RÉSUMÉ
Polyglutamine (polyQ) expansion of proteins can trigger protein misfolding and amyloid-like aggregation, which thus lead to severe cytotoxicities and even the respective neurodegenerative diseases. However, why polyQ aggregation is toxic to cells is not fully elucidated. Here, we took the fragments of polyQ-expanded (PQE) ataxin-7 (Atx7) and huntingtin (Htt) as models to investigate the effect of polyQ aggregates on the cellular proteostasis of endogenous ataxin-3 (Atx3), a protein that frequently appears in diverse inclusion bodies. We found that PQE Atx7 and Htt impair the cellular proteostasis of Atx3 by reducing its soluble as well as total Atx3 level but enhancing formation of the aggregates. Expression of these polyQ proteins promotes proteasomal degradation of endogenous Atx3 and accumulation of its aggregated form. Then we verified that the co-chaperone HSJ1 is an essential factor that orchestrates the balance of cellular proteostasis of Atx3; and further discovered that the polyQ proteins can sequester HSJ1 into aggregates or inclusions in a UIM domain-dependent manner. Thereby, the impairment of Atx3 proteostasis may be attributed to the sequestration and functional loss of cellular HSJ1. This study deciphers a potential mechanism underlying how PQE protein triggers proteinopathies, and also provides additional evidence in supporting the hijacking hypothesis that sequestration of cellular interacting partners by protein aggregates leads to cytotoxicity or neurodegeneration.
Sujet(s)
Ataxine-3/métabolisme , Protéines du choc thermique HSP40/métabolisme , Chaperons moléculaires/métabolisme , Maladies neurodégénératives/métabolisme , Peptides/métabolisme , Agrégats de protéines/génétique , Agrégation pathologique de protéines/métabolisme , Homéostasie protéique/génétique , Protéines de répression/métabolisme , Amyloïde/métabolisme , Protéines amyloïdogènes/métabolisme , Ataxine-3/composition chimique , Ataxine-3/génétique , Cellules HEK293 , Humains , Protéine huntingtine/métabolisme , Corps d'inclusion/métabolisme , Espace intracellulaire/métabolisme , Proteasome endopeptidase complex/métabolisme , Agrégation pathologique de protéines/génétique , Domaines protéiques/génétique , Protéolyse , Protéines de répression/composition chimique , Protéines de répression/génétique , Transduction du signal/génétique , Solubilité , TransfectionRÉSUMÉ
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.
Sujet(s)
Ataxine-3/composition chimique , Maladie de Machado-Joseph/génétique , Pliage des protéines , Ataxine-3/génétique , Humains , Simulation de dynamique moléculaire , Mutation , Peptides/composition chimique , Peptides/génétique , Conformation des protéinesRÉSUMÉ
The throughput level currently reached by automatic liquid handling and assay monitoring techniques is expected to facilitate the discovery of new modulators of enzyme activity. Judicious and dependable ways to interpret vast amounts of information are, however, required to effectively answer this challenge. Here, the 3-point method of kinetic analysis is proposed as a means to significantly increase the hit success rates and decrease the number of falsely identified compounds (false positives). In this post-Michaelis-Menten approach, each screened reaction is probed in three different occasions, none of which necessarily coincide with the initial period of constant velocity. Enzymology principles rather than subjective criteria are applied to identify unwanted outliers such as assay artifacts, and then to accurately distinguish true enzyme modulation effects from false positives. The exclusion and selection criteria are defined based on the 3-point reaction coordinates, whose relative positions along the time-courses may change from well to well or from plate to plate, if necessary. The robustness and efficiency of the new method is illustrated during a small drug repurposing screening of potential modulators of the deubiquinating activity of ataxin-3, a protein implicated in Machado-Joseph disease. Apparently, intractable Z factors are drastically enhanced after (1) eliminating spurious results, (2) improving the normalization method, and (3) increasing the assay resilience to systematic and random variability. Numerical simulations further demonstrate that the 3-point analysis is highly sensitive to specific, catalytic, and slow-onset modulation effects that are particularly difficult to detect by typical endpoint assays.
