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Henry L Paulson - One of the best experts on this subject based on the ideXlab platform.
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differential toxicity of Ataxin 3 isoforms in drosophila models of spinocerebellar ataxia type 3
Neurobiology of Disease, 2019Co-Authors: Sean L Johnson, Henry L Paulson, Jessica R. Blount, Wei-ling Tsou, Kozeta Libohova, Bedri Ranxhi, Sokol V. TodiAbstract:Abstract The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein Ataxin-3, a deubiquitinase. Two major full-length isoforms of Ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds Ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each Ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic Ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.
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Ataxin 3 links nod2 and tlr2 mediated innate immune sensing and metabolism in myeloid cells
bioRxiv, 2019Co-Authors: Thomas P Chapman, Henry L Paulson, Daniele Corridoni, Seiji Shiraishi, Sumeet Pandey, Anna Aulicino, Simon Wigfield, Maria Do Carmo Costa, Marie L Thezenas, Roman FischerAbstract:Abstract The interplay between NOD2 and TLR2 following recognition of components of the bacterial cell wall peptidoglycan is well established, however their role in redirecting metabolic pathways in myeloid cells to degrade pathogens and mount antigen presentation remains unclear. We show NOD2 and TLR2 mediate phosphorylation of the deubiquitinase Ataxin-3 via RIPK2 and TBK1. In myeloid cells Ataxin-3 associates with the mitochondrial cristae protein MIC60, and is required for oxidative phosphorylation. Depletion of Ataxin-3 leads to impaired induction of mitochondrial reactive oxygen species (mROS) and defective bacterial killing. A mass spectrometry analysis of NOD2/TLR2 triggered Ataxin-3 deubiquitination targets revealed immunometabolic regulators, including HIF-1α and LAMTOR1 that may contribute to these effects. Thus, we define how Ataxin-3 plays an essential role in NOD2 and TLR2 sensing and effector functions in myeloid cells. Significance Statement In recent years it has become clear that cross-talk between metabolic and immune pathways is central to the regulation of host defence. This interplay appears of particular importance in myeloid cells including dendritic cells and macrophages, but it is unclear how two of their key bacterial sensors NOD2 and TLR2 influence metabolism. Here, we define how NOD2/TLR2 signal in myeloid cells to drive optimal mitochondrial functioning required for bacterial destruction. We uncover a new role for Ataxin-3, a deubiquitinase required for non-selective autophagy, in this pathway. We provide a non-biased analysis of Ataxin-3 targets generating evidence for a role in deubiquitination of metabolic mediators during myeloid cell differentiation that will provide an important basis for further study.
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interaction of the polyglutamine protein Ataxin 3 with rad23 regulates toxicity in drosophila models of spinocerebellar ataxia type 3
Human Molecular Genetics, 2017Co-Authors: Joanna R Sutton, Henry L Paulson, Jessica R. Blount, Wei-ling Tsou, Kozeta Libohova, Matthew K Scaglione, Maria Do Carmo Costa, Gnanada S Joshi, Sokol V. TodiAbstract:Polyglutamine (polyQ) repeat expansion in the deubiquitinase Ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of Ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the Ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic Ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic Ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic Ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the Ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.
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the de ubiquitinating enzyme Ataxin 3 does not modulate disease progression in a knock in mouse model of huntington disease
Journal of Huntington's disease, 2013Co-Authors: Li Zeng, Sara J Tallaksengreene, Bo Wang, Roger L Albin, Henry L PaulsonAbstract:Ataxin-3 is a deubiquitinating enzyme (DUB) that participates in ubiquitin-dependent protein quality control pathways and, based on studies in model systems, may be neuroprotective against toxic polyglutamine proteins such as the Huntington's disease (HD) protein, huntingtin (htt). HD is one of at least nine polyglutamine neurodegenerative diseases in which disease-causing proteins accumulate in ubiquitin-positive inclusions within neurons. In studies crossing mice null for Ataxin-3 to an established HD knock-in mouse model (HdhQ200), we tested whether loss of Ataxin-3 alters disease progression, perhaps by impairing the clearance of mutant htt or the ubiquitination of inclusions. While loss of Ataxin-3 mildly exacerbated age-dependent motor deficits, it did not alter inclusion formation, ubiquitination of inclusions or levels of mutant or normal htt. Ataxin-3, itself a polyglutamine-containing protein with multiple ubiquitin binding domains, was not observed to localize to htt inclusions. Changes in neurotransmitter receptor binding known to occur in HD knock-in mice also were not altered by the loss of Ataxin-3, although we unexpectedly observed increased GABAA receptor binding in the striatum of HdhQ200 mice, which has not previously been noted. Finally, we confirmed that CNS levels of hsp70 are decreased in HD mice as has been reported in other HD mouse models, regardless of the presence or absence of Ataxin-3. We conclude that while Ataxin-3 may participate in protein quality control pathways, it does not critically regulate the handling of mutant htt or contribute to major features of disease pathogenesis in HD.
