Kaleidoscopic protein–protein interactions in the life and death of ataxin-1: new strategies against protein aggregation
Authors: Cesira de Chiara, Annalisa Pastore
Journal: Trends in Neurosciences (2014)

•Ataxin-1 (Atx1) is the protein responsible for spinocerebellar ataxia type 1 (SCA1).•Normal function and anomalous aggregation are competing pathways.•Protein–protein interactions protect Atx1 from aggregation and misfolding.•This knowledge can be exploited in drug development. Understanding how proteins protect themselves from aberrant aggregation is of primary interest for understanding basic biology, protein biochemistry, and human disease. We discuss the paradigmatic example of ataxin-1 (Atx1), the protein responsible for neurodegenerative spinocerebellar ataxia type 1 (SCA1). This disease is part of the increasing family of pathologies caused by protein aggregation and misfolding. We discuss the importance of protein–protein interactions not only in the nonpathological function of Atx1 but also in protecting the protein from aggregation and misfolding. The lessons learned from Atx1 may lead to a more general understanding of the cell's protective strategies against aggregation. The obtained knowledge may suggest a new perspective for designing specific therapeutic strategies for the cure of misfolding diseases. Understanding how proteins protect themselves from aberrant aggregation is of primary interest for understanding basic biology, protein biochemistry, and human disease. We discuss the paradigmatic example of ataxin-1 (Atx1), the protein responsible for neurodegenerative spinocerebellar ataxia type 1 (SCA1). This disease is part of the increasing family of pathologies caused by protein aggregation and misfolding. We discuss the importance of protein–protein interactions not only in the nonpathological function of Atx1 but also in protecting the protein from aggregation and misfolding. The lessons learned from Atx1 may lead to a more general understanding of the cell's protective strategies against aggregation. The obtained knowledge may suggest a new perspective for designing specific therapeutic strategies for the cure of misfolding diseases. The increasing realization that protein aggregation plays a predominant role in the development of a number of diseases parallels an increasing interest in the mechanisms that protect proteins from this often unwanted phenomenon. Although much has been said about these protection mechanisms, and several possible strategies that the cell may take in combating protein aggregation have been suggested [1Cohen F.E. Kelly J.W. Therapeutic approaches to protein-misfolding diseases.Nature. 2003; 426: 905-909Crossref PubMed Scopus (730) Google Scholar, 2Ehrnhoefer D.E. et al.EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1141) Google Scholar, 3Shao J. Diamond M.I. Polyglutamine diseases: emerging concepts in pathogenesis and therapy.Hum. Mol. Genet. 2007; 16: R115-R123Crossref PubMed Scopus (215) Google Scholar, 4Silva J.L. et al.Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.Acc. Chem. Res. 2010; 43: 271-279Crossref PubMed Scopus (98) Google Scholar], our knowledge of the topic remains limited. One of the most interesting hypotheses is that interactions with other cellular partners could have a leading role in preventing protein aggregation [3Shao J. Diamond M.I. Polyglutamine diseases: emerging concepts in pathogenesis and therapy.Hum. Mol. Genet. 2007; 16: R115-R123Crossref PubMed Scopus (215) Google Scholar, 5Boeddrich A. et al.An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.EMBO J. 2006; 25: 1547-1558Crossref PubMed Scopus (130) Google Scholar]. Several examples seem to support this hypothesis. It has, for instance, been observed that binding to ligands, including nucleic acids, prevents misfolding of the oncogene p53 [4Silva J.L. et al.Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.Acc. Chem. Res. 2010; 43: 271-279Crossref PubMed Scopus (98) Google Scholar]. Similarly, the protein ataxin-3 has been found to be protected from aggregation when interacting with the natural cellular partner ubiquitin [6Masino L. et al.Functional interactions as a survival strategy against abnormal aggregation.FASEB J. 2011; 25: 45-54Crossref PubMed Scopus (66) Google Scholar]. These examples in turn suggest that normal function and aberrant aggregation are competing pathways [7Pastore A. Temussi P.A. The two faces of Janus: functional interactions and protein aggregation.Curr. Opin. Struct. Biol. 2012; 22: 30-37Crossref PubMed Scopus (55) Google Scholar]. In this review, we discuss the paradigmatic example of Atx1. This protein provides a unique illustration of how protein–protein interactions can sort the 'fate' of a protein. Atx1 is responsible for SCA1 (OMIM #164400) [8Hamosh A. et al.Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders.Nucleic Acids Res. 2005; 33: D514-D517Crossref PubMed Scopus (2003) Google Scholar] (for a definition of ataxia, see the Glossary). SCA1 is a late onset autosomal dominant neurodegenerative disorder characterized by cerebellar ataxia and associated with varying degrees of oculomotor abnormalities, pyramidal and extrapyramidal features, peripheral neuropathy, and cognitive impairment [9Donato S.D. et al.Spinocerebellar ataxia type 1.Handb. Clin. Neurol. 2012; 103: 399-421Crossref PubMed Scopus (28) Google Scholar]. Until a link with disease was established, Atx1 had largely been overlooked. However, over the last decade or so, a substantial amount of research into the structure and function of Atx1 has started to provide a clearer picture of the cellular role of this protein [10de Chiara C. Pastore A. Polyglutamine diseases and neurodegeneration: the example of ataxin-1.in: Brnjas-Kraljevic J. Pifat-Mrzljak G. Supramolecular Structure and Function 10. Springer, 2011: 87-99Crossref Google Scholar, 11Orr H.T. Cell biology of spinocerebellar ataxia.J. Cell Biol. 2012; 197: 167-177Crossref PubMed Scopus (124) Google Scholar]. In the following sections, we review our current knowledge of Atx1 and place these observations within the context of the protection mechanisms against aggregation. We show how the normal function of the protein, together with its unusual structural properties, determine its cellular function/dysfunction and how aggregation is mediated by multiple regions acting in cooperation. We believe that this example may open a new perspective for the study of SCA1 and other misfolding diseases and eventually suggest a strategy for a specific cure. Atx1 is, together with the above mentioned ataxin-3, a member of the family of proteins that contain a polyglutamine (polyQ) tract and are implicated in genetic neurodegenerative diseases [12Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (172) Google Scholar] (Figure 1A) . These pathologies are caused by the anomalous expansion of a polymorphic tract of polyQ, which, when above a threshold of approximately 37 repeats, causes aggregation that is ultimately associated with cell toxicity and neuronal death [12Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (172) Google Scholar] (Figure 1B). Although rare and diversified in symptoms, these diseases are dominant and currently incurable. Several lines of evidence suggest that polyQ expansion is the necessary event for disease development: attachment of a polyQ stretch to an otherwise healthy protein is sufficient to cause toxicity; interruption of the polyQ tract by even one non-glutamine amino acid appreciably slows down disease and the age at disease onset correlates inversely with the number of uninterrupted polyQ repeats [13Menon R.P. et al.The role of interruptions in polyQ in the pathology of SCA1.PLoS Genet. 2013; 9: e1003648Crossref PubMed Scopus (47) Google Scholar]. It has however been recognized that, in addition to polyQ, other regions significantly contribute to the aggregation process. An increasing interest in studying Atx1 stems not only from the desire of finding a cure for SCA1 but also because, being one of the small members of the polyQ family, Atx1 is an excellent model for understanding the behavior of the whole family [14de Chiara, C. and Pastore, A. (2011) Prediction and experimental detection of structural and functional motifs in intrinsically unfolded proteins. In Selected Works in Bioinformatics (Xia, X., ed.), pp. 81-102, InTechGoogle Scholar]. Atx1 is a predominantly nuclear, ubiquitous protein well conserved in vertebrates [15Klement I.A. et al.Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice.Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar]. Human Atx1 is 816 amino acids long although its length can increase depending on the length of the polyQ tract, which varies from six to circa 44 glutamine repeats in the normal population and up to about 83 uninterrupted glutamines in SCA1 patients [10de Chiara C. Pastore A. Polyglutamine diseases and neurodegeneration: the example of ataxin-1.in: Brnjas-Kraljevic J. Pifat-Mrzljak G. Supramolecular Structure and Function 10. Springer, 2011: 87-99Crossref Google Scholar, 12Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (172) Google Scholar, 13Menon R.P. et al.The role of interruptions in polyQ in the pathology of SCA1.PLoS Genet. 