Sujet(s)
Activateurs d'enzymes/pharmacologie , Antienzymes/pharmacologie , Enzymes/composition chimique , Tests de criblage à haut débit , Artéfacts , Ataxine-3/composition chimique , Coumarines/composition chimique , Enzymes de désubiquitinylation/composition chimique , Découverte de médicament/méthodes , Repositionnement des médicaments , Activateurs d'enzymes/composition chimique , Antienzymes/composition chimique , Humains , Cinétique , Protéines de répression/composition chimique , Sensibilité et spécificité , Ubiquitine/composition chimiqueRÉSUMÉ
Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3's non-polyglutamine domains in disease, we utilized a new, allelic series of Drosophila melanogaster. We found that ataxin-3 pathogenicity is saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. Our studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration.
Sujet(s)
Ataxine-3 , Protéines de Drosophila/métabolisme , Protéines du choc thermique HSC70/métabolisme , Peptides , Ubiquitine/métabolisme , Motifs d'acides aminés , Animaux , Ataxine-3/composition chimique , Ataxine-3/génétique , Ataxine-3/métabolisme , Ataxine-3/toxicité , Drosophila , Protéines de Drosophila/composition chimique , Protéines du choc thermique HSC70/composition chimique , Humains , Larve/métabolisme , Maladie de Machado-Joseph/génétique , Peptides/composition chimique , Peptides/génétique , Peptides/métabolisme , Peptides/toxicité , Ubiquitine/composition chimiqueRÉSUMÉ
The 26S proteasome is a large (~2.5 MDa) protein complex consisting of at least 33 different subunits and many other components, which form the ubiquitin proteasomal system (UPS), an ATP-dependent protein degradation system in the cell. UPS serves as an essential component of the cellular protein surveillance machinery, and its dysfunction leads to cancer, neurodegenerative and immunological disorders. Importantly, the functions and regulations of proteins are governed by the combination of ordered regions, intrinsically disordered protein regions (IDPRs) and molecular recognition features (MoRFs). The structure-function relationships of UPS components have not been identified completely; therefore, in this study, we have carried out the functional intrinsic disorder and MoRF analysis for potential neurodegenerative disease and anti-cancer targets of this pathway. Our report represents the presence of significant intrinsic disorder and disorder-based binding regions in several UPS proteins, such as extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), proteasome-associated polyubiquitin receptors (ADRM1 and PSMD4), deubiquitinating enzymes (DUBs) (ATXN3 and USP14), and ubiquitinating enzymes (E2 (UBE2R2) and E3 (STUB1) enzyme). We believe this study will have implications for the conformation-specific roles of different regions of these proteins. This will lead to a better understanding of the molecular basis of UPS-associated diseases.