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valosin containing protein vcp p97 is an activator of wild type Ataxin 3
PLOS ONE, 2012Co-Authors: Mario N Laco, Luisa Cortes, Henry L Paulson, Sue M Travis, Cristina A RegoAbstract: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.
Sokol V. Todi - One of the best experts on this subject based on the ideXlab platform.
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differential toxicity of Ataxin 3 isoforms in drosophila models of spinocerebellar ataxia type 3
Neurobiology of Disease, 2019Co-Authors: Sean L Johnson, Henry L Paulson, Jessica R. Blount, Wei-ling Tsou, Kozeta Libohova, Bedri Ranxhi, Sokol V. TodiAbstract:Abstract The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein Ataxin-3, a deubiquitinase. Two major full-length isoforms of Ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds Ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each Ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic Ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.
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toxicity and aggregation of the polyglutamine disease protein Ataxin 3 is regulated by its binding to vcp p97 in drosophila melanogaster
Neurobiology of Disease, 2018Co-Authors: Gorica Ristic, Kozeta Libohova, Joanna R Sutton, Sokol V. TodiAbstract:Among the nine dominantly inherited, age-dependent neurodegenerative diseases caused by abnormal expansion in the polyglutamine (polyQ) repeat of otherwise unrelated proteins is Spinocerebellar Ataxia Type 3 (SCA3). SCA3 is caused by polyQ expansion in the deubiquitinase (DUB), Ataxin-3. Molecular sequelae related to SCA3 remain unclear. Here, we sought to understand the role of protein context in SCA3 by focusing on the interaction between this DUB and Valosin-Containing Protein (VCP). VCP is bound directly by Ataxin-3 through an arginine-rich area preceding the polyQ repeat. We examined the importance of this interaction in Ataxin-3-dependent degeneration in Drosophila melanogaster. Our assays with new isogenic fly lines expressing pathogenic Ataxin-3 with an intact or mutated VCP-binding site show that disrupting the Ataxin-3-VCP interaction delays the aggregation of the toxic protein in vivo. Importantly, early on flies that express pathogenic Ataxin-3 with a mutated VCP-binding site are indistinguishable from flies that do not express any SCA3 protein. Also, reducing levels of VCP through RNA-interference has a similar, protective effect to mutating the VCP-binding site of pathogenic Ataxin-3. Based on in vivo pulse-chases, aggregated species of Ataxin-3 are highly stable, in a manner independent of VCP-binding. Collectively, our results highlight an important role for the Ataxin-3-VCP interaction in SCA3, based on a model that posits a seeding effect from VCP on pathogenic Ataxin-3 aggregation and subsequent toxicity.
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interaction of the polyglutamine protein Ataxin 3 with rad23 regulates toxicity in drosophila models of spinocerebellar ataxia type 3
Human Molecular Genetics, 2017Co-Authors: Joanna R Sutton, Henry L Paulson, Jessica R. Blount, Wei-ling Tsou, Kozeta Libohova, Matthew K Scaglione, Maria Do Carmo Costa, Gnanada S Joshi, Sokol V. TodiAbstract:Polyglutamine (polyQ) repeat expansion in the deubiquitinase Ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of Ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the Ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic Ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic Ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic Ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the Ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.
