Human Molecular Genetics, 2000, Vol. 9, No. 12 1795-1803
© 2000 Oxford University Press
Ataxin-3, the MJD1 gene product, interacts with the two human homologs of yeast DNA repair protein RAD23, HHR23A and HHR23B
1Laboratory for CAG Repeat Diseases, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan and 2Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan
Received 13 March 2000; Revised and Accepted 16 May 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Machado–Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The mutant ataxin-3 forms intranuclear inclusions in cultured cells as well as in diseased human brain and also causes cell death in transfected cells. However, the normal function of ataxin-3 remains unknown. To explore the function of ataxin-3, we used the two-hybrid system to screen for the protein(s) that interacts with ataxin-3. We found that ataxin-3 interacts with two human homologs of the yeast DNA repair protein RAD23, HHR23A and HHR23B. Furthermore, we confirmed that ataxin-3 interacts with the ubiquitin-like domain at the N-terminus of the HHR23 proteins, which is important for nucleotide excision repair; however, ataxin-3 does not interact with ubiquitin, implying that ataxin-3 might be functionally associated with the HHR23 proteins through this specific interaction. The normal and mutant ataxin-3 proteins show no difference in their ability to bind to the HHR23 proteins. However, in 293 cells HHR23A is recruited to intranuclear inclusions formed by the mutant ataxin-3 through its interaction with ataxin-3. These results suggest that this interaction is associated with the normal function of ataxin-3 and that some functional abnormality of the HHR23 proteins might exist in MJD.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Machado–Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder which is characterized clinically by cerebellar ataxia, pyramidal and extrapyramidal signs, peripheral neuropathy and ophthalmoplegia (1). The gene for MJD has been mapped to the long arm of chromosome 14 (14q24.3–q32.1) (2), cloned and designated MJD1. The MJD1 gene product, called ataxin-3, has unstable CAG repeats located near the C-terminus (3). In normal individuals the number of repeats ranges between 13 and 36, whereas in affected individuals this range is expanded to 62–82 (3,4). The MJD1 gene is widely expressed in the brain of normal and affected individuals, preferentially in neurons (5). Ataxin-3 is a protein of unknown function, with a predicted molecular weight of 42 kDa (3). Both the normal and mutant ataxin-3 proteins are expressed ubiquitously. In brain, certain regions, such as the striatum, express ataxin-3 in a limited subset of neurons (6). Both the normal and mutant forms of ataxin-3 are expressed at similar levels in MJD lymphoblastoid cell lines (7). Studies have shown that ataxin-3 is located in the cytoplasm (6) or in both the cytoplasm and nucleus (7). Furthermore, a potential nuclear localization signal has been identified in normal ataxin-3 and that study also showed that ataxin-3 is transported into the nucleus and associated with the nuclear matrix in COS-7 as well as neuroblastoma cells (8). CSM14.1 cells expressing recombinant normal ataxin-3 or mutant ataxin-3 show cytoplasmic and strong nuclear staining (9). Moreover, overexpression of expanded full-length ataxin-3 causes cell death and the formation of intranuclear inclusions (9). An expanded polyglutamine fragment of ataxin-3 also leads to cell death (10) and the formation of intranuclear inclusions (11,12).
Although MJD is one of eight neurodegenerative disorders caused by an expansion of CAG repeats in the affected gene and by an increase in the length of the corresponding polyglutamine domain in the gene-encoded protein (13), the physiological and biochemical functions of ataxin-3 remain unknown.
