The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291Δ). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291Δ strongly reduced the growth rate in the early stages (2–4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291Δ on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular β-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.     

Temperature profoundly affects ataxin-3 fibrillogenesis

Ataxin-3 (AT3) triggers spinocerebellar ataxia type 3 when it carries a polyglutamine stretch expanded beyond a critical threshold. By Fourier transform infrared spectroscopy and atomic force microscopy we previously showed that a normal (AT3Q24) and an expanded (AT3Q55) variant were capable of evolving into oligomers and protofibrils at 37 °C, whereas only the expanded form generated irreversibly aggregated fibrils that also were associated with a network of side-chain glutamine hydrogen bonding [Natalello et al. (2011) PLoS One. 6:e18789]. We report here that AT3Q24, when gradually heated up to 85 °C, undergoes aggregation similar to that observed at 37 °C; in contrast, AT3Q55 only generates large, amorphous aggregates. We propose a possible interpretation of the mechanism by which temperature affects the outcome of fibrillogenesis.     

The two-stage pathway of ataxin-3 fibrillogenesis involves a polyglutamine-independent step

The aggregation of ataxin-3 is associated with spinocerebellar ataxia type 3, which is characterized by the formation of intraneuronal aggregates. However, the mechanism of aggregation is currently not well understood. Ataxin-3 consists of a folded Josephin domain followed by two ubiquitin-interacting motifs and a C-terminal polyglutamine tract, which in the non-pathological form is less than 45 residues in length. We demonstrate that ataxin-3 with 64 glutamines (at(Q64)) undergoes a two-stage aggregation. The first stage involves formation of SDS-soluble aggregates, and the second stage results in formation of SDS-insoluble aggregates via the poly(Q) region. Both these first and second stage aggregates display typical amyloid-like characteristics. Under the same conditions at(Q15) and at(QHQ) undergo a single step aggregation event resulting in SDS-soluble aggregates, which does not involve the polyglutamine tract. These aggregates do not convert to the SDS-insoluble form. These observations demonstrate that ataxin-3 has an inherent capacity to aggregate through its non-polyglutamine domains. However, the presence of a pathological length polyglutamine tract introduces an additional step resulting in formation of a highly stable amyloid-like aggregate.     

Towards a structural understanding of the fibrillization pathway in Machado-Joseph's disease: trapping early oligomers of non-expanded ataxin-3

Machado-Joseph's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under near-physiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in beta-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin-3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph's disease, as well as valuable diagnostic markers in disease settings.     

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.     

Suppression of polyglutamine toxicity by the yeast Sup35 prion domain in Drosophila

The propensity of proteins to form beta-sheet-rich amyloid fibrils is related to a variety of biological phenomena, including a number of human neurodegenerative diseases and prions. A subset of amyloidogenic proteins forms amyloid fibrils through glutamine/asparagine (Q/N)-rich domains, such as pathogenic polyglutamine (poly(Q)) proteins involved in neurodegenerative disease, as well as yeast prions. In the former, the propensity of an expanded poly(Q) tract to abnormally fold confers toxicity on the respective protein, leading to neuronal dysfunction. In the latter, Q/N-rich prion domains mediate protein aggregation important for epigenetic regulation. Here, we investigated the relationship between the pathogenic ataxin-3 protein of the human disease spinocerebellar ataxia type 3 (SCA3) and the yeast prion Sup35, using Drosophila as a model system. We found that the capacity of the Sup35 prion domain to mediate protein aggregation is conserved in Drosophila. Although select yeast prions enhance poly(Q) toxicity in yeast, the Sup35N prion domain suppressed poly(Q) toxicity in the fly. Suppression required the oligopeptide repeat of the Sup35N prion domain, which is critical for prion properties in yeast. These results suggest a trans effect of prion domains on pathogenic poly(Q) disease proteins in a multicellular environment and raise the possibility that Drosophila may allow studies of prion mechanisms.     

Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73–96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided.Polyglutamine (polyQ)¹ diseases comprise a group of hereditary neurodegenerative disorders in which expansion of polyQ stretches within their causative proteins induces protein aggregation and the formation of polyQ-containing neuronal aggregates (1). The mechanisms by which expanded polyQ regions contribute to aggregation and disease are not well understood. In all cases, polyQ length is negatively correlated with the age of onset of the disease (2), but the various polyQ disorders are associated with different neurodegenerative symptoms and affect different regions of the brain (3). Several of the polyQ proteins, including ataxin-3 (atx-3) (4) and huntingtin (5), have been shown to aggregate in vitro through a complex multidomain misfolding pathway (6) in which flanking domain aggregation precedes polyQ aggregation. Increasing evidence also suggests a key role for misfolding of flanking regions in the process of polyQ aggregation in vivo (7 –10). Thus, as the proteins have no sequence similarity other than in their polyQ regions, flanking domain content may be significant in determining the disease state and neuronal-specific selectivity. Given that there is growing support to suggest that the toxic entities in polyQ diseases are the soluble oligomers and assembly intermediates, rather than the fibrillar aggregates (11), effective therapeutics may be generated by targeting flanking domain interactions (12) rather than targeting the polyQ region itself. An enhanced understanding of the molecular mechanisms of assembly of polyQ proteins is required, as is a greater comprehension of the effects of polyQ length on the structure, dynamics, aggregation propensity, and oligomerisation pathway of the flanking domains. Here, we set out to determine the influence of an expanded polyQ tract on each stage of atx-3 aggregation by harnessing the power of mass-spectrometry-based approaches to identify and characterize assembly mechanisms (13, 14).Atx-3 consists of a structured N-terminal Josephin domain (JD), which has ubiquitin protease activity (15) and an intrinsically disordered C-terminal region, the latter containing several ubiquitin-interacting motifs (UIMs) followed by the polyQ tract and a variable region (16) (Fig. 2A). In vivo, expansion of the polyQ stretch beyond ca. 55 glutamine residues results in Machado–Joseph disease (17). Consistent with this, atx-3 with a polyQ tract beyond ca. 55 glutamine residues aggregates into amyloid-like fibrils rapidly in vitro (18). Aggregation proceeds by means of a two-stage pathway (4): the first stage resulting in the production of SDS-sensitive, short, curvilinear, protofibrils, and the second producing long-straight and SDS-resistant mature fibrils. The first stage involves self-association of the JD (19) and occurs in all atx-3 variants whether or not they contain a polyQ region of nonpathological length (nonexpanded, 12–40 glutamine residues (17)), an expanded polyQ region of disease length (polyQ-expanded, 55–84 glutamine residues (17)), or are devoid of a polyQ region (20). The second stage occurs only in polyQ-expanded atx-3 and involves hydrogen bonding between side-chains in the polyQ region (21), which renders aggregation irreversible.Open in a separate windowFig. 2.Limited proteolysis of protofibrils and mature fibrils. (A) Schematic illustrations of atx-3(14Q) (left) and atx-3(78Q) (right) with amino acid residue numbers for each domain shown. Mass spectra obtained following limited proteolysis with trypsin of (B) atx-3(14Q) protofibrils (C) atx-3(78Q) protofibrils and (D) atx-3(78Q) mature fibrils. Mass spectra of (left) the depolymerized fibrillar material are contrasted with those obtained from analysis of (right) the soluble products of proteolysis. Asterisks represent species observed in the pellet fraction that were also observed in supernatant samples ((-16)-45⁴⁺ and (-16)-47⁴⁺, respectively). Peaks identified as containing the polyQ tract are highlighted in pink, while those representing QBP-1 are highlighted in orange.Despite the fact that the first stage of atx-3 aggregation does not require the polyQ tract, aggregation of polyQ-expanded atx-3 occurs more rapidly (with a shorter lag time) than aggregation of nonexpanded atx-3 (4, 20). The precise molecular mechanism for this observation has yet to be elucidated. An initial hypothesis was that polyQ expansion destabilizes atx-3, allowing the JD to adopt misfolded, aggregation-prone conformations more readily (15, 18). However, a study comparing atx-3 constructs with polyQ regions of different lengths showed that polyQ expansion does not affect the folding/unfolding kinetics or thermodynamic stability of the JD (22). Consequently, it has been postulated that the expanded polyQ tract may perturb the structure of the JD without affecting its stability (20).We set out to address why aggregation occurs more rapidly in atx-3 with an expanded polyQ tract by studying monomeric, oligomeric, and fibril structures for atx-3 with a pathological length polyQ tract of 77 glutamines with a single, naturally occurring, lysine residue in the fourth position (named atx-3(78Q)); atx-3 with a nonpathological length polyQ tract of 13 glutamines (also with a single lysine residue in the fourth position) (atx-3(14Q)); and the isolated JD. Results from a combination of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), limited proteolysis, fluorescence spectroscopy, and transmission electron microscopy (TEM) analyses confirm that protofibrils of these three atx-3 constructs are formed through equivalent processes and reveal that the resulting protofibril cores are similar, if not identical. Limited proteolysis experiments combined with MS analyses provide evidence that an expanded polyQ tract alters the conformational dynamics of the JD, exposing its aggregation-prone region more frequently than in its nonexpanded counterparts, rationalizing the enhanced aggregation potential of the polyQ-expanded protein. Finally, oligomers populated en route to fibrils are examined by ESI-IMS-MS and a model for oligomer growth is provided. Together these results reveal how polyQ length affects each stage of atx-3 aggregation and demonstrate how different MS-based techniques can provide information about each stage of the aggregation mechanism.     

