 30 °C, and pulled down with Ni-Sepharose. Bound materials were eluted with buffer containing 300 mM imidazole, and analyzed by Western blotting. It was revealed that both CDC-48s are capable of binding to both GST-HttQ20 and GST-HttQ53 (Fig. 3A,B). These results also demonstrated that the interaction of CDC-48s with Htt exon1 does not depend on the length of polyQ repeats. Furthermore, to determine whether CDC-48s interact with a polyQ stretches or a region outside the polyQ stretches in Htt, we investigated the interaction of another polyQ-expanded protein, truncated ataxin2-Q56 fused with GST. HttQ20 and HttQ53 contain polyQ regions flanked by non-polyQ regions of 17 and 50 residues, whereas truncated ataxin2-Q56 contains a polyQ repeat of 56 residues franked by non-polyQ regions of only 8 and 9 residues. It is revealed that both CDC-48s interact with GST-ataxin2-Q56 as well as GST-HttQ (Fig. 3). It is most likely to conclude that CDC-48s interact with the polyQ region in both Htt exon1 and ataxin2 fragments.


Figure 4
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  		Figure 4  In vitro preparation of polyQ-dependent aggregates. (A) Schematic representation of the recombinant GST-HttQn (n = 20, 53) protein. PP is the cleavage site for PreScission protease. (B) Time course of HttQ53 aggregation in the presence or absence of CDC-48.2 at room temperature. The concentrations of HttQ53 and CDC-48.2 were 12 µM. The aggregate formation of HttQ53 was detected by turbidity at 405 nm. (C) Aggregated HttQ53 spotted on freshly cleaved mica was observed under the atomic force microscope.

 

Figure 3
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  		Figure 3  Interaction of CDC-48s with Htt exon1 and truncated ataxin2 fragments. GST-HttQ and GST-ataxin2-Q56 were incubated with his-tagged CDC-48.1 or CDC-48.2 for 30 min at 30 °C, isolated with Ni-Sepharose, and analyzed by immunoblotting with anti-GST antibody and anti-p97 antibody (Sasagawa et al. 2007). The interactions between CDC-48.1 and HttQ (A), CDC-48.2 and HttQ (B), CDC-48.1 and truncated ataxin2-Q56 (C), and CDC-48.2 and truncated ataxin2-Q56 (D) were shown.

 
Direct role of CDC-48.1 and CDC-48.2 suppressing polyQ aggregation

Cleavage of purified GST-HttQ53 between the GST and HttQ53 domains by PreScission protease led to the aggregate formation of HttQ53, which was detectable by the increment of turbidity at 405 nm (Fig. 4B). We used atomic force microscopy (AFM) to characterize the structure of HttQ53 aggregates, which were prepared by the treatment of GST-HttQ53 with PreScission protease for 12 h at 30 °C. As shown in Fig. 4C, the AFM observation revealed the accumulation of fibrillar aggregates as well as amorphous aggregates. In contrast, the aggregate formation was not detected after cleavage of the HttQ20 domain from GST (data not shown).

In order to investigate whether CDC-48s suppress the HttQ53 aggregation, we briefly analyzed the time course of aggregate formation of HttQ53 by measuring turbidity at 405 nm in the presence or absence of CDC-48.2 (Fig. 4B). The aggregate formation of HttQ53 was apparently delayed in the presence of equimolar concentration of CDC-48.2, indicating that CDC-48s directly suppress the aggregate formation of polyQ-expanded proteins as well as chemically-denatured rhodanese. It has been reported that disease-causative proteins, such as Aβ, {alpha}-synuclein, and polyQ-expanded proteins, form SDS-insoluble, fibrillar aggregates (Rochet & Lansbury 2000). In order to investigate the effect of CDC-48s on the formation of SDS-insoluble aggregates, we monitored the aggregate formation of Htt by filter trap assay, in which SDS-soluble aggregates were not trapped on a 200-nm pore-sized membrane, but SDS-insoluble aggregates were trapped after boiling in the presence of SDS (Scherzinger et al. 1997; Muchowski et al. 2000). In the absence of CDC-48s, or in the presence of bovine serum albumin (BSA) as a control, SDS-insoluble aggregates were detected at 10–12 h after cleavage. When the equimolar concentration of CDC-48.1 or CDC-48.2 relative to HttQ53 co-existed in the reaction mixture, aggregates were detectable at 14–16 h after cleavage (Fig. 5A), indicating that CDC-48s clearly retarded the SDS-insoluble aggregate formation of HttQ53. We also analyzed effects of concentrations of CDC-48s on the aggregate formation, which were determined at the 14-h time point. The aggregate formation of HttQ53 was inversely correlated with the amounts of CDC-48s (Fig. 5B). Especially, in the presence of threefold excess of CDC-48s, little amount of SDS-insoluble aggregates of HttQ53 were detected. The suppression effect was observed in the presence of either ATP, ADP or ATP{gamma}S, or even in the absence of nucleotides (Fig. 5C), indicating that it is nucleotide-independent.