To explore the normal function of ataxin-3, we undertook a yeast two-hybrid screen to isolate proteins that interact directly with ataxin-3. We report here that two human homologs of the yeast DNA repair protein RAD23, HHR23A and HHR23B, interact with both normal and mutant ataxin-3. Furthermore, we confirmed that both HHR23A and HHR23B interact with ataxin-3 through their N-terminal ubiquitin-like (UBL) domain. Interestingly, HHR23A co-localizes with aggregates formed by mutant ataxin-3 and thus may participate in pathological processes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Isolation of HHR23A by yeast two-hybrid screen
Using ataxin-3 as bait, a human fetal brain cDNA library was screened by sequential transformation. Of ~2 x 106 resulting transformants, four were true positives, identified by retransformation into yeast cells harboring pAS2-1-MJD-CAG20, pAS2-1-MJD-CAG68 or pAS2-1-MJD-CAG80 and growth on SD-Trp-Leu-His selective plates with 5 mM 3-amino-1,2,4-triazole (3-AT) and a positive result in ß-galactosidase (ß-gal) filter assays. The plasmids of these four yeast clones were isolated and sequenced. One of the plasmids, named pACT2-EB202, carrying an ~1.3 kb cDNA insert, was confirmed to contain the full-length HHR23A cDNA. The full-length HHR23A protein interacted specifically with ataxin-3-Q20, ataxin-3-Q68 and ataxin-3-Q80, but did not interact with the GAL4-binding domain (GAL4-BD) expressed by the empty GAD4-BD vector pAS2-1 (Fig. 1A). The three other clones are currently being characterized.
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In humans there are two genes, HHR23A and HHR23B, with sequence homology to Saccharomyces cerevisiae RAD23 (14). Both HHR23A and HHR23B have a UBL domain at the N-terminus and a xeroderma pigmentosum group C (XPC)-binding domain near the C-terminus (15). Since HHR23A interacts with ataxin-3, we decided to examine whether HHR23B does as well. To this end, we cloned the full-length HHR23B gene from a human fetal spleen cDNA library and tested the binding of HHR23B and ataxin-3 using the two-hybrid system. The results showed that HHR23B also interacts with ataxin-3 (Fig. 1B). Liquid ß-gal assays showed that there is no difference in this interaction dependent on polyglutamine length (Fig. 1).
To determine which domain of ataxin-3 interacts with the HHR23 proteins, we created several deletion mutants of normal ataxin-3 (Fig. 2). We examined the interaction of these deletion mutants of ataxin-3 with the HHR23 proteins using the two-hybrid system. Both HHR23A and HHR23B interact with the N-terminal fragment of ataxin-3, which does not contain polyglutamine (Fig. 2).
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In vitro interaction of the HHR23 proteins with normal and mutant ataxin-3
To assess the specificity of the interaction between ataxin-3 and the HHR23 proteins, we evaluated the binding of glutathione S-transferase (GST)–HHR23A with normal and mutant recombinant ataxin-3 or native ataxin-3 in vitro. Recombinant ataxin-3 containing 20, 68 or 80 glutamines expressed by vector pET-21a(+) in Escherichia coli was tested for binding with GST alone or GST–HHR23A coupled to glutathione–agarose beads. All recombinant ataxin-3-Q20, ataxin-3-Q68 and ataxin-3-Q80 specifically interacted with GST–HHR23A, but not with GST alone (Fig. 3A). In addition, a pull-down assay was performed. HHR23A also pulled down both the normal and mutant native ataxin-3 at similar levels to lymphoblastoid cell lines (Fig. 3B). Similar results were obtained using 293 cells or human control and MJD patient brain samples (Fig. 3C). Moreover, HHR23B also interacted with ataxin-3 in vitro (data not shown).
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Ataxin-3 interacts specifically with the UBL domain of the HHR23 proteins, but not with ubiquitin
To determine which domain in the HHR23 proteins is required for interaction with ataxin-3, we generated different N- and C-terminal deletion mutants of the HHR23 proteins in vector pGEX-5x-1 (Fig. 4A). These deletion mutants of the HHR23 proteins were tested for interaction with ataxin-3 in GST pull-down assays. Expression of these deletion mutants of GST–HHR23A and GST–HHR23B was examined using Coomassie Brilliant Blue R-250 staining of proteins separated by SDS–PAGE (Fig. 4B). The supernatants of 293 cell extracts were tested for binding with GST–HHR23 and associated deletion mutants of GST fusions coupled to glutathione–agarose beads. Constructs containing the UBL domain of HHR23A and HHR23B were sufficient for strong interaction with ataxin-3; however, deletion of the HHR23A and HHR23B UBL domains completely abolished HHR23A–ataxin-3 and HHR23B–ataxin-3 interaction (Fig. 4C). Therefore, the UBL domain of HHR23A and HHR23B is sufficient and required for this interaction.
