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.     

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.     

The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains

Ubiquitin chain complexity in cells is likely regulated by a diverse set of deubiquitinating enzymes (DUBs) with distinct ubiquitin chain preferences. Here we show that the polyglutamine disease protein, ataxin-3, binds and cleaves ubiquitin chains in a manner suggesting that it functions as a mixed linkage, chain-editing enzyme. Ataxin-3 cleaves ubiquitin chains through its amino-terminal Josephin domain and binds ubiquitin chains through a carboxyl-terminal cluster of ubiquitin interaction motifs neighboring the pathogenic polyglutamine tract. Ataxin-3 binds both Lys(48)- or Lys(63)-linked chains yet preferentially cleaves Lys(63) linkages. Ataxin-3 shows even greater activity toward mixed linkage polyubiquitin, cleaving Lys(63) linkages in chains that contain both Lys(48) and Lys(63) linkages. The ubiquitin interaction motifs regulate the specificity of this activity by restricting what can be cleaved by the protease domain, demonstrating that linkage specificity can be determined by elements outside the catalytic domain of a DUB. These findings establish ataxin-3 as a novel DUB that edits topologically complex chains.     

JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

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.     

Structural and functional analysis of ataxin-2 and ataxin-3.

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin-2 and ataxin-3, respectively. To provide a rationale for further functional experiments, we explored the protein architectures of ataxin-2 and ataxin-3. Using structure-based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners. Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNA-binding Lsm domain of ataxin-2 and the deubiquitinating Josephin domain of ataxin-3. We also speculate about distant evolutionary relationships of ubiquitin-binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions.     

Valosin-Containing Protein (VCP/p97) Is an Activator of Wild-Type Ataxin-3

Alterations in the ubiquitin-proteasome system (UPS) have been reported in several neurodegenerative disorders characterized by protein misfolding and aggregation, including the polylgutamine diseases. Machado-Joseph disease (MJD) or Spinocerebellar Ataxia type 3 is caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene, which encodes a 42 kDa deubiquitinating enzyme (DUB), ataxin-3. We investigated ataxin-3 deubiquitinating activity and the functional relevance of ataxin-3 interactions with two proteins previously described to interact with ataxin-3, hHR23A and valosin-containing protein (VCP/p97). We confirmed ataxin-3 affinity for both hHR23A and VCP/p97. hHR23A and ataxin-3 were shown to co-localize in discrete nuclear foci, while VCP/p97 was primarily cytoplasmic. hHR23A and VCP/p97 recombinant proteins were added, separately or together, to normal and expanded ataxin-3 in in vitro deubiquitination assays to evaluate their influence on ataxin-3 activity. VCP/p97 was shown to be an activator specifically of wild-type ataxin-3, exhibiting no effect on expanded ataxin-3, In contrast, we observed no significant alterations in ataxin-3 enzyme kinetics or substrate preference in the presence of hHR23A alone or in combination with VCP. Based on our results we propose a model where ataxin-3 normally functions with its interactors to specify the cellular fate of ubiquitinated proteins.     

Characterization of the Conformational Fluctuations in the Josephin Domain of Ataxin-3

As for a variety of other molecular recognition processes, conformational fluctuations play an important role in the cleavage of polyubiquitin chains by the Josephin domain of ataxin-3. The interaction between Josephin and ubiquitin appears to be mediated by the motions of α-helical hairpin that is unusual among deubiquitinating enzymes. Here, we characterized the conformational fluctuations of the helical hairpin by incorporating NMR measurements as replica-averaged restraints in molecular dynamics simulations, and by validating the results by small-angle x-ray scattering measurements. This approach allowed us to define the extent of the helical hairpin motions and suggest a role of such motions in the recognition of ubiquitin.     