Figure 5
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  		Figure 5  Suppression of HttQ53 aggregation by CDC-48s monitored by filter-trap assay. (A) Time course of HttQ53 aggregation in the presence of BSA, CDC-48.1 or CDC-48.2 at 30 °C. The concentrations of HttQ53, CDC-48s and BSA were 4 µM. SDS-insoluble, heat-stable aggregates were trapped as described in Experimental procedures, and detected by anti-c-Myc antibody. (B) HttQ53 aggregation assays were performed in the presence of different concentrations of CDC-48s. Reactions were stopped at 14 h, and the aggregates of HttQ53 were analyzed as described in (A). (C) HttQ53 aggregation assays were performed in the absence and presence of 1 mM nucleotide (ATP, ADP, ATP{gamma}S or AMP-PNP). The concentrations of HttQ53 and CDC-48s were 4 µM. RS indicates ATP regeneration system (8.8 mM creatine phosphate and 10 units/mL creatine kinase). Reactions were stopped at 14 h, and the aggregates of HttQ53 were analyzed as described in (A). (D) Effect of timing of addition of CDC-48 to aggregation reaction. CDC-48s were added into the aggregation reaction at 0, 4 or 8 h after cleavage of GST-HttQ53. The concentrations of HttQ53 and CDC-48s were 4 µM. Reactions were stopped at 14 h, and aggregates were detected as described in (A).

 
It is known that there are two steps in the fibrillar formation; nucleation step and fibrillar growth step. We then examined which step CDC-48s suppress. CDC-48s were added into the reaction mixture at 0, 4 or 8 h after cleavage of GST-HttQ53, and the HttQ53 aggregates were monitored at 14 h. CDC-48s suppressed the aggregate formation of HttQ53 even when they were added to the reaction mixture at the 8-h time point, although the suppression was more effective when they were added at an earlier time point (Fig. 5D). It should be noted that the HttQ53 aggregates were detectable at 10–12 h after the reaction started as mentioned above (Fig. 5A). Therefore, CDC-48s seem to suppress at least the fibrillar growth, because the nucleates formed after 8 h were not probably detected as aggregates at 14 h. Taken into account the fact that the effect of suppression was more effective when they were added at 0 h, it is likely that CDC-48s affect the nucleation reaction as well as fibrillar growth of HttQ53.