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Since ataxin-3 interacts with the UBL domain of the HHR23 proteins, we wondered whether ataxin-3 binds to any proteins containing a homolog of ubiquitin. To examine this, we produced a GST–ubiquitin fusion to test for binding of ataxin-3 and ubiquitin. The results clearly show that ataxin-3 interacts with the UBL domain of the HHR23 proteins but not with ubiquitin (Fig. 5). Only under conditions of extremely high concentrations of ubiquitin, nearly 20 times more than the UBL domain of the HHR23 proteins, was an interaction between ataxin-3 and ubiquitin detected (data not shown).
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Co-immunoprecipitation of ataxin-3 and HHR23A
To further characterize the in vivo interaction of ataxin-3 and the HHR23 proteins, we co-transfected 293 cells with pEGFP-N1 vectors expressing EGFP or HHR23A–EGFP and pREP10-MJD vectors expressing ataxin-Q20 or ataxin-Q80. When HHR23A–EGFP was immunoprecipitated from the supernatants of cell lysates with anti-GFP antibody, co-transfected ataxin-3 was co-immunoprecipitated (Fig. 6). However, no ataxin-3 was co-immunoprecipitated by anti-GFP antibody in 293 cells co-transfected with vectors expressing EGFP and ataxin-3 (Fig. 6). These results suggest that both the normal and mutant ataxin-3 specifically interact with HHR23A in vivo.
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HHR23A co-localizes with ataxin-3 and its mutant aggregates
Since the HHR23 proteins interacted with ataxin-3 in vitro and in vivo, we further examined the intracellular localization of these proteins. Vectors expressing EGFP, ataxin-3-Q20– EGFP, ataxin-3-Q80–EGFP and ataxin-3-Q130–EGFP were transfected into 293 cells. Ataxin-3-Q20–EGFP and ataxin-3-Q80–EGFP showed a ubiquitous localization in transfected cells, being stronger in the nuclei (Fig. 7B and C). Nuclear aggregates formed by ataxin-3-Q130 were observed and the diffusely distributed ataxin-3-Q130 appeared to fade from the nucleus after formation of intranuclear inclusions (Fig. 7D). The HHR23 proteins have been shown previously to have a clear nuclear localization and appeared to be absent from nucleoli (16). Our results also showed HHR23A to be localized diffusely in the nucleus with the exception of the nucleoli, bearing a similar cellular localization to ataxin-3 (Fig. 7E). In 293 cells co-transfected with ataxin-3–EGFP and HHR23A–v5, these two proteins were observed to be co-localized (Fig. 8). Moreover, when the mutant ataxin-3 formed aggregates in the nucleus, the diffuse nuclear distribution of HHR23A was also abolished and HHR23A co-localized with the aggregates of mutant ataxin-3 (Fig. 8). Seventeen percent of aggregates formed by mutant ataxin-3 showed co-localization with HHR23A (data from three transfections).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Using the two-hybrid system to screen for ataxin-3-binding protein(s), we have identified two ataxin-3-interacting proteins, namely HHR23A and HHR23B, which are human homologs of the yeast DNA repair protein RAD23. The interaction of ataxin-3 with the HHR23 proteins is highly specific and has been verified using several methods for detecting protein–protein interactions. To date, several proteins that interact with polyglutamine tract-containing disease gene products have been identified: huntingtin-associated protein (HAP1) (17), huntingtin-interacting protein (HIP1) (18) and SH3GL3, a protein containing an SH3 domain (19), interacting with huntingtin; IRSp53, a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (20) and the atrophin-1-interacting proteins (AIPs), which contain a WW domain (21), interacting with atrophin-1; cerebellar leucine-rich acidic nuclear protein (LANP), interacting with ataxin-1 (22). To some extent, these interacting proteins imply some function of their interacting polyglutamine tract disease gene products. However, to date, ataxin-3 is a protein of unknown function. The present study is the first to identify ataxin-3-interacting proteins, namely the HHR23 proteins.