Sujet(s)
Protéines intrinsèquement désordonnées/composition chimique , Tumeurs/métabolisme , Maladies neurodégénératives/métabolisme , Proteasome endopeptidase complex/composition chimique , Ubiquitination , Protéines adaptatrices de la transduction du signal/composition chimique , Protéines adaptatrices de la transduction du signal/métabolisme , Ataxine-3/composition chimique , Ataxine-3/métabolisme , Protéines associées à l'autophagie/composition chimique , Protéines associées à l'autophagie/métabolisme , Humains , Protéines et peptides de signalisation intracellulaire/composition chimique , Protéines et peptides de signalisation intracellulaire/métabolisme , Protéines intrinsèquement désordonnées/métabolisme , Proteasome endopeptidase complex/métabolisme , Liaison aux protéines , Pliage des protéines , Cartes d'interactions protéiques , Protéines de liaison à l'ARN/composition chimique , Protéines de liaison à l'ARN/métabolisme , Protéines de répression/composition chimique , Protéines de répression/métabolisme , Ubiquitin thiolesterase/composition chimique , Ubiquitin thiolesterase/métabolisme , Ubiquitin-activating enzymes/composition chimique , Ubiquitin-activating enzymes/métabolisme , Ubiquitin-protein ligases/composition chimique , Ubiquitin-protein ligases/métabolismeRÉSUMÉ
DNA methylation has recently been linked to transcriptional dysregulation and neuronal dysfunction in polyglutamine (polyQ) disease. This study aims to determine whether (CAG)n expansion in ATXN3 perturbs DNA methylation status and affects gene expression. We analyzed DNA methylation throughout the genome using reduced representation bisulfite sequencing (RRBS) and confirmed the results using MethylTarget sequencing. Dynamic changes in DNA methylation, transcriptional and translational levels of specific genes were detected using BSP, qRT-PCR and western blot. In total, 135 differentially methylated regions (DMRs) were identified between SCA3/MJD and WT mouse cerebellum. KEGG analysis revealed differentially methylated genes involved in amino acid metabolism, Hedgehog signaling pathway, thyroid cancer, tumorigenesis and other pathways. We focused on DMRs that were directly associated with gene expression. On this basis, we further assessed 7 genes, including 13 DMRs, for DNA methylation validation and gene expression. We found that the methylation status of the DMRs of En1 and Nkx2-1 was negatively associated with their transcriptional and translational levels and that alteration of the DNA methylation status of DMRs and the corresponding transcription occurred before dyskinesia in SCA3/MJD mice. These results revealed novel DNA methylation-regulated genes, En1 and Nkx2-1, which may be useful for understanding the pathogenesis of SCA3/MJD.
Sujet(s)
Ataxine-3 , Méthylation de l'ADN/génétique , Régulation de l'expression des gènes/génétique , Protéines de répression , Animaux , Ataxine-3/composition chimique , Ataxine-3/génétique , Ataxine-3/métabolisme , Expansion de séquence répétée de l'ADN/génétique , Humains , Mâle , Souris , Souris transgéniques , Peptides/génétique , Protéines de répression/composition chimique , Protéines de répression/génétique , Protéines de répression/métabolismeRÉSUMÉ
Drug discovery frequently relies on the kinetic analysis of physicochemical reactions that are at the origin of the disease state. Amyloid fibril formation has been extensively investigated in relation to prevalent and rare neurodegenerative diseases, but thus far no therapeutic solution has directly arisen from this knowledge. Other aggregation pathways producing smaller, hard-to-detect soluble oligomers are increasingly appointed as the main reason for cell toxicity and cell-to-cell transmissibility. Here we show that amyloid fibrillation kinetics can be used to unveil the protein oligomerization state. This is illustrated for human insulin and ataxin-3, two model proteins for which the amyloidogenic and oligomeric pathways are well characterized. Aggregation curves measured by the standard thioflavin-T (ThT) fluorescence assay are shown to reflect the relative composition of protein monomers and soluble oligomers measured by nuclear magnetic resonance (NMR) for human insulin, and by dynamic light scattering (DLS) for ataxin-3. Unconventional scaling laws of kinetic measurables were explained using a single set of model parameters consisting of two rate constants, and in the case of ataxin-3, an additional order-of-reaction. The same fitted parameters were used in a discretized population balance that adequately describes time-course measurements of fibril size distributions. Our results provide the opportunity to study oligomeric targets using simple, high-throughput compatible, biophysical assays.
Sujet(s)
Amyloïde/composition chimique , Protéines amyloïdogènes/composition chimique , Ataxine-3/composition chimique , Insuline/composition chimique , Amyloïdose/génétique , Amyloïdose/anatomopathologie , Humains , Cinétique , Multimérisation de protéines , ThermodynamiqueRÉSUMÉ
Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and ß-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of ß-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.