CDC-48.1 and CDC48.2 modulate oligomers of HttQ53 during aggregate formation

Recently, soluble oligomers rather than mature amyloid fibrils are considered to play an important role in pathogenesis of neurodegenerative disorders (Caughey & Lansbury 2003; Stefani & Dobson 2003; Klein et al. 2004; Haass & Selkoe 2007; Nagai et al. 2007). In order to elucidate the effect of CDC-48s on the oligomeric states of HttQ53, we investigated the molecular size of HttQ53 oligomers during the aggregate formation by density gradient assay. The reaction mixtures incubated for 12 h at 30 °C were applied onto linear sucrose gradients, and ultracentrifuged for 16 h at 16 °C. Samples were fractionated, and blotted on nitrocellulose membranes. Then, HttQ53 and CDC-48s were detected by anti-c-Myc antibody and anti-p97 antibody, respectively. In the presence of BSA as a control, there were two peaks of HttQ53; one is at smaller than 25 kDa, and the other approximately 70–150 kDa (Fig. 6A,B). Because the molecular mass of the HttQ53 fragment is calculated to be 17 kDa, the smaller and larger peaks seem to represent monomers and oligomers of HttQ53, respectively. Interestingly, in the presence of CDC-48s, another novel peak corresponding to 300–500 kDa appeared in addition to these two peaks. All these peaks have molecular sizes different from that of CDC-48s; broader and smaller than CDC-48s. There are two possibilities for the newly appeared 300–500 kDa oligomer; a complex of HttQ53 and CDC-48s or a new oligomeric state of HttQ53 modulated by CDC-48s. The former possibility seems unlikely, because the peak of the new 300–500 kDa oligomer was broader and significantly smaller than that of CDC-48s of 500–600 kDa, which corresponds to a hexamer of approximately 560 kDa, and the absence of Htt fragments did not affect the peak position of CDC-48s (data not shown). This notion is also supported by the fact that CDC-48s do not exist in the bottom fraction containing the HttQ53 aggregates. Therefore, it is likely that the newly appeared 300–500 kDa oligomer is a CDC-48-modulated form of HttQ53.


Figure 6
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  		Figure 6  Density gradient analysis of molecular sizes of HttQ53 and CDC-48s during aggregation reaction. Sucrose density gradient centrifugation of HttQ53 (A) and Htt20 (C) in the presence of BSA, CDC-48.1 or CDC-48.2 was performed and samples were fractionated from top to bottom. Thyroglobulin (669 kDa), catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa) and chymotrypsin (25 kDa) are used as molecular weight standard. Blotted membranes of (A) and (C) were densitometrically quantified and shown in (B) and (D), respectively.

 
We also investigated the oligomeric states of HttQ20. HttQ20 was mainly present in the top fraction, indicating that HttQ20 did not form oligomers (Fig. 6C,D). CDC-48s did not promote the oligomer formation of HttQ20. The peak position of CDC-48s in the presence of HttQ20 was approximately 500–600 kDa, which was identical to that of CDC-48s in the presence of HttQ53 or the absence of Htt fragments. These results support that CDC-48s and Htt fragments do not form a stable complex.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
p97 has been suggested to work as a pathological effector for polyQ diseases from in vivo experiments (Hirabayashi et al. 2001; Higashiyama et al. 2002; Nollen et al. 2004; Yamanaka et al. 2004), but how p97 regulates the polyQ aggregation is unclear. In this study, we investigated effects of C. elegans p97 homologs, CDC-48.1 and CDC-48.2, on the polyQ aggregation in vitro. We show here that CDC-48s directly target to HttQ53, and regulate the aggregate formation of HttQ53.

We found that CDC-48s interact with Htt fragments directly (Fig. 3). However, some contradictory results have been reported for the interaction between polyQ-expanded proteins and p97. Boeddrich et al. (2006) have reported that mammalian p97 binds to ataxin-3 (Machado Joseph disease protein), another disease-related polyQ-expanded protein, but not to Htt fragments in vitro. The interaction between p97 and ataxin-3 is mainly mediated by an arginine/lysine-rich region in ataxin-3, but p97 still affects the aggregate formation of ataxin-3 lacking the arginine/lysine-rich region. Our results show that CDC-48s bind to Htt exon1 and truncated ataxin2 fragments, suggesting that CDC-48s interact with polyQ repeats directly. Because the interaction is very weak, however, it might be difficult to detect in a different experimental procedure. Alternatively, it might be because of the difference of p97, mammalian p97 and C. elengas CDC-48s. However, Hirabayashi et al. have reported that p97 prepared by an in vitro transcription and translation system binds to ataxin-3 depending on the length of polyQ repeats (Hirabayashi et al. 2001). Our result indicates that the interaction of CDC-48s with Htt fragments is independent of the length of polyQ repeats. They detected the interaction in the presence of cell extracts, whereas we used purified proteins in the present study. The interaction between p97 and polyQ-expanded proteins may be modulated by a factor(s) such as adaptors of p97.