RAD23 is one of the yeast proteins involved in nucleotide excision repair (NER), bearing an N-terminal UBL domain that is required for NER (23). In humans there are two homologs of yeast RAD23, HHR23A and HHR23B (14). Both have an XPC-binding domain and a UBL domain. The HHR23 proteins execute DNA repair through interaction with XPC (24). Furthermore, only a small fraction of HHR23B complexed with XPC is necessary for NER and there is excess endogenous HHR23B protein in vivo (16,25). Thus, HHR23B is believed to have additional functions to NER in vivo (16,25). The subcellular localization of the HHR23 proteins is primarily nuclear (16) and, in our results and several other studies, the immunoreactivity of ataxin-3 is also stronger in the nucleus (8,9). The similarity in subcellular localization of ataxin-3 and the HHR23 proteins suggests that these proteins interact physiologically and that the biological function of ataxin-3 may be related to this interaction. Moreover, we found no difference in the ability of the HHR23 proteins to interact with both normal and mutant ataxin-3. We also found that the N-terminus of ataxin-3, which does not contain a polyglutamine tract, interacts with the HHR23 proteins. These results suggest that these interactions are most likely associated with some normal function(s) of ataxin-3.
Although aside from NER the functions of the HHR23 proteins are unclear, their subcellular distribution is homogeneous localization in the nucleus but absence from the nucleoli (16). In the present study we also showed that overexpressed HHR23A is localized in the nucleus. However, cells co-transfected with HHR23A and ataxin-3-Q130 demonstrated an altered distribution pattern of HHR23A, with abolition of the homogeneous distribution and co-localization with the aggregates formed by ataxin-3-Q130. These results are similar to the phenomena observed in SCA1, namely that LANP, which by itself does not form nuclear aggregates, is recruited to subnuclear structures through its interaction with ataxin-1 (21). We also co-transfected a C-terminal fragment of mutant ataxin-3, encoding amino acids 249–341 and containing 130 glutamines, and HHR23A into N2a and 293 cells. Surprisingly, HHR23A was also recruited to the aggregates formed by this fragment in these cell lines (data not shown). It has been reported that a deletion mutant form of ataxin-3 that encodes the N-terminal 80% of the ataxin-3 polypepetide without a polyglutamine tract co-localized to the inclusions formed by a C-terminal fragment of ataxin-3 containing 78 glutamine repeats, suggesting that N-terminal regions of ataxin-3 may interact with the C-terminus of the protein where the glutamine repeats reside (12). Considering this report and our results on the interaction domain of ataxin-3, we suggest that recruitment of the HHR23 proteins may be due to recruited endogenous full-length ataxin-3. Furthermore, when we co-transfected HHR23A–v5 and mutant truncated N-terminal huntingtin (tNhtt) with EGFP into 293 cells, HHR23A failed to be recruited to the aggregates formed by mutant tNhtt (data not shown). When we transfected HHR23A–v5 into an N2a stable inducible cell line (26), which expresses tNhtt-Q150–EGFP after induction with muristerone A, no HHR23A co-localized with the aggregates formed by tNhtt-Q150 (data not shown). These results suggest that the HHR23 proteins specifically interact with ataxin-3, but not with any polyglutamine tracts.
The HHR23 proteins have also been implicated in other processes, such as involvement in the proteolytic pathway in cells (27) and regulation of cell cycle progression (28,29). Although no difference in the ability of HHR23A to bind to normal or mutant ataxin-3 was observed, the recruitment of HHR23A to the aggregates formed by mutant ataxin-3 may affect any of the aforementioned functions of the HHR23 proteins.
