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.     

Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein

Spinocerebellar Ataxia Type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases that are all characterized by progressive neuronal dysfunction and the presence of neuronal inclusions containing aggregated polyQ protein, suggesting that protein misfolding is a key part of this disease. Ataxin-3, the causative protein of SCA3, contains a globular, structured N-terminal domain (the Josephin domain) and a flexible polyQ-containing C-terminal tail, the repeat-length of which modulates pathogenicity. It has been suggested that the fibrillogenesis pathway of ataxin-3 begins with a non-polyQ-dependent step mediated by Josephin domain interactions, followed by a polyQ-dependent step. To test the involvement of the Josephin domain in ataxin-3 fibrillogenesis, we have created both pathogenic and nonpathogenic length ataxin-3 variants with a stabilized Josephin domain, and have both stabilized and destabilized the isolated Josephin domain. We show that changing the thermodynamic stability of the Josephin domain modulates ataxin-3 fibrillogenesis. These data support the hypothesis that the first stage of ataxin-3 fibrillogenesis is caused by interactions involving the non-polyQ containing Josephin domain and that the thermodynamic stability of this domain is linked to the aggregation propensity of ataxin-3.     

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.     

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.     

A major role for side-chain polyglutamine hydrogen bonding in irreversible ataxin-3 aggregation

The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an anti-oligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm(-1) in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry.     

NEDD8: a new ataxin-3 interactor

Machado-Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate.     

The Machado–Joseph disease‐associated form of ataxin‐3 impacts dynamics of clathrin‐coated pits

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin‐3 is the main cause of neurodegeneration in Machado–Joseph disease. Ataxin‐3 contains an N‐terminal catalytic domain, called Josephin domain, and a highly aggregation‐prone C‐terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin‐mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin‐3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin‐3 could impact CME. Here, we report that both the wild‐type and the expanded ataxin‐3 reduce transferrin internalization and expanded ataxin‐3 impacts dynamics of clathrin‐coated pits (CCPs) by reducing CCP nucleation and increasing short‐lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.     

Crystal structure of a Josephin-ubiquitin complex: evolutionary restraints on ataxin-3 deubiquitinating activity

The Josephin domain is a conserved cysteine protease domain found in four human deubiquitinating enzymes: ataxin-3, the ataxin-3-like protein (ATXN3L), Josephin-1, and Josephin-2. Josephin domains from these four proteins were purified and assayed for their ability to cleave ubiquitin substrates. Reaction rates differed markedly both among the different proteins and for different substrates with a given protein. The ATXN3L Josephin domain is a significantly more efficient enzyme than the ataxin-3 domain despite their sharing 85% sequence identity. To understand the structural basis of this difference, the 2.6 Å x-ray crystal structure of the ATXN3L Josephin domain in complex with ubiquitin was determined. Although ataxin-3 and ATXN3L adopt similar folds, they bind ubiquitin in different, overlapping sites. Mutations were made in ataxin-3 at selected positions, introducing the corresponding ATXN3L residue. Only three such mutations are sufficient to increase the catalytic activity of the ataxin-3 domain to levels comparable with that of ATXN3L, suggesting that ataxin-3 has been subject to evolutionary restraints that keep its deubiquitinating activity in check.     

Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology.     

d-Polyglutamine Amyloid Recruits l-Polyglutamine Monomers and Kills Cells

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.     

Identification of human proteins that modify misfolding and proteotoxicity of pathogenic ataxin-1

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.     

Structural and functional analysis of the Josephin domain of the polyglutamine protein ataxin-3

Ataxin-3 belongs to the family of polyglutamine proteins, which are associated with nine different neurodegenerative disorders. Relatively little is known about the structural and functional properties of ataxin-3, and only recently have these aspects of the protein begun to be explored. We have performed a preliminary investigation into the conserved N-terminal domain of ataxin-3, termed Josephin. We show that Josephin is a monomeric domain which folds into a globular conformation and possesses ubiquitin protease activity. In addition, we demonstrate that the presence of the polyglutamine region of the protein does not alter the structure of the protein. However, its presence destabilizes the Josephin domain. The implications of these data in the pathogenesis of polyglutamine repeat proteins are discussed.     

A Highlights from MBoC Selection: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases, spinocerebellar ataxia type 3 (SCA3) and Huntington’s disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain, is diminished when polyQ repeat proteins (ataxin-3/huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation, resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates, rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in a transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyQ aggregates.     