Sujet(s)
Ataxine-3/composition chimique , Protéines de Drosophila/composition chimique , Drosophila melanogaster/génétique , Facteurs de transcription Forkhead/composition chimique , Neurones/métabolisme , Peptides/composition chimique , Ataxies spinocérébelleuses/génétique , Séquence d'acides aminés , Animaux , Ataxine-3/génétique , Ataxine-3/métabolisme , Comportement animal , Sites de fixation , Noyau de la cellule/métabolisme , Modèles animaux de maladie humaine , Protéines de Drosophila/génétique , Protéines de Drosophila/métabolisme , Drosophila melanogaster/métabolisme , Facteurs de transcription Forkhead/génétique , Facteurs de transcription Forkhead/métabolisme , Humains , Mutation , Neurones/ultrastructure , Peptides/génétique , Peptides/métabolisme , Liaison aux protéines , Structure en hélice alpha , Structure en brin bêta , Motifs et domaines d'intéraction protéique , Ataxies spinocérébelleuses/métabolisme , Ataxies spinocérébelleuses/anatomopathologieRÉSUMÉ
An expansion of the polyglutamine (polyQ) tract within the deubiquitinase ataxin-3 protein is believed to play a role in a neurodegenerative disorder. Ataxin-3 contains a Josephin catalytic domain and a polyQ tract that renders it intrinsically prone to aggregate, and thus full-length protein is difficult to characterize structurally by high-resolution methods. We established a robust protocol for expression and purification of wild-type and expanded ataxin-3, presenting 19Q and 74Q, respectively. Both proteins are monodisperse as assessed by analytical size exclusion chromatography. Initial biophysical characterization was performed, with apparent transition melting temperature of expanded ataxin-3 lower than the wild-type counterpart. We further characterize the molecular envelope of wild-type and expanded polyQ tract in ataxin-3 using small angle X-ray scattering (SAXS). Characterization of protein-protein interactions between ataxin-3 and newly identified binding partners will benefit from our protocol.
Sujet(s)
Ataxine-3/composition chimique , Maladie de Machado-Joseph/génétique , Peptides/composition chimique , Protéines recombinantes/composition chimique , Protéines de répression/composition chimique , Ataxine-3/biosynthèse , Ataxine-3/génétique , Ataxine-3/isolement et purification , Chromatographie sur gel/méthodes , Clonage moléculaire , Escherichia coli/génétique , Escherichia coli/métabolisme , Expression des gènes , Vecteurs génétiques/composition chimique , Vecteurs génétiques/métabolisme , Humains , Agranulocytes/métabolisme , Agranulocytes/anatomopathologie , Maladie de Machado-Joseph/métabolisme , Maladie de Machado-Joseph/anatomopathologie , Modèles moléculaires , Peptides/métabolisme , Domaines protéiques , Pliage des protéines , Structure secondaire des protéines , Protéines recombinantes/biosynthèse , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification , Protéines de répression/biosynthèse , Protéines de répression/génétique , Protéines de répression/isolement et purification , Diffusion aux petits angles , Diffraction des rayons XRÉSUMÉ
The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.
Sujet(s)
Amyloïde/effets des médicaments et des substances chimiques , Ataxine-3/métabolisme , Agrégats de protéines/effets des médicaments et des substances chimiques , Protéines de répression/métabolisme , Trifluoroéthanol/pharmacologie , Ataxine-3/composition chimique , Dichroïsme circulaire , Humains , Conformation moléculaire , Simulation de dynamique moléculaire , Peptides/métabolisme , Conformation des protéines/effets des médicaments et des substances chimiques , Domaines protéiques/effets des médicaments et des substances chimiques , Stabilité protéique/effets des médicaments et des substances chimiques , Structure secondaire des protéines/effets des médicaments et des substances chimiques , Structure tertiaire des protéines/effets des médicaments et des substances chimiques , Protéines de répression/composition chimiqueRÉSUMÉ
It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions that can both contribute to function. Although equally interesting, the disordered regions are difficult to study, because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. NMR spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, a task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe the structural properties of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. Ataxin-3 is a 42-kDa protein containing a globular N-terminal Josephin domain and a C-terminal tail that comprises 13 polyglutamine repeats within a low complexity region. We developed a strategy that allowed us to achieve 87% assignment of the NMR spectrum using a mixed protocol based on high-dimensionality, high-resolution experiments and different labeling schemes. Thanks to the almost complete spectral assignment, we proved that the C-terminal tail is flexible, with extended helical regions, and interacts only marginally with the rest of the protein. We could also, for the first time to our knowledge, observe the structural propensity of the polyglutamine repeats within the context of the full-length protein and show that its structure is stabilized by the preceding region.