Ataxin-3 is subject to autolytic cleavage

The protein ataxin-3 is responsible for spinocerebellar ataxia type 3, a neurodegenerative disease triggered when the length of a stretch of consecutive glutamines exceeds a critical threshold. Different physiologic roles have been suggested for this protein. More specifically, recent papers have shown that the highly conserved N-terminal Josephin domain of ataxin-3 binds ubiquitin and has ubiquitin hydrolase activity, thanks to a catalytic device specific to cysteine proteases. This article shows that the protein also has autoproteolytic activity, sustained by the same residues responsible for the ubiquitin hydrolase activity. The autolytic activity was abolished when these residues, i.e. Cys14 and His119, were replaced by noncatalytic ones. Furthermore, we found that pretreatment of the protein with tosyl l-phenylalanine chloromethyl ketone also abolished this activity, and that this site-specific reagent covalently bound His119, findings supported by MS experiments. MS also allowed us to establish that the attack was aspecific, as cleavage sites were observed at the carboxyl side of apolar, acidic and polar uncharged residues, clustered in the C-terminal, unstructured domain of the protein. In contrast, the Josephin domain was preserved from attack. We propose that the autolytic activity reported here may play a role in pathogenesis, as fragments carrying expanded polyglutamines are thought to be significantly more toxic than the whole protein.     

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.     

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.     

Polyglutamine tracts regulate autophagy

Expansions of polyglutamine (polyQ) tracts in different proteins cause 9 neurodegenerative conditions, such as Huntington disease and various ataxias. However, many normal mammalian proteins contain shorter polyQ tracts. As these are frequently conserved in multiple species, it is likely that some of these polyQ tracts have important but unknown biological functions. Here we review our recent study showing that the polyQ domain of the deubiquitinase ATXN3/ataxin-3 enables its interaction with BECN1/beclin 1, a key macroautophagy/autophagy initiator. ATXN3 regulates autophagy by deubiquitinating BECN1 and protecting it from proteasomal degradation. Interestingly, expanded polyQ tracts in other polyglutamine disease proteins compete with the shorter ATXN3 polyQ stretch and interfere with the ATXN3-BECN1 interaction. This competition results in decreased BECN1 levels and impaired starvation-induced autophagy, which phenocopies the loss of autophagic function mediated by ATXN3. Our findings describe a new autophagy-protective mechanism that may be altered in multiple neurodegenerative diseases.     

Effects of the enlargement of polyglutamine segments on the structure and folding of ataxin-2 and ataxin-3 proteins

Spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3) are two common autosomal-dominant inherited ataxia syndromes, both of which are related to the unstable expansion of trinucleotide CAG repeats in the coding region of the related ATXN2 and ATXN3 genes, respectively. The poly-glutamine (poly-Q) tract encoded by the CAG repeats has long been recognized as an important factor in disease pathogenesis and progress. In this study, using the I-TASSER method for 3D structure prediction, we investigated the effect of poly-Q tract enlargement on the structure and folding of ataxin-2 and ataxin-3 proteins. Our results show good agreement with the known experimental structures of the Josephin and UIM domains providing credence to the simulation results presented here, which show that the enlargement of the poly-Q region not only affects the local structure of these regions but also affects the structures of functional domains as well as the whole protein. The changes observed in the predicted models of the UIM domains in ataxin-3 when the poly-Q track is enlarged provide new insights on possible pathogenic mechanisms.     

Regulation of proteolysis by human deubiquitinating enzymes

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein–protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USPs) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN +/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

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.     

Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP

The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating, and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination, ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or?similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In?addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3.     

Structural modeling of ataxin-3 reveals distant homology to adaptins

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3.     

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.     

Structure validation of the Josephin domain of ataxin-3: Conclusive evidence for an open conformation

The availability of new and fast tools in structure determination has led to a more than exponential growth of the number of structures solved per year. It is therefore increasingly essential to assess the accuracy of the new structures by reliable approaches able to assist validation. Here, we discuss a specific example in which the use of different complementary techniques, which include Bayesian methods and small angle scattering, resulted essential for validating the two currently available structures of the Josephin domain of ataxin-3, a protein involved in the ubiquitin/proteasome pathway and responsible for neurodegenerative spinocerebellar ataxia of type 3. Taken together, our results demonstrate that only one of the two structures is compatible with the experimental information. Based on the high precision of our refined structure, we show that Josephin contains an open cleft which could be directly implicated in the interaction with polyubiquitin chains and other partners.