2013; 9: e1003648Crossref PubMed Scopus (47) Google Scholar] (Figure 2A) . Prediction of the structure of Atx1 based on the amino acid sequence indicates that Atx1 is predominantly an intrinsically unstructured protein [14de Chiara, C. and Pastore, A. (2011) Prediction and experimental detection of structural and functional motifs in intrinsically unfolded proteins. In Selected Works in Bioinformatics (Xia, X., ed.), pp. 81-102, InTechGoogle Scholar]. The only globular domain present in the protein, named AXH (SMART SM00536), is an independently folded motif of approximately 120 amino acids that was identified on the bases of sequence conservation across species and secondary structure prediction [16de Chiara C. et al.The AXH module: an independently folded domain common to ataxin-1 and HBP1.FEBS Lett. 2003; 551: 107-112Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. According to the human Atx1 sequence (UniProtKB/Swiss-Prot No. P54253 +1), this domain spans residues 567–689 [16de Chiara C. et al.The AXH module: an independently folded domain common to ataxin-1 and HBP1.FEBS Lett. 2003; 551: 107-112Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. The long N-terminal region 1–566 contains the low complexity polymorphic polyQ tract starting at residue 197 and one of the two experimentally verified phosphorylation sites [14de Chiara, C. and Pastore, A. (2011) Prediction and experimental detection of structural and functional motifs in intrinsically unfolded proteins. In Selected Works in Bioinformatics (Xia, X., ed.), pp. 81-102, InTechGoogle Scholar]. The polyQ stretch is expected to be unstructured as shown by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy [17Masino L. et al.Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins.FEBS Lett. 2002; 513: 267-272Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. The C terminus (residues 690–816) is also predicted to be unstructured and contains several linear motifs, either clustered in proximity or including the phosphorylable S776, which are extremely important for protein localization [15Klement I.A. et al.Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice.Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar, 18Lai S. et al.14-3-3 Binding to ataxin-1(ATXN1) regulates its dephosphorylation at Ser-776 and transport to the nucleus.J. Biol. Chem. 2011; 286: 34606-34616Crossref PubMed Scopus (47) Google Scholar] and recognition of biological partners [19Emamian E.S. et al.Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice.Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 20de Chiara C. et al.Phosphorylation of s776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors.PLoS ONE. 2009; 4: e8372Crossref PubMed Scopus (48) Google Scholar]. The intrinsic tendency of Atx1 to degradation and aggregation even when in its non-expanded form has so far hampered the possibility of expressing and purifying the recombinant full-length protein in an intact mono-disperse form suitable for a structural characterization of its conformational ensemble. However, it was possible to produce the AXH domain in isolation and to investigate its structure both in the crystal and in solution. The crystal structure revealed the open barrel typical of an oligonucleotide-binding (OB) fold, a structural motif involved in nucleic acid and protein recognition [21Chen Y.W. et al.The structure of the AXH domain of spinocerebellar ataxin-1.J. Biol. Chem. 2004; 279: 3758-3765Crossref PubMed Scopus (50) Google Scholar]. The crystal asymmetric unit contains a dimer of dimers (Figure 2B). The individual protomers are arranged in an antiparallel fashion with the N termini sandwiched between the dimer interface. The dimer structure resembles two left hands (the protomers) touching each other in an antiparallel fashion so that the two thumbs (corresponding to the N termini) are close in space and parallel (Figure 2C). The protomers are nonsymmetric having the first approximately 20 N-terminal amino acids of the individual protomers adopting different conformations and thus defining asymmetric dimer interfaces. The unusually variable structure of the N terminus of the domain makes the AXH a unique case of a protein 'chameleon', that is, a protein that is able to adopt distinct conformations [21Chen Y.W. et al.The structure of the AXH domain of spinocerebellar ataxin-1.J. Biol. Chem. 2004; 279: 3758-3765Crossref PubMed Scopus (50) Google Scholar, 22de Chiara C. et al.Self-assembly and conformational heterogeneity of the AXH domain of ataxin-1: an unusual example of a chameleon fold.Biophys. J. 2013; 104: 1304-1313Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. The presence of a dimeric AXH domain