Sujet(s)
Ataxine-3/composition chimique , Séquence d'acides aminés , Ataxine-3/génétique , Mutation , Résonance magnétique nucléaire biomoléculaire , Domaines protéiques , Pliage des protéines , SolutionsRÉSUMÉ
OBJECTIVE: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the most common dominantly inherited ataxia. Despite advances in understanding this CAG repeat/polyglutamine expansion disease, there are still no therapies to alter its progressive fatal course. Here, we investigate whether an antisense oligonucleotide (ASO) targeting the SCA3 disease gene, ATXN3, can prevent molecular, neuropathological, electrophysiological, and behavioral features of the disease in a mouse model of SCA3. METHODS: The top ATXN3-targeting ASO from an in vivo screen was injected intracerebroventricularly into early symptomatic transgenic SCA3 mice that express the full human disease gene and recapitulate key disease features. Following a single ASO treatment at 8 weeks of age, mice were evaluated longitudinally for ATXN3 suppression and rescue of disease-associated pathological changes. Mice receiving an additional repeat injection at 21 weeks were evaluated longitudinally up to 29 weeks for motor performance. RESULTS: The ATXN3-targeting ASO achieved sustained reduction of polyglutamine-expanded ATXN3 up to 8 weeks after treatment and prevented oligomeric and nuclear accumulation of ATXN3 up to at least 14 weeks after treatment. Longitudinal ASO therapy rescued motor impairment in SCA3 mice, and this rescue was associated with a recovery of defects in Purkinje neuron firing frequency and afterhyperpolarization. INTERPRETATION: This preclinical study established efficacy of ATXN3-targeted ASOs as a disease-modifying therapeutic strategy for SCA3. These results support further efforts to develop ASOs for human clinical trials in this polyglutamine disease as well as in other dominantly inherited disorders caused by toxic gain of function. Ann Neurol 2018;83:64-77.
Sujet(s)
Ataxine-3/composition chimique , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Maladie de Machado-Joseph/traitement médicamenteux , Oligonucléotides antisens/usage thérapeutique , Potentiels d'action/effets des médicaments et des substances chimiques , Potentiels d'action/génétique , Facteurs âges , Animaux , Apoptose/effets des médicaments et des substances chimiques , Apoptose/génétique , Ataxine-3/génétique , Encéphale/effets des médicaments et des substances chimiques , Encéphale/métabolisme , Protéines de liaison au calcium/métabolisme , Modèles animaux de maladie humaine , Comportement d'exploration/effets des médicaments et des substances chimiques , Femelle , Régulation de l'expression des gènes/génétique , Gliose/traitement médicamenteux , Gliose/étiologie , Maladie de Machado-Joseph/génétique , Maladie de Machado-Joseph/anatomopathologie , Maladie de Machado-Joseph/physiopathologie , Mâle , Souris , Souris transgéniques , Protéines des microfilaments/métabolisme , Activité motrice/effets des médicaments et des substances chimiques , Activité motrice/génétique , Mutation/génétique , Cellules de Purkinje/effets des médicaments et des substances chimiques , Cellules de Purkinje/anatomopathologie , Protéines de liaison à l'ARN/métabolismeRÉSUMÉ