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the deubiquitinase Ataxin 3 requires rad23 and dnaj 1 for its neuroprotective role in drosophila melanogaster
Neurobiology of Disease, 2015Co-Authors: Wei-ling Tsou, Jessica R. Blount, Aaron A. Burr, Michelle Ouyang, Ryan R Hosking, Joanna R Sutton, Sokol V. TodiAbstract:Ataxin-3 is a deubiquitinase and polyglutamine (polyQ) disease protein with a protective role in Drosophila melanogaster models of neurodegeneration. In the fruit fly, wild-type Ataxin-3 suppresses toxicity from several polyQ disease proteins, including a pathogenic version of itself that causes spinocerebellar ataxia type 3 and pathogenic huntingtin, which causes Huntington's disease. The molecular partners of Ataxin-3 in this protective function are unclear. Here, we report that Ataxin-3 requires its direct interaction with the ubiquitin-binding and proteasome-associated protein, Rad23 (known as hHR23A/B in mammals) in order to suppress toxicity from polyQ species in Drosophila. According to additional studies, Ataxin-3 does not rely on autophagy or the proteasome to suppress polyQ-dependent toxicity in fly eyes. Instead this deubiquitinase, through its interaction with Rad23, leads to increased protein levels of the co-chaperone DnaJ-1 and depends on it to protect against degeneration. Through DnaJ-1, our data connect Ataxin-3 and Rad23 to protective processes involved with protein folding rather than increased turnover of toxic polyQ species.
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Ubiquitin-binding site 2 of Ataxin-3 prevents its proteasomal degradation by interacting with Rad23
Nature communications, 2014Co-Authors: Jessica R. Blount, Wei-ling Tsou, Gorica Ristic, Aaron A. Burr, Michelle Ouyang, Holland Galante, K. Matthew Scaglione, Sokol V. TodiAbstract:Polyglutamine repeat expansion in Ataxin-3 causes neurodegeneration in the most common dominant ataxia, spinocerebellar ataxia type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how Ataxin-3 is degraded. Here we show that, unlike most proteins, Ataxin-3 turnover does not require its ubiquitination, but is regulated by ubiquitin-binding site 2 (UbS2) on its N terminus. Mutating UbS2 decreases Ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of Ataxin-3 protein. Importantly, reducing Rad23 suppresses Ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance Ataxin-3 degradation for SCA3 therapy.
Luís Pereira De Almeida - One of the best experts on this subject based on the ideXlab platform.
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Ataxin 3 phosphorylation decreases neuronal defects in spinocerebellar ataxia type 3 models
Journal of Cell Biology, 2016Co-Authors: Carlos A Matos, Clévio Nóbrega, Luís Pereira De Almeida, Susana R Louros, Bruno Almeida, Elisabete Ferreiro, Jorge Valero, Sandra Macedoribeiro, Ana Luisa CarvalhoAbstract:Different neurodegenerative diseases are caused by aberrant elongation of repeated glutamine sequences normally found in particular human proteins. Although the proteins involved are ubiquitously distributed in human tissues, toxicity targets only defined neuronal populations. Changes caused by an expanded polyglutamine protein are possibly influenced by endogenous cellular mechanisms, which may be harnessed to produce neuroprotection. Here, we show that Ataxin-3, the protein involved in spinocerebellar ataxia type 3, also known as Machado-Joseph disease, causes dendritic and synapse loss in cultured neurons when expanded. We report that S12 of Ataxin-3 is phosphorylated in neurons and that mutating this residue so as to mimic a constitutive phosphorylated state counters the neuromorphologic defects observed. In rats stereotaxically injected with expanded Ataxin-3–encoding lentiviral vectors, mutation of serine 12 reduces aggregation, neuronal loss, and synapse loss. Our results suggest that S12 plays a role in the pathogenic pathways mediated by polyglutamine-expanded Ataxin-3 and that phosphorylation of this residue protects against toxicity.
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Re-establishing Ataxin-2 downregulates translation of mutant Ataxin-3 and alleviates Machado-Joseph disease.