The UBL domain of RAD23 is important for NER function (22) and deletion of this domain impairs NER. Recently, RAD23 has been shown to interact with yeast 26S proteasomes through its UBL domain (27,30). The activity of ATPase in the 19S regulatory complex of the proteasome is required for NER and mutations in a 19S regulatory complex ATPase can result in increased UV sensitivity (31). These results suggest that the interaction between proteasomes and the UBL domain of RAD23 is associated with NER. Furthermore, the two human homologs of RAD23, the HHR23 proteins, interact with the S5a subunit of the 26S proteasome directly through their UBL domains (27). HHR23B interacts with the 26S proteasome in HeLa cell extracts and has an inhibitory effect on the degradation of [125I]lysozyme in rabbit reticulocyte lysate, suggesting that part of the HHR23 proteins is involved in the proteolytic pathway in cells (27). Our results revealed that ataxin-3 interacts specifically with the UBL domain of the HHR23 proteins, but not with ubiquitin. Therefore, ataxin-3 may also be involved in NER or the proteolytic pathway through physiologically and functionally interacting with the UBL domain of HHR23. Another possibility is that this interaction increases the susceptibility of neurons to the degeneration caused by mutant ataxin-3, through the interaction of the HHR23 proteins with the 26S proteasome, which subsequently affects the proteolytic pathway in certain neurons, although our studies have not yet addressed this possibility. Future research will focus on determining the potential relationship between ataxin-3 and the function(s) of the HHR23 proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid constructs
The normal MJD1 gene (3) containing 20 CAG repeats was inserted into the BamHI and SalI sites of pGEX-5x-1 (Amersham Pharmacia Biotech, Uppsala, Sweden) (7). The mutant MJD1 gene was created by RT–PCR using the primers MJD1956F and MJD2877R (Table 1) and mRNA which had been extracted from the brains of MJD patients. BglII–StuI fragments of MJD1 containing 68 or 80 CAG repeats were excised from the PCR products and exchanged with normal MJD1 in pGEX-5x-1. Full-length MJD1 constructs containing 20, 68 or 80 CAG repeats were excised from the pGEX-5x-1 vector and subcloned into the GAL4-BD vector pAS2-1 via BamHI and SalI sites to generate the plasmids pAS2-1-MJD-CAG20, pAS2-1-MJD-CAG68 and pAS2-1-MJD-CAG80. A human fetal brain cDNA library was then constructed in the GAL4-activation domain (GAL4-AD) vector pACT2 (Clontech, Palo Alto, CA). The pACT2-HHR23A gene was obtained from a two-hybrid screen. The HHR23B gene was obtained by PCR using primers w16 and w17 from a human fetal spleen cDNA library (Stratagene, La Jolla, CA) and pACT2-HHR23B was created by subcloning the PCR product into the pACT2 vector at the BamHI and XhoI sites. pAS2-1-MJD(1–531), which encodes a fusion of the N-terminus of ataxin-3 (amino acids 1–177) was created by digesting pAS2-1-MJD-20Q with PstI to remove the C-terminus of MJD1 and ligating the vector again. To create the constructs pAS2-1-MJD399–747, which encodes a fusion of the middle fragment of ataxin-3 (amino acids 133–249), and pAS2-1-MJD399–1071, which encodes a fusion of the C-terminus of ataxin-3 (amino acids 133–357), we cut out the fragment of MJD1 gene from pAS2-1-MJD-20Q with EcoRI and SalI and subcloned this fragment into pBluescript II SK(+). From this construct we cut out the fragment encoding amino acids 133–249 of ataxin-3 with SmaI and BglII and subcloned it into the SmaI and BamHI sites of vector pAS2-1 to create pAS2-1-MJD(399–747). We also cut out the C-terminus of MJD1 encoding amino acids 133–357 from this construct and subcloned it into vector pAS2-1 to create pAS2-1-MJD(399–1071).
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The following vectors, described elsewhere (32,33), were from Clontech: pVA3-1, which encodes a fusion of murine p53 protein (amino acids 72–390) with the GAL4-BD, and pTD1-1, which encodes SV40 large T antigen (amino acids 87–708) fused with the GAL4-AD, served as positive controls; pLAM5'-1, which encodes a fusion protein of human lamin C protein (amino acids 66–230) with the GAL4-BD, was the negative control. pET-21a(+)-MJD-CAG20, pET-21a(+)-MJD-CAG68 and pET-21a(+)-MJD-CAG80 were generated by subcloning full-length MJD1 from plasmids pAS2-1-MJD-CAG20, pAS2-1-MJD-CAG68 and pAS2-1-MJD-CAG80, respectively, via the NdeI and SalI sites, into pET-21a(+) (Novagen, Madison, WI). pGEX-5x-1-HHR23A and pGEX-5x-1-HHR23B were obtained by subcloning full-length HHR23A or HHR23B from pACT2-HHR23A or pACT2-HHR23B into pGEX-5x-1 at the BamHI and XhoI sites. Deletion mutants of the HHR23A and HHR23B genes were created by PCR using pACT2-HHR23A and pACT2-HHR23B as the templates. Plasmid pGEX-HHR23A(1–231) was created by subcloning the product from PCR with primers s1 and w50 into vector pGEX-5x-1 at the BamHI and XhoI sites; pGEX-HHR23A(1–82) was created using primers s1 and w13; pGEX-HHR23A(232–363) using primers w59 and w4; pGEX-HHR23A(83–363) using primers w14 and w4; pGEX-HHR23B(1–275) using primers w16 and w52; pGEX-HHR23B(1–80) using primers w16 and w51; pGEX-HHR23B(276–409) using primers w60 and w17; pGEX-HHR23A(81–409) using primers w61 and w17.