How do CDC-48s suppress the aggregate formation of HttQ53? CDC-48s affect the nucleation step of the aggregate formation of HttQ53, because (i) CDC-48s interacted with monomeric Htt fragments (Fig. 3), (ii) the aggregation suppression was more effective when they were added at an earlier time point (Fig. 5D), and (iii) CDC-48s changed the oligomeric states of HttQ53 (Fig. 6A,B). Furthermore, CDC-48s also suppressed the fibrillar growth of HttQ53, because CDC-48s suppressed the aggregate formation of HttQ53 even when they were added to the reaction mixture at 8 h after the reaction started (Fig. 5D). Although it is known that during the cycle of ATP hydrolysis p97 undergoes conformational changes and applies tensions to the bound substrate, leading to the disassembly or unfolding of substrates, we found that p97 does not need ATP for the suppression of protein aggregation, which is consistent with previous findings on p97 (Thoms 2002; Song et al. 2007). In contrast, it has been reported that Hsp70 and Hsp40 suppress the protein aggregation in an ATP-dependent manner (Minami et al. 1996). In the presence of ATP, Hsp70 and Hsp40 affected the nucleation step of polyQ aggregation reaction (Jana et al. 2000; Schaffar et al. 2004; Behrends et al. 2006), and Hsp70 and Hsp40 suppressed SDS-insoluble amyloid fibril formation and instead allowed the formation of amorphous, SDS-soluble aggregates (Muchowski et al. 2000). To test whether CDC-48s also can change the morphology of HttQ53 aggregates like Hsp70 and Hsp40, we observed HttQ53 aggregates formed in the presence or absence of CDC-48s using AFM. However, we found no difference in morphology of aggregated HttQ53 between in the presence and absence of CDC-48s (data not shown). The mechanism of p97 on the aggregation suppression may be different from that of Hsp70 and Hsp40.

Recently, soluble oligomers rather than mature fibrils have been shown to be principal pathogenic species in the neurodegenerative diseases (Caughey & Lansbury 2003; Stefani & Dobson 2003; Haass & Selkoe 2007). For example, less cell death has been observed when large aggregates of Htt with expanded polyQ are present in cells than when only soluble Htt is present without these inclusions (Arrasate et al. 2004). In this study, we demonstrate that CDC-48s modulate the oligomeric states of HttQ53 during the aggregate formation. In the absence of CDC-48s, monomers and 70–150 kDa oligomers were detected by density gradient centrifugation, whereas 300–500 kDa oligomers as well as monomers and 70–150 kDa oligomers were detected in the presence of CDC-48s (Fig. 6). In the previous study, similar sizes of oligomers were observed in yeast in which polyQ-expanded proteins were expressed (Schaffar et al. 2004). HttQ96 assembled into 70–120 kDa oligomers, and these oligomers bound with a transcription factor, TBP, in the nucleus, suggesting that the observed oligomers are cytotoxic. It has also been reported that mammalian chaperonin TRiC/CCT modulated the oligomeric states of polyQ-expanded proteins in cooperation with Hsp70 and Hsp40, and prevented the toxicity of polyQ-expanded proteins (Behrends et al. 2006; Kitamura et al. 2006). HttQ110-GFP fusion protein formed approximately 200 kDa oligomers in yeast and inhibited the growth, whereas co-expression of TRiC/CCT with HttQ110-GFP counteracted the growth defect and promoted the assembly of the larger oligomers of 300–500 kDa of HttQ110-GFP (Behrends et al. 2006). CDC-48s also promoted the large HttQ53 oligomer formation of approximately 300–500 kDa (Fig. 6). Although we have not analyzed the toxicity of these oligomers observed in this study, p97 might prevent toxicity of polyQ-expanded proteins by modulating its oligomeric states like TRiC/CCT. Taken these results together, it is likely that p97 plays a protective role in polyQ disorders as a molecular chaperone.