Brain : a journal of neurology, 2015Co-Authors: Clévio Nóbrega, Hirokazu Hirai, Sara Carmo-silva, David Albuquerque, Ana Vasconcelos-ferreira, Udaya-geetha Vijayakumar, Liliana Mendonça, Luís Pereira De AlmeidaAbstract:Machado-Joseph disease is a progressive neurodegenerative disorder associated with the polyQ-expanded Ataxin-3 (encoded by ATXN3), for which no therapy is available. With the aim of clarifying the mechanism of neurodegeneration, we hypothesized that the abnormally long polyQ tract would interact aberrantly with Ataxin-2 (encoded by ATXN2), another polyQ protein whose function has recently been linked to translational regulation. Using patient's samples and cellular and animal's models we found that in Machado-Joseph disease: (i) Ataxin-2 levels are reduced; and (ii) its subcellular localization is changed towards the nucleus. Restoring Ataxin-2 levels by lentiviral-mediated overexpression: (i) reduced mutant Ataxin-3 levels; and (ii) rescued behaviour defects and neuropathology in a transgenic mouse model of Machado-Joseph disease. Conversely (i) mutating the Ataxin-2 motif that enables binding to its natural interactor and translation activator poly(A)-binding protein; or (ii) overexpressing poly(A)-binding protein, had opposite effects, increasing mutant Ataxin-3 translation and aggregation. This work suggests that in Machado-Joseph disease, mutant Ataxin-3 drives an abnormal reduction of Ataxin-2 levels, which overactivates poly(A)-binding protein, increases translation of mutant Ataxin-3 and other proteins and aggravates Machado-Joseph disease. Re-establishment of Ataxin-2 levels reduces mutant Ataxin-3 and alleviates Machado-Joseph disease pathogenesis opening a new avenue for therapeutic intervention in this and potentially other polyQ disorders.
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calpain inhibition reduces Ataxin 3 cleavage alleviating neuropathology and motor impairments in mouse models of machado joseph disease
Human Molecular Genetics, 2014Co-Authors: Ana Teresa Simoes, Nélio Gonçalves, Rui Jorge Nobre, Carlos B Duarte, Luís Pereira De AlmeidaAbstract:Machado-Joseph Disease (MJD) is the most prevalent autosomal dominantly inherited cerebellar ataxia. It is caused by an expanded CAG repeat in the ATXN3 gene, which translates into a polyglutamine tract within the Ataxin-3 protein. Present treatments are symptomatic and do not prevent disease progression. As calpain overactivation has been shown to contribute to mutant Ataxin-3 proteolysis, translocation to the nucleus, inclusions formation and neurodegeneration, we investigated the potential role of calpain inhibition as a therapeutic strategy to alleviate MJD pathology. For this purpose, we administered orally the calpain inhibitor BDA-410 to a lentiviral mouse model of MJD. Western-blot and immunohistochemical analysis revealed the presence of N- and C-terminal mutant Ataxin-3 fragments and the colocalization of large inclusions with cleaved caspase-3 in the mice brain. Oral administration of the calpain inhibitor BDA-410 decreased both fragments formation and full-length Ataxin-3 levels, reduced aggregation of mutant Ataxin-3 and prevented cell injury and striatal and cerebellar degeneration. Importantly, in correlation with the preserved cerebellar morphology, BDA-410 prevented motor behavioural deficits. In conclusion, BDA-410 alleviates Machado-Joseph neuropathology and may therefore be an effective therapeutic option for MJD.
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silencing mutant Ataxin 3 rescues motor deficits and neuropathology in machado joseph disease transgenic mice
PLOS ONE, 2013Co-Authors: Clévio Nóbrega, Hirokazu Hirai, David Albuquerque, Isabel Onofre, Nicole Déglon, Isabel Nascimentoferreira, Luís Pereira De AlmeidaAbstract:Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – Ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant Ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant Ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.
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calpastatin mediated inhibition of calpains in the mouse brain prevents mutant Ataxin 3 proteolysis nuclear localization and aggregation relieving machado joseph disease
Brain, 2012Co-Authors: Ana T Simões, Nicole Déglon, Nélio Gonçalves, Arnulf Koeppen, Sebastian Kügler, Carlos Bandeira Duarte, Luís Pereira De AlmeidaAbstract:Machado–Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the Ataxin 3 protein, which upon proteolysis may trigger Machado–Joseph disease. We investigated the role of calpains in the generation of toxic Ataxin 3 fragments and pathogenesis of Machado–Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado–Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant Ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of Ataxin 3, calpastatin overexpression modified the subcellular localization of mutant Ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant Ataxin 3 expression. Our findings are the first in vivo proof that mutant Ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado–Joseph disease. * Abbreviations : AAV : adeno-associated virus DARPP-32 : dopamine- and cyclic AMP-regulated neuronal phosphoprotein GFP : green fluorescent protein
Annalisa Pastore - One of the best experts on this subject based on the ideXlab platform.