The ubiquitin gene was obtained by PCR using primers ubiquitin-F and ubiquitin-R from a human fetal spleen cDNA library (Clontech) and pGEX-ubiquitin was created by subcloning the PCR product into pGEX-5x-1 at the BamHI and SalI sites. pcDNA3.1/v5-His-HHR23A and pEGFP-N1-HHR23A expression vectors were created by subcloning the PCR product from pACT2-HHR23A using pACT2-primer 2 and primer w69. pEGFP-N1-MJD-CAG20 and pEGFP-N1-MJD-CAG80 were created by excising the MJD1 gene at the BamHI and StuI sites of pAS2-1-MJD-CAG20 and pAS2-1-MJD-CAG80 and subcloning the resultant fragments into vector pEGFP-N1 (Clontech) at the BglII and SmaI sites. Using the instability of CAG in this construct, we obtained a further expanded construct containing 130 CAG repeats in vector pEGFP-N1. pEGFP-N1-130Q, which expresses a mutant C-terminal fragment of ataxin-3, was created by excising a fragment from the MJD1 gene with BglII and StuI and subcloning it into vector pEGFP-N1 at the BglII and SmaI sites. pREP10-MJD-CAG20 and pREP10-MJD-CAG80 were created by excising the MJD1 gene from pAS2-1-MJD-CAG20 and pAS2-1-MJD-CAG80 at the BamHI and SalI sites and subcloning the resultant fragments into vector pREP10 (Invitrogen, Carlsbad, CA) at the BamHI and XhoI sites. The fidelity of all constructs was confirmed by sequencing.
Two-hybrid screen
The MATCHMARKER Two-Hybrid System 2 (Clontech) was used in this experiment. All experiments were performed in yeast strain CG-1945. Plasmids pAS2-1-MJD-CAG20, pAS2-1-MJD-CAG68 and pAS2-1-MJD-CAG80 were transformed into CG-1945 cells using the lithium acetate method according to the manual and the transformants then tested for the absence of autoactivation of the lacZ and HIS3 reporter genes and the absence of yeast cell toxicity. For the library screen, a single colony of CG-1945 cells transformed with pAS2-1-MJD-CAG20, pAS2-1-MJD-CAG68 or pAS2-1-MJD-CAG80 was grown overnight in SD-Trp medium and then transformed with a human fetal brain cDNA library constructed in vector pACT2 (Clontech). Approximately 2 x 106 transformants were plated on SD-Trp-Leu-His selective plates, with 5 mM 3-AT used to limit false positives caused by leakage of HIS3 protein expression. After incubation at 30°C for 5–7 days, all clones were subjected to ß-gal filter assays and patched on SD-Trp-Leu-His selective plates with 5 mM 3-AT for further analysis. Plasmid DNAs were isolated from the ß-gal-positive clones by growth in SD-Leu medium followed by transformation into E.coli HB101. The plasmids extracted from HB101 cells were first subjected to restriction enzyme analysis and those with library inserts then retransformed into CG-1945 cells harboring pAS2-1-MJD-CAG20, pAS2-1-MJD-CAG68 or pAS2-1-MJD-CAG80. The cells were selected on SD-Trp-Leu-His plates with 5 mM 3-AT. The resulting transformants that were positive in ß-gal filter assays were sequenced around the junction of the GAL4-AD and the inserted target protein using pACT2 primer 2 or reverse insert primer. In order to characterize binding between ataxin-3 and HHR23B, plasmids pACT2-HHR23B and pAS2-1-MJDs were co-transformed into yeast and the cells grown on selection plates. The transformants were also examined by ß-gal filter assays. For liquid ß-gal assays the yeast cells were cultured overnight in SD-Trp-Leu selection medium until the cells were in mid log phase (OD600 of 1 ml = 0.5–0.8). The cells were collected and assays were performed using o-nitrophenyl-ß-D-galactopyranoside (ONPG) (Sigma, St Louis, MO) as substrate according to the protocol.