Conformational Behavior and Aggregation of Ataxin-3 in SDS

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases all characterized by the presence of intraneuronal inclusions that contain aggregated protein. Aggregation of ataxin-3, the causative protein of SCA3, has been well characterized in vitro, with both pathogenic and non-pathogenic length ataxin-3 undergoing fibrillogenesis. However, only ataxin-3 containing an expanded polyQ tract leads to SCA3. Therefore other cellular factors, not present in previous in vitro studies, may modulate aggregation during disease. The interactions between fibrillar species and cell membranes have been characterized in a number of amyloid diseases, including Huntington’s Disease, and these interactions affect aggregation and toxicity. We have characterized the effects of the membrane mimetic sodium dodecyl sulfate (SDS) on ataxin-3 structure and aggregation, to show that both micellar and non-micellar SDS have differing effects on the two stages of ataxin-3 aggregation. We also demonstrate that fibrillar ataxin-3 binds phospholipids, in particular phosphorylated phosphotidylinositols. These results highlight the effect of intracellular factors on the ataxin-3 misfolding landscape and their implications in SCA3 and polyQ diseases in general are discussed.     

Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat–containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.     

Temperature-dependent, irreversible formation of amyloid fibrils by a soluble human ataxin-3 carrying a moderately expanded polyglutamine stretch (Q36)

The protein ataxin-3 is responsible for Machado-Joseph disease/spinocerebellar ataxia type 3, a neurodegenerative disorder caused by the presence of an expanded polyglutamine tract. A previous investigation [Bevivino, A. E., and Loll, P. J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11955-11960] showed that a nonexpanded ataxin-3 (Q27) was fully soluble, whereas an expanded form (Q78) gave rise to amyloid fibrils. Here, we report investigations on three forms of ataxin-3 (i.e., human nonexpanded (Q26), moderately expanded (Q36) ataxins-3, and the murine protein (Q6)). Far-UV circular dichroism spectra at room temperature were substantially similar, with a relatively high helical content. On heating to 96 degrees C, human Q26 and murine proteins did not display large structural changes, nor did they undergo any precipitation, which highlights their amazing heat-resistance. In contrast, human Q36 ataxin-3 underwent a progressive increase in the beta-sheet and a concomitant decrease in helical content when the temperature was shifted from 37 to 80 degrees C, followed by the irreversible formation of aggregates above 80 degrees C. They were shown to consist of amyloid fibrils, as supported by both electron microscopy images and the typical spectral shift displayed by Congo red when it was added to the protein at growing temperatures. We also found that protein precipitation could be prevented by mixing the dye with Q36 ataxin-3 prior to heating, which also confirms that the precipitates do represent authentic amyloid fibrils. In contrast, other compounds structurally related to Congo red did not exert significant effects. Our observations suggest that the temperature of the observed transition is inversely related to the length of the expansion. Finally, we suggest that antiamyloidogenic compounds might be selected on the basis of their ability to block or retard human Q36 ataxin-3 precipitation on heat-treatment.     

Different ataxin-3 amyloid aggregates induce intracellular Ca2 + deregulation by different mechanisms in cerebellar granule cells

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca^2 + levels and the abnormal Ca^2 + signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca^2 + responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca^2 + response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.     

Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease, is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated disease genes. Polyglutamine disorders are characterized by disease protein misfolding and aggregation; often within the nuclei of affected neurons. Although the precise mechanism of polyglutamine-mediated cell death remains elusive, evidence suggests that proteolysis of polyglutamine disease proteins by caspases contributes to pathogenesis. Using cellular models we now show that the endogenous spinocerebellar ataxia type-3 disease protein, ataxin-3, is proteolyzed in apoptotic paradigms, resulting in the loss of full-length ataxin-3 and the corresponding appearance of an approximately 28-kDa fragment containing the glutamine repeat. Broad-spectrum caspase inhibitors block ataxin-3 proteolysis and studies suggest that caspase-1 is a primary mediator of cleavage. Site-directed mutagenesis experiments eliminating three, six or nine potential caspase cleavage sites in the protein suggest redundancy in the site(s) at which cleavage can occur, as previously described for other disease proteins; but also map a major cleavage event to a cluster of aspartate residues within the ubiquitin-binding domain of ataxin-3 near the polyglutamine tract. Finally, caspase-mediated cleavage of expanded ataxin-3 resulted in increased ataxin-3 aggregation, suggesting a potential role for caspase-mediated proteolysis in spinocerebellar ataxia type-3 pathogenesis.     