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Capturing the Conformational Ensemble of the Mixed Folded Polyglutamine Protein Ataxin-3
Structure (London England : 1993), 2020Co-Authors: Alessandro Sicorello, Bartosz Różycki, Petr V. Konarev, Dmitri I. Svergun, Annalisa PastoreAbstract:Summary Ataxin-3 is a deubiquitinase involved in protein quality control and other essential cellular functions. It preferentially interacts with polyubiquitin chains of four or more units attached to proteins delivered to the ubiquitin-proteasome system. Ataxin-3 is composed of an N-terminal Josephin domain and a flexible C terminus that contains two or three ubiquitin-interacting motifs (UIMs) and a polyglutamine tract, which, when expanded beyond a threshold, leads to protein aggregation and misfolding and causes spinocerebellar ataxia type 3. The high-resolution structure of the Josephin domain is available, but the structural and dynamical heterogeneity of Ataxin-3 has so far hindered the structural description of the full-length protein. Here, we characterize non-expanded and expanded variants of Ataxin-3 in terms of conformational ensembles adopted by the proteins in solution by jointly using experimental data from nuclear magnetic resonance and small-angle X-ray scattering with coarse-grained simulations. Our results pave the way to a molecular understanding of polyubiquitin recognition.
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The Josephin domain determines the morphological and mechanical properties of Ataxin-3 fibrils.
Biophysical journal, 2011Co-Authors: Laura Masino, Giuseppe Nicastro, Alfonso De Simone, Lesley J. Calder, Justin E. Molloy, Annalisa PastoreAbstract: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.
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activity and cellular functions of the deubiquitinating enzyme and polyglutamine disease protein Ataxin 3 are regulated by ubiquitination at lysine 117
Journal of Biological Chemistry, 2010Co-Authors: Sokol V. Todi, Jessica R. Blount, Annalisa Pastore, Matthew K Scaglione, Venkatesha Basrur, Kevin P Conlon, Kojo S J Elenitobajohnson, Henry L PaulsonAbstract:Deubiquitinating enzymes (DUbs) play important roles in many ubiquitin-dependent pathways, yet how DUbs themselves are regulated is not well understood. Here, we provide insight into the mechanism by which ubiquitination directly enhances the activity of Ataxin-3, a DUb implicated in protein quality control and the disease protein in the polyglutamine neurodegenerative disorder, Spinocerebellar Ataxia Type 3. We identify Lys-117, which resides near the catalytic triad, as the primary site of ubiquitination in wild type and pathogenic Ataxin-3. Further studies indicate that ubiquitin-dependent activation of Ataxin-3 at Lys-117 is important for its ability to reduce high molecular weight ubiquitinated species in cells. Ubiquitination at Lys-117 also facilitates the ability of Ataxin-3 to induce aggresome formation in cells. Finally, structure-function studies support a model of activation whereby ubiquitination at Lys-117 enhances Ataxin-3 activity independent of the known ubiquitin-binding sites in Ataxin-3, most likely through a direct conformational change in or near the catalytic domain.
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understanding the role of the josephin domain in the polyub binding and cleavage properties of Ataxin 3
PLOS ONE, 2010Co-Authors: Giuseppe Nicastro, Henry L Paulson, Sokol V. Todi, Ezgi Karaca, Alexandre M J J Bonvin, Annalisa PastoreAbstract: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.
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josephin domain of Ataxin 3 contains two distinct ubiquitin binding sites
Biopolymers, 2009Co-Authors: Giuseppe Nicastro, Laura Masino, Alfonso De Simone, Veronica Esposito, Rajesh P Menon, Franca Fraternali, Annalisa PastoreAbstract:Joseph-Machado is an incurable neurodegenerative disease caused by toxic aggregation of Ataxin-3, a ubiquitin-specific cysteine protease, involved in the ubiquitin-proteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. The enzymatic site resides in the N-terminal josephin domain of Ataxin-3. We have characterized the ubiquitin-binding properties of josephin and showed that, unexpectedly, josephin contains two contiguous but distinct ubiquitin-binding sites. One is close to the enzymatic cleft and exploits an induced fit mechanism, which involves a flexible helical hairpin; the other overlaps with the site involved in recognition of HHR23B, a protein involved in delivering proteolytic substrates to the proteasome. To gain a structural description of the system, we had to overcome the nontrivial problem of dealing with a weak ternary complex. This was done by designing josephin mutants, which retain only one binding site and by characterizing the complexes with complementary computational and experimental techniques. The presence of two ubiquitin-binding sites explains how Ataxin-3 binds poly-ubiquitin chains and provides new insights into the molecular mechanism of ubiquitin recognition.