In vitro binding assay
An aliquot containing 20 µg of protein from the soluble fraction of E.coli cell lysates expressing GST–HHR23A, GST–HHR23B or GST–ubiquitin was incubated with 20 µl of glutathione–agarose beads (1:1 slurry) (Amersham Pharmacia Biotech) for 20 min at room temperature. After washing three times with phosphate-buffered saline (PBS), beads bound with GST–HHR23A or GST–HHR23B were incubated with 50 µg of protein from the supernatants of E.coli crude extracts containing recombinant ataxin-3-Q20, ataxin-3-Q68 or ataxin-3-Q80 expressed from pET-21a(+)-MJD-CAG20, pET-21a(+)-MJD-CAG68 or pET-21a(+)-MJD-CAG80, respectively, or the supernatant from lymphoblastoid cell lines, 293 cells, control and MJD brains in 0.25 ml of HNTG buffer (20 mM HEPES–KOH, pH 7.5, 100 mM NaCl, 0.1% Triton X-100 and 10% glycerol) (19) for 1 h at 4°C. After incubation the beads were washed four times with 1 ml of HNTG buffer to remove unbound protein. Bound proteins were eluted from the beads by boiling in SDS sample buffer and detected by immunoblot analysis using a monoclonal anti-ataxin-3 antibody, MJ2-5-3 (7).
Cell culture and transfection
293 cells cultured overnight in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Rockville, MD) medium containing 10% calf serum (CS) were washed with Opti-MEM and then transfected with expressing vectors using LipofectAMINE 2000 reagent (Gibco) in Opti-MEM medium without serum. The same volume of DMEM containing 10% CS was added to the culture medium 6 h after transfection. After 48 h the transfected cells were observed using an inverted system microscope (Olympus, Tokyo, Japan) or used for immunofluorescence staining, immunoblot analysis or immunoprecipitation.
Immunofluorescence
Cells transfected with pcDNA3.1/v5-His-HHR23A or co-transfected with pEGFP-N1-MJDs and pcDNA3.1/v5-His-HHR23A were fixed in PBS with 4% paraformaldehyde for 5 min and then treated with 0.25% Triton X-100 for 5 min. After blocking in 4% bovine serum albumin in PBS for 1 h, the cells were incubated with monoclonal anti-v5 antibody (1:2000; Invitrogen) overnight at 4°C. After washing with PBS the cells were incubated with Cy3-labeled goat anti-mouse antibodies (1:800) (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. Sections were then examined using a confocal microscope (Fluoview) (Olympus).
Immunoprecipitation
293 cells co-transfected with pEGFP-N1 or pEGFP-N1-HHR23A and pREP10-MJD-CAG20 or pREP10-MJD-CAG80 were collected 48 h after transfection. The cells were sonicated in TSPI buffer (50 mM Tris–HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 µM Pefabloc SC and 10 µg/ml pepstain) containing 1% NP-40. Cellular debris was removed by centrifugation at 16 000 g for 15 min at 4°C. The supernatants were then preincubated with protein G–Sepharose (Amersham Pharmacia Biotech) for 2 h at 4°C and then incubated with monoclonal anti-GFP antibody (Boehringer Mannheim, Mannheim, Germany) for 1 h at 4°C. After incubation protein G–Sepharose was used for precipitation. The beads were washed with TSPI buffer four times and then eluted with SDS sample buffer for immunoblot analysis.
Immunoblot analysis
Proteins were separated by 10% SDS–PAGE or 5–20% gradient SDS–PAGE and then transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). The primary antibodies used were MJ2-5-3 antibody (7) and monoclonal anti-GFP antibody. Sheep anti-mouse IgG-HRP antibody (Amersham Pharmacia Biotech) was used as the secondary antibody. The proteins were visualized using an ECL detection kit (Amersham Pharmacia Biotech).
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ACKNOWLEDGEMENT |
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This work was partly supported by a grant from the Ministry of Health and Welfare.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 48 467 9702; Fax: +81 48 462 4796; Email: nukina@brain.riken.go.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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