also in solution was demonstrated by analytical ultracentrifugation studies, which showed that the predominant species in the low micromolar range of concentrations is dimeric, although in co-presence with higher molecular species [16de Chiara C. et al.The AXH module: an independently folded domain common to ataxin-1 and HBP1.FEBS Lett. 2003; 551: 107-112Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 21Chen Y.W. et al.The structure of the AXH domain of spinocerebellar ataxin-1.J. Biol. Chem. 2004; 279: 3758-3765Crossref PubMed Scopus (50) Google Scholar]. In agreement with these data, small angle X-ray scattering (SAXS) confirmed the existence of a complex equilibrium between monomeric, dimeric, and tetrameric species in solution [22de Chiara C. et al.Self-assembly and conformational heterogeneity of the AXH domain of ataxin-1: an unusual example of a chameleon fold.Biophys. J. 2013; 104: 1304-1313Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. Taken together, these findings indicate a strong tendency of the AXH domain to dimerize and suggest that the dimerization observed for the AXH could account for the known self-association of the full-length protein observed in the cell [23Burright E.N. et al.Identification of a self-association region within the SCA1 gene product, ataxin-1.Hum. Mol. Genet. 1997; 6: 513-518Crossref PubMed Scopus (57) Google Scholar]. Not long after the SCA1 gene was identified [24Banfi S. et al.Identification and characterization of the gene causing type 1 spinocerebellar ataxia.Nat. Genet. 1994; 7: 513-520Crossref PubMed Scopus (316) Google Scholar], it was discovered that expanded Atx1 has to enter the nucleus of Purkinje cells to become pathogenic [15Klement I.A. et al.Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice.Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar]. Subsequently, analysis of a transgenic mouse model for SCA1 showed that gene expression is dysregulated at a very early stage of the disease, when neurological hallmarks are still undetectable [25Lin X. et al.Polyglutamine expansion down-regulates specific neuronal genes before pathologic changes in SCA1.Nat. Neurosci. 2000; 3: 157-163Crossref PubMed Scopus (408) Google Scholar, 26Serra H.G. et al.Gene profiling links SCA1 pathophysiology to glutamate signaling in Purkinje cells of transgenic mice.Hum. Mol. Genet. 2004; 13: 2535-2543Crossref PubMed Scopus (148) Google Scholar]. Several transcriptional corepressors were also identified as modulators of the Atx1-mediated eye phenotype in a genetic screen in Drosophila [27Fernandez-Funez P. et al.Identification of genes that modify ataxin-1-induced neurodegeneration.Nature. 2000; 408: 101-106Crossref PubMed Scopus (551) Google Scholar]. Overall, these observations suggested that Atx1 is involved in transcription regulation and that SCA1 pathogenesis could be the result of alterations in gene expression, rather than or in addition to the gain of function mechanism triggered by polyQ expansion. Using a general read-out assay for repression of transcription it was shown that both full-length Atx1 and the isolated AXH domain repress transcription when tethered to DNA [28Tsai C.C. et al.Ataxin 1, a SCA1 neurodegenerative disorder protein, is functionally linked to the silencing mediator of retinoid and thyroid hormone receptors.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4047-4052Crossref PubMed Scopus (124) Google Scholar, 29de Chiara C. et al.Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.J. Mol. Biol. 2005; 354: 883-893Crossref PubMed Scopus (70) Google Scholar]. However, interaction with DNA must be mediated by other transcription factors because crosslinking experiments excluded a direct binding between Atx1 and DNA [29de Chiara C. et al.Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.J. Mol. Biol. 2005; 354: 883-893Crossref PubMed Scopus (70) Google Scholar]. Atx1 has been described as modulating the function of several transcriptional regulators, often by means of direct protein–protein interactions. The partners identified so far include polyQ binding protein-1 (PQBP1) [30Okazawa H. et al.Interaction between mutant ataxin-1 and PQBP-1 affects transcription and cell death.Neuron. 2002; 34: 701-713Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar], the mediator of retinoid and thyroid hormone receptors SMRT/SMRTER [28Tsai C.C. et al.Ataxin 1, a SCA1 neurodegenerative disorder protein, is functionally linked to the silencing mediator of retinoid and thyroid hormone receptors.