Some AAA+ family proteins such as bacterial ClpB and yeast Hsp104 which have two AAA+ ATPase domains per subunit like p97, have been shown to display disaggregation activities (Mogk et al. 1999; Lee et al. 2004). ClpB and Hsp104 can disaggregate and remodel aggregated proteins together with the DnaK/Hsp70 chaperone system in an ATP-dependent manner. Recently, it has been reported that mammalian p97 is involved in the clearance of pre-formed polyQ aggregates and other abnormal protein aggregates in vivo (Kobayashi et al. 2007). We have analyzed effects of CDC-48s on preformed polyQ aggregates in vitro, but they have not displayed any disaggregation activities so far (data not shown). It is, however, still possible that CDC-48s may disaggregate preformed polyQ aggregates in cooperation with other chaperones such as Hsp70 and Hsp40, and this remains elusive.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmid construction

The entire open reading frames for CDC-48s were amplified by PCR from cDNA clones yk670b4 and yk588f3 (generous gifts from Dr Y. Kohara, National Institute of Genetics, Japan) using primers with SmaI and XhoI restriction sites for cdc-48.1 and BglII and XhoI for cdc-48.2. The amplified fragments were ligated into the transfer vector pAcHLT-B (BD Biosciences, La Jolla, CA) to express recombinant proteins with N-terminal His6-tag in the insect Sf9 cell. Site-directed mutagenesis was carried out by using QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Constructs were confirmed by DNA sequencing. Plasmid expressing GST-Htt, pGEX-HttQ20 and pGEX-HttQ53, were kindly provided by Dr U. F. Hartl (Muchowski et al. 2000). Plasmid expressing GST-ataxin2-Q56 was constructed by inserting the truncated ataxin2-Q56 sequence from pT-SCA2-Q56-GFP (a generous gift from Dr O. Onodera) (Nozaki et al. 2001) into pGEX-6p-3.

Protein purification

Recombinant CDC-48 proteins were expressed in insect cells. The transfer plasmids described above were used to transfect Sf9 cells together with helper viruses (BD Biosciences), and thus recombinant baculoviruses were obtained. Sf9 cells were infected with each recombinant virus and harvested. Cells were suspended in buffer A (20 mM Tris–HCl (pH 7.4), 500 mM NaCl, 2 mM MgCl2, 0.1 mM ATP, 20 mM imidazole, 20% (w/v) glycerol), and disrupted using sonicator. Soluble cell extract was then loaded onto a 5-mL HiTrap Chelating column (GE Healthcare, Little Chalfont, UK). The column was washed with buffer A containing 70 mM imidazole, and His-tagged recombinant proteins were then eluted with buffer A containing 300 mM imidazole. Eluted fractions thus obtained were analyzed by SDS-PAGE visualized with Coomassie brilliant blue staining. Protein concentration was determined with the Bradford assay using BSA as a standard.

GST-HttQ53, GST-HttQ20 and GST-ataxin2-Q56 were expressed in Escherichia coli BL21 (DE3). Cells were disrupted by sonication, and soluble fractions were incubated with glutathione Sepharose 4B (GE Healthcare) for 1 h at 4 °C. GST-HttQ53, GST-HttQ20 and GST-ataxin2-Q56 were eluted with 20 mM Tris–HCl (pH 8.0), 20% glycerol, and 10 mM reduced glutathione. Samples were analyzed by SDS-PAGE as described above.

ATPase assay

ATPase activity was determined using the malachite green assay (Chan et al. 1986). Release of inorganic phosphate was monitored spectrophotometrically at 660 nm (DU530 spectrohotometer, Beckman). Standards were prepared by dissolving KH2PO4 in buffer as inorganic phosphate.

Size exclusion column chromatography

The oligomeric state of CDC-48s was analyzed by size exclusion column chromatography using Superose 6 10/300GL column (GE Healthcare). Samples were applied to the column in a buffer consisting of 50 mM Tris–HCl (pH 7.4), 500 mM NaCl, 2 mM MgCl2, 5 mM β-mercaptoethanol and 20% glycerol. Proteins eluted were monitored by absorbance at 280 nm.