Jessica R. Blount - One of the best experts on this subject based on the ideXlab platform.
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differential toxicity of Ataxin 3 isoforms in drosophila models of spinocerebellar ataxia type 3
Neurobiology of Disease, 2019Co-Authors: Sean L Johnson, Henry L Paulson, Jessica R. Blount, Wei-ling Tsou, Kozeta Libohova, Bedri Ranxhi, Sokol V. TodiAbstract:Abstract The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein Ataxin-3, a deubiquitinase. Two major full-length isoforms of Ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds Ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each Ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic Ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.
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interaction of the polyglutamine protein Ataxin 3 with rad23 regulates toxicity in drosophila models of spinocerebellar ataxia type 3
Human Molecular Genetics, 2017Co-Authors: Joanna R Sutton, Henry L Paulson, Jessica R. Blount, Wei-ling Tsou, Kozeta Libohova, Matthew K Scaglione, Maria Do Carmo Costa, Gnanada S Joshi, Sokol V. TodiAbstract:Polyglutamine (polyQ) repeat expansion in the deubiquitinase Ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of Ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the Ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic Ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic Ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic Ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the Ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.
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the deubiquitinase Ataxin 3 requires rad23 and dnaj 1 for its neuroprotective role in drosophila melanogaster
Neurobiology of Disease, 2015Co-Authors: Wei-ling Tsou, Jessica R. Blount, Aaron A. Burr, Michelle Ouyang, Ryan R Hosking, Joanna R Sutton, Sokol V. TodiAbstract:Ataxin-3 is a deubiquitinase and polyglutamine (polyQ) disease protein with a protective role in Drosophila melanogaster models of neurodegeneration. In the fruit fly, wild-type Ataxin-3 suppresses toxicity from several polyQ disease proteins, including a pathogenic version of itself that causes spinocerebellar ataxia type 3 and pathogenic huntingtin, which causes Huntington's disease. The molecular partners of Ataxin-3 in this protective function are unclear. Here, we report that Ataxin-3 requires its direct interaction with the ubiquitin-binding and proteasome-associated protein, Rad23 (known as hHR23A/B in mammals) in order to suppress toxicity from polyQ species in Drosophila. According to additional studies, Ataxin-3 does not rely on autophagy or the proteasome to suppress polyQ-dependent toxicity in fly eyes. Instead this deubiquitinase, through its interaction with Rad23, leads to increased protein levels of the co-chaperone DnaJ-1 and depends on it to protect against degeneration. Through DnaJ-1, our data connect Ataxin-3 and Rad23 to protective processes involved with protein folding rather than increased turnover of toxic polyQ species.
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Ubiquitin-binding site 2 of Ataxin-3 prevents its proteasomal degradation by interacting with Rad23
Nature communications, 2014Co-Authors: Jessica R. Blount, Wei-ling Tsou, Gorica Ristic, Aaron A. Burr, Michelle Ouyang, Holland Galante, K. Matthew Scaglione, Sokol V. TodiAbstract:Polyglutamine repeat expansion in Ataxin-3 causes neurodegeneration in the most common dominant ataxia, spinocerebellar ataxia type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how Ataxin-3 is degraded. Here we show that, unlike most proteins, Ataxin-3 turnover does not require its ubiquitination, but is regulated by ubiquitin-binding site 2 (UbS2) on its N terminus. Mutating UbS2 decreases Ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of Ataxin-3 protein. Importantly, reducing Rad23 suppresses Ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance Ataxin-3 degradation for SCA3 therapy.
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Ubiquitination regulates the neuroprotective function of the deubiquitinase Ataxin-3 in vivo.
The Journal of biological chemistry, 2013Co-Authors: Wei-ling Tsou, Jessica R. Blount, Aaron A. Burr, Michelle Ouyang, K. Matthew Scaglione, Sokol V. TodiAbstract:Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB Ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with Ataxin-3 with only Lys-117 present, Ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates Ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that Ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.