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4047-4052Crossref PubMed Scopus (124) Google Scholar], the repressor Capicua (CIC) [31Lam Y.C. et al.ATAXIN-1 interacts with the repressor Capicua in its native complex to cause SCA1 neuropathology.Cell. 2006; 127: 1335-1347Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar], the transcription factors Senseless/Gfi-1 [32Tsuda H. et al.The AXH domain of Ataxin-1 mediates neurodegeneration through its interaction with Gfi-1/Senseless proteins.Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar] and Sp1 [33Goold R. et al.Down-regulation of the dopamine receptor D2 in mice lacking ataxin 1.Hum. Mol. Genet. 2007; 16: 2122-2134Crossref PubMed Scopus (52) Google Scholar], transcriptional complexes like Tip60-RORα [34Serra H.G. et al.RORalpha-mediated Purkinje cell development determines disease severity in adult SCA1 mice.Cell. 2006; 127: 697-708Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar], LANP-E4F [35Cvetanovic M. et al.The role of LANP and ataxin 1 in E4F-mediated transcriptional repression.EMBO Rep. 2007; 8: 671-677Crossref PubMed Scopus (46) Google Scholar] and HDAC-MEF2 [36Bolger T.A. et al.The neurodegenerative disease protein ataxin-1 antagonizes the neuronal survival function of myocyte enhancer factor-2.J. Biol. Chem. 2007; 282: 29186-29192Crossref PubMed Scopus (35) Google Scholar], and, more recently, CBF1, a Notch signaling pathway transcription factor [37Tong X. et al.Ataxin-1 and Brother of ataxin-1 are components of the Notch signalling pathway.EMBO Rep. 2011; 12: 428-435Crossref PubMed Scopus (52) Google Scholar]. Several of these interactions map to the AXH domain (Figure 2A) and have been shown to be positively or negatively affected by expansion of the polyQ with consequent perturbation of the associated transcriptional pathway. In addition to a role in transcriptional regulation [10de Chiara C. Pastore A. Polyglutamine diseases and neurodegeneration: the example of ataxin-1.in: Brnjas-Kraljevic J. Pifat-Mrzljak G. Supramolecular Structure and Function 10. Springer, 2011: 87-99Crossref Google Scholar, 12Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (172) Google Scholar], Atx1 is an RNA binding protein. It recognizes RNA homo-polymers in a manner that depends on the length of the polyQ tract [38Yue S. et al.The spinocerebellar ataxia type 1 protein, ataxin-1, has RNA-binding activity that is inversely affected by the length of its polyglutamine tract.Hum. Mol. Genet. 2001; 10: 25-30Crossref PubMed Scopus (128) Google Scholar]. The AXH domain was also identified as the region responsible for RNA homo-polymer binding, exhibiting the same preference as the full-length Atx1 [16de Chiara C. et al.The AXH module: an independently folded domain common to ataxin-1 and HBP1.FEBS Lett. 2003; 551: 107-112Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. Further support for a role of Atx1 in RNA metabolism is provided by investigations of the protein interactome, most of which consists of proteins involved in RNA binding. Atx1 was shown to recruit the mRNA export factor TAP/NXF1 in an RNA-dependent manner, suggesting a role in processing and/or exporting specific mRNAs to the cytoplasm [39Irwin S. et al.RNA association and nucleocytoplasmic shuttling by ataxin-1.J. Cell Sci. 2005; 118: 233-242Crossref PubMed Scopus (94) Google Scholar]. Interestingly, the majority of the RNA binding proteins thereafter identified as Atx1 binding partners, that is, RBM9/FOX2, A2BP/FOX-1 [40Lim J. et al.A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.Cell. 2006; 125: 801-814Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar], RBM17/SPF45 and U2AF65 [41Lim J. et al.Opposing effects of polyglutamine expansion on native protein complexes contribute to SCA1.Nature. 2008; 452: 713-718Crossref PubMed Scopus (260) Google Scholar], are splicing factors. In the case of RBM17 and U2AF65, the interaction was mapped onto the short linear UHM ligand motif (ULM) in the C terminus of Atx1, a sequence known to be involved in recognition of the splicing factor U2AF (U2 auxiliary factor) homology motif (UHM) domain [20de Chiara C. et al.Phosphorylation of s776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors.PLoS ONE. 2009; 4: e8372Crossref PubMed Scopus (48) Google Scholar]. It was also shown that Atx1 has a positive effect on U2AF65 splicing [20de Chiara C. et al.Phosphorylation of s776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors.PLoS ONE. 2009; 4: e8372Crossref PubMed Scopus (48) Google Scholar] and that overexpression of Atx1 positively affects splicing of transcripts of the other polyQ-containing protein ataxin-2 by RBM9/FOX2 [42Welzel F. et al.FOX-2 dependent splicing of ataxin-2 transcript is affected by ataxin-1 overexpression.PLoS ONE. 2012; 7: e37985Crossref PubMed Scopus (7) Google Scholar]. These findings strongly suggest a specific role of Atx1 in pre-mRNA processing. Although polyQ expansion plays a predominant role in disease, it has been demonstrated that polyQ expansion is a necessary but not sufficient condition for disease development and that regions either flanking or sequentially distant from the polyQ tract contribute to protein aggregation [12Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (172) Google Scholar]. One such region is the AXH domain [29de Chiara C. et al.Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.J. Mol. Biol. 2005; 354: 883-893Crossref PubMed Scopus (70) Google Scholar] and, C terminal to it, the stretch that contains phosphorylable S776 and the nuclear localization signal (NLS). These motifs seem to be particularly important for the formation of inclusions and SCA1 progression [12Zoghbi H.Y. Orr H.T. Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1.J. Biol. Chem. 2009; 284: 7425-7429Crossref PubMed Scopus (172) Google Scholar, 15Klement I.A. et al.Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice.Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar, 19Emamian E.S. et al.Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice.Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 20de Chiara C. et al.Phosphorylation of s776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors.PLoS ONE. 2009; 4: e8372Crossref PubMed Scopus (48) Google Scholar, 29de Chiara C. et al.Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.J. Mol. Biol. 2005; 354: 883-893Crossref PubMed Scopus (70) Google Scholar, 43Skinner P.J. et al.Amino acids in a region of ataxin-1 outside of the polyglutamine tract influence the course of disease in SCA1 transgenic mice.Neuromol. Med. 2002; 1: 33-42Crossref PubMed Scopus (25) Google Scholar]. For example, it was demonstrated that the isolated AXH domain possesses an intrinsic ability to aggregate and that, in vitro, the domain spontaneously forms fibers even in the absence of destabilizing conditions, whereas in eukaryotic cells its propensity to multimerization positively influences the aggregation of the expanded full-length protein [29de Chiara C. et al.Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.J. Mol. Biol. 2005; 354: 883-893Crossref PubMed Scopus (70) Google Scholar]. Thus, it has been suggested that, in addition to polyQ, AXH may act as a second aggregation 'hotspot'. The possibility of forming dimers, tetramers, and higher molecular species observed at the level of the isolated AXH seems to be on-pathway to fiber formation. This could explain why overexpression of non-expanded Atx1 (i.e., 30 glutamines) in flies and mice causes phenotypes similar to those caused by overexpression of Drosophila Atx1, which lacks the polyQ tract, but different from those observed for polyQ peptides [27Fernandez-Funez P. et al.Identification of genes that modify ataxin-1-induced neurodegeneration.Nature. 2000; 408: 101-106Crossref PubMed Scopus (551) Google Scholar, 32Tsuda H. et al.The AXH domain of Ataxin-1 mediates neurodegeneration through its interaction with Gfi-1/Senseless proteins.Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar]. The evidence of an involvement of AXH as an independent aggregation hotspot is so compelling as to allow Zohgbi and coworkers to suggest that 'the AXH domain but not the expanded polyQ tract could be necessary to generate the Atx1 gain-of-function phenotype in flies' and be required for SCA1 pathogenesis [32Tsuda H. et al.The AXH domain of Ataxin-1 mediates neurodegeneration through its interaction with Gfi-1/Senseless proteins.Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar]. Therefore, the pathological behavior of Atx1 is a consequence of a complex crosstalk between several different regions. This is not the first example of a protein with multiple aggregation sites comprising both intrinsically unfolded and globular domains [44Saunders H.M. et al.Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein.Protein Sci. 2011; 20: 1675-1681Crossref PubMed Scopus (33) Google Scholar]. Protein aggregation has been suggested to be a competing pathway of normal function and therefore of functional protein–protein interactions [7Pastore A. Temussi P.A. The two faces of Janus: functional interactions and protein aggregation.Curr. Opin. Struct. Biol. 2012; 22: 30-37Crossref PubMed Scopus (55) Google Scholar] (Figure 3A) . This statement appears to hold true for Atx1; although the subject is still debated, SCA1 appears to be better explained by a loss of function caused by alterations of native pro