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.     

Swelling behavior and structural characteristics of wheat gluten polypeptide films

Wheat gluten films were subjected to controlled thermomechanical treatments to increase the percentage of aggregated sodium dodecyl sulfate (SDS)-insoluble gluten protein, the aggregation reaction being disulfide bonding. The rheological properties of the films were measured under immersion in water, where wheat gluten films are stable and show only slight swelling. The equilibrium swelling of the gluten films in water decreased with the increase of the percentage of SDS-insoluble protein aggregates, and the frequency the independent shear modulus increased sharply with increasing percentage of SDS-insoluble aggregates. Both findings confirm that disulfide bonding between gluten proteins is the predominant cross-linking reaction in the system. A relationship between shear modulus and aggregated protein compatible with a power law (of exponent 3) suggests the existence of a protein network at a molecular scale. However, the classical Flory-Rehner model failed to describe the relationship between the plateau modulus and the gluten volume fraction (a very drastic increase, compatible with a power law of an exponent of about 14). This result shows that gluten cannot be described as an entangled polymer network. The interpretation of both relationships is a network of mesoscale particles which in turn have a fractal inner structure (with a fractal dimension close to 3).     

Mechanisms of ataxin-3 misfolding and fibril formation: kinetic analysis of a disease-associated polyglutamine protein

The polyglutamine diseases are a family of nine proteins where intracellular protein misfolding and amyloid-like fibril formation are intrinsically coupled to disease. Previously, we identified a complex two-step mechanism of fibril formation of pathologically expanded ataxin-3, the causative protein of spinocerebellar ataxia type-3 (Machado-Joseph disease). Strikingly, ataxin-3 lacking a polyglutamine tract also formed fibrils, although this occurred only via a single-step that was homologous to the first step of expanded ataxin-3 fibril formation. Here, we present the first kinetic analysis of a disease-associated polyglutamine repeat protein. We show that ataxin-3 forms amyloid-like fibrils by a nucleation-dependent polymerization mechanism. We kinetically model the nucleating event in ataxin-3 fibrillogenesis to the formation of a monomeric thermodynamic nucleus. Fibril elongation then proceeds by a mechanism of monomer addition. The presence of an expanded polyglutamine tract leads subsequently to rapid inter-fibril association and formation of large, highly stable amyloid-like fibrils. These results enhance our general understanding of polyglutamine fibrillogenesis and highlights the role of non-poly(Q) domains in modulating the kinetics of misfolding in this family.     

Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.     

The Josephin domain determines the morphological and mechanical properties of ataxin-3 fibrils

Fibrillar aggregation of the protein ataxin-3 is linked to the inherited neurodegenerative disorder Spinocerebellar ataxia type 3, a member of the polyQ expansion disease family. We previously reported that aggregation and stability of the nonpathological form of ataxin-3, carrying an unexpanded polyQ tract, are modulated by its N-terminal Josephin domain. It was also shown that expanded ataxin-3 aggregates via a two-stage mechanism initially involving Josephin self-association, followed by a polyQ-dependent step. Despite this recent progress, however, the exact mechanism of ataxin-3 fibrilization remains elusive. Here, we have used electron microscopy, atomic force microscopy, and other biophysical techniques to characterize the morphological and mechanical properties of nonexpanded ataxin-3 fibrils. By comparing aggregates of ataxin-3 and of the isolated Josephin domain, we show that the two proteins self-assemble into fibrils with markedly similar features over the temperature range 37–50°C. Estimates of persistence length and Young's modulus of the fibrils reveal a great flexibility. Our data indicate that, under physiological conditions, during early aggregation Josephin retains a nativelike secondary structure but loses its enzymatic activity. The results suggest a key role of Josephin in ataxin-3 fibrillar aggregation.     

Compromised mitochondrial complex II in models of Machado-Joseph disease

Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.     

Compromised mitochondrial complex II in models of Machado-Joseph disease

Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.