Analysis of aggregation of polyQ-expanded protein in vitro

GST-HttQ53 and GST-HttQ20 were incubated with PreScission protease (BD Bioscience) in 20 mM Tris–HCl (pH 8.5), 50 mM KCl, 20 mM β-mercaptoethanol, 30% glycerol, and 0.05% Triton X-100. CDC-48.1, CDC-48.2 or BSA was added together with the protease. Aggregate formation of HttQ53 was monitored spectroscopically at 405 nm or filter-trap assay. In filter-trap assay, reactions were stopped by the addition of 20-time volume of SDS sample buffer (50 mM Tris–HCl (pH 6.8), 2% SDS, 350 mM β-mercaptoethanol and 10% glycerol), followed by heating at 98 °C for 30 min. SDS-insoluble aggregates were trapped on a cellulose acetate membrane (0.2 µm pore size), and detected with anti-c-Myc antibody (monoclonal antibody MC045, Nacalai tesque).

Analysis of aggregation with denaturant-induced unfolded rhodanese

Rhodanese from bovine liver (Sigma, St Louis, MO) was unfolded in 50 mM Tris–HCl (pH 8.0), 6 M guanidine hydrochloride and 1 mM DTT. Unfolded rhodanese was diluted 50-fold (a final concentration of rhodanese, 1.2 µM) into the aggregation assay buffer (50 mM Tris–HCl (pH 8.0), 3 mM MgCl2, 0.01% Triton X-100, and 40% glycerol), and its aggregation was monitored spectroscopically at 320 nm in a cuvette with 1-cm path length. When indicated, CDC-48.1, CDC-48.2 or BSA was added to the reaction at a final concentration of 1.2 µM as hexamer.

Density gradient analysis

GST-HttQ53 and GST-HttQ20 were incubated for 12 h at 30 °C with PreScission protease in the presence of CDC-48.1, CDC-48.2 or BSA. The reaction mixtures were applied onto linear sucrose gradients (10 mL, 10%–40% sucrose in 50 mM Tris–HCl (pH 8.0)), and centrifuged at 40 000 x rpm for 16 h at 16 °C. After centrifugation, fractions were collected from top of the gradients, and blotted on the nitrocellulose membrane. Htt fragments were detected by anti-c-Myc antibody (monoclonal antibody MC045, Nacalai tesque), and CDC-48s were detected by anti-p97 antibody (Sasagawa et al. 2007).

AFM imaging

AFM was used to analyze the morphology of HttQ53 aggregates. A sample for AFM imaging was prepared by a casting method spotted on cleaved mica. Approximately 5 min later, the sample was washed with 10–15 mL of water, and completely dried at room temperature. The AFM measurements were performed in the tapping imaging mode in air at room temperature using a NanoScope IIIa (Digital Instruments Inc., Tonawanda, NY). For the tapping mode measurements, Si cantilevers (Pointprobe NCH, NanoWorld) were used. All the images were collected in the ‘height mode’ whereas maintaining a constant force. In order to minimize drift effects, the system was calibrated for 1–2 h using a standard mica sample. The images were taken in ambient conditions, and at a scan frequency of 1 Hz. The image shown in this paper was flattened and plane-fitted without further manipulation.


   Acknowledgements
 
We thank Dr Y. Kohara for gifting of cDNA clones, Dr U. F. Hartl for plasmids expressing Htt fragments, Dr O. Onodera for plasmid expressing truncated ataxin2-Q56, Ms C. Ichinose and Y. Kawata for secretarial assistance, and Ms S. Fukunaga for technical assistance. This work was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), by grants from the Ministry of Education, Culture, Science, Sports and Technology of Japan, by the Core Research for Evolutional Science and Technology (CREST) program grant from the Japan Science and Technology Agency (JST). S.N. is a Research Fellow of the JSPS.


   Footnotes
 
Communicated by: Keiji Tanaka

* Correspondence: ogura{at}gpo.kumamoto-u.ac.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 11 May 2008





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