Novel Polyglutamine Model Uncouples Proteotoxicity from Aging

Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia. In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins. We are interested in elucidating the underlying mechanisms of toxicity of the protein ataxin-3, in which a polyglutamine expansion is the genetic determinant for Machado-Joseph Disease (MJD), also referred to as spinocerebellar ataxia 3 (SCA3). To this end, we have developed a novel model for ataxin-3 protein aggregation, by expressing a disease-related polyglutamine-containing fragment of ataxin-3 in the genetically tractable body wall muscle cells of the model system C. elegans. Here, we demonstrate that this ataxin-3 fragment aggregates in a polyQ length-dependent manner in C. elegans muscle cells and that this aggregation is associated with cellular dysfunction. However, surprisingly, this aggregation and resultant toxicity was not influenced by aging. This is in contrast to polyglutamine peptides alone whose aggregation/toxicity is highly dependent on age. Thus, the data presented here not only describe a new polyglutamine model, but also suggest that protein context likely influences the cellular interactions of the polyglutamine-containing protein and thereby modulates its toxic properties.     

Expanded polyglutamines impair synaptic transmission and ubiquitin-proteasome system in Caenorhabditis elegans

Polyglutamine (polyQ) expansion in many proteins, including huntingtin and ataxin-3, is pathogenic and responsible for neuronal dysfunction and degeneration. Although at least nine neurodegenerative diseases are caused by expanded polyQ, the pathogenesis of these diseases is still not well understood. In the present study, we used Caenorhabditis elegans to study the molecular mechanism of polyQ-mediated toxicity. We expressed full-length and truncated ataxin-3 with different lengths of polyQ in the nervous system of C. elegans. We show that expanded polyQ interrupts synaptic transmission, and induces swelling and aberrant branching of neuronal processes. Using an ubiquitinated fluorescence reporter construct, we also showed that polyQ aggregates impair the ubiquitin-proteasome system in C. elegans. These results may provide information for further understanding the pathogenesis of polyQ diseases.     

An apparent core/shell architecture of polyQ aggregates in the aging Caenorhabditis elegans neuron

Huntington''s disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein which results in its abnormal aggregation in the nervous system. Huntingtin aggregates are linked to toxicity and neuronal dysfunction, but a comprehensive understanding of the aggregation mechanism in vivo remains elusive. Here, we examine the morphology of polyQ aggregates in Caenorhabditis elegans mechanosensory neurons as a function of age using confocal and fluorescence lifetime imaging microscopy. We find that aggregates in young worms are mostly spherical with homogenous intensity, but as the worm ages aggregates become substantially more heterogeneous. Most prominently, in older worms we observe an apparent core/shell morphology of polyQ assemblies with decreased intensity in the center. The fluorescence lifetime of polyQ is uniform across the aggregate indicating that the dimmed intensity in the assembly center is most likely not due to quenching or changes in local environment, but rather to displacement of fluorescent polyQ from the central region. This apparent core/shell architecture of polyQ aggregates in aging C. elegans neurons contributes to the diverse landscape of polyQ aggregation states implicated in Huntington''s disease.     

Josephin domain-containing proteins from a variety of species are active de-ubiquitination enzymes

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by the presence of an extended polyglutamine stretch (polyQ) in the unstructured C-terminus of the human ataxin-3 (AT3) protein. The structured N-terminal Josephin domain (JD) of AT3 is conserved within a novel family of potential ubiquitin proteases, the JD-containing proteins, which are sub-divided into two groups termed ataxins and Josephins. These AT3 orthologs are encoded by the genomes of organisms ranging from Plasmodium falciparum to humans, with most species possessing more than one homolog. While Josephins consist of JDs alone, ataxins contain additional functional domains that may influence their enzyme activity. Here, we show that the enzyme activity of human AT3 (hAT3) is not affected by the length of polyQ in its C-terminus, even when it is in the range associated with SCA3. We also show that JDs of all human proteins with homology to AT3 and its homologs from various species possess de-ubiquitination activity. These results establish JD-containing proteins as a novel family of active de-ubiquitination enzymes with wide phylogenic distribution.     

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.     

Structural features and domain organization of huntingtin fibrils

Misfolding and aggregation of huntingtin is one of the hallmarks of Huntington disease, but the overall structure of these aggregates and the mechanisms by which huntingtin misfolds remain poorly understood. Here we used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study the structural features of huntingtin exon 1 (HDx1) containing 46 glutamine residues in its polyglutamine (polyQ) region. Despite some residual structuring in the N terminus, we find that soluble HDx1 is highly dynamic. Upon aggregation, the polyQ domain becomes strongly immobilized indicating significant tertiary or quaternary packing interactions. Analysis of spin-spin interactions does not show the close contact between same residues that is characteristic of the parallel, in-register structure commonly found in amyloids. Nevertheless, the same residues are still within 20 Å of each other, suggesting that polyQ domains from different molecules come into proximity in the fibrils. The N terminus has previously been found to take up a helical structure in fibrils. We find that this domain not only becomes structured, but that it also engages in tertiary or quaternary packing interactions. The existence of spin-spin interactions in this region suggests that such contacts could be made between N-terminal domains from different molecules. In contrast, the C-terminal domain is dynamic, contains polyproline II structure, and lacks pronounced packing interactions. This region must be facing away from the core of the fibrils. Collectively, these data provide new constraints for building structural models of HDx1 fibrils.