Autosomal recessive juvenile Parkinsonism (ARJP) is an inherited neurodegenerative disease in which 50% of affected individuals harbor mutations in the gene encoding the E3 ligase parkin. Parkin regulates the mitochondrial recycling pathway, which is induced by oxidative stress. In its native state, parkin is auto-inhibited by its N-terminal ubiquitin-like (Ubl) domain, which blocks the binding site for an incoming E2â¼ubiquitin conjugate, needed for parkin's ubiquitination activity. Parkin is activated via phosphorylation of Ser-65 in its Ubl domain by PTEN-induced putative kinase 1 (PINK1) and a ubiquitin molecule phosphorylated at a position equivalent to Ser-65 in parkin. Here we have examined the underlying molecular mechanism of phosphorylation of parkin's Ubl domain carrying ARJP-associated substitutions and how altered phosphorylation modulates parkin activation and ubiquitination. We found that three substitutions in the Ubl domain (G12R, R33Q, and R42P) significantly decrease PINK1's ability to phosphorylate the Ubl domain. We noted that two basic loss-of-function substitutions (R33Q and R42P) are close to acidic patches in the proposed PINK1-parkin interface, indicating that ionic interactions at this site may be important for efficient parkin phosphorylation. Increased auto-ubiquitination with unique ubiquitin chain patterns was observed for two other Ubl domain substitutions (G12R and T55I), suggesting that these substitutions, along with phosphorylation, increase parkin degradation. Moreover, Ubl domain phosphorylation decreased its affinity for the potential effector protein ataxin-3, which edits ubiquitin chain building by parkin. Overall, our work provides a framework for the mechanisms of parkin's loss-of-function, indicating an interplay between ARJP-associated substitutions and phosphorylation of its Ubl domain.
Sujet(s)
Mutation faux-sens , Syndromes parkinsoniens/enzymologie , Ubiquitin-protein ligases/composition chimique , Substitution d'acide aminé , Ataxine-3/composition chimique , Ataxine-3/génétique , Humains , Syndromes parkinsoniens/génétique , Phosphorylation/génétique , Domaines protéiques , Protéines de répression/composition chimique , Protéines de répression/génétique , Ubiquitin-protein ligases/génétique , Ubiquitin-protein ligases/métabolisme , Ubiquitines/composition chimique , Ubiquitines/génétiqueRÉSUMÉ
The expansion of a trinucleotide (CAG) repeat, translated into a polyglutamine expanded sequence in the protein encoded by the MJD gene, was identified over 20 years ago as the causative mutation in a severe neurodegenerative disorder originally diagnosed in individuals of Portuguese ancestry. This incapacitating disease, called Machado-Joseph disease or spinocebellar ataxia type 3, is integrated into a larger group of neurodegenerative disorders-the polyglutamine expansion disorders-caused by extension of a CAG repeat in the coding sequence of otherwise unrelated genes. These diseases are generally linked with the appearance of intracellular inclusions , which despite having a controversial role in disease appearance and development represent a characteristic common fingerprint in all polyglutamine-related disorders. Although polyglutamine expansion is an obvious trigger for neuronal dysfunction, the role of the different domains of these complex proteins in the function and aggregation properties of the carrier proteins is being uncovered in recent studies. In this review the current knowledge about the structural and functional features of full-length ataxin-3 protein will be discussed. The intrinsic conformational dynamics and interplay between the globular and intrinsically disordered regions of ataxin-3 will be highlighted, and a perspective picture of the role of known ataxin-3 post-translational modifications on regulating ataxin-3 aggregation and function will be drawn.
Sujet(s)
Ataxine-3 , Simulation de dynamique moléculaire , Maturation post-traductionnelle des protéines , Protéines de répression , Ataxine-3/composition chimique , Ataxine-3/génétique , Ataxine-3/métabolisme , Humains , Maladie de Machado-Joseph/génétique , Maladie de Machado-Joseph/métabolisme , Maladie de Machado-Joseph/anatomopathologie , Peptides , Protéines de répression/composition chimique , Protéines de répression/génétique , Protéines de répression/métabolisme , Expansion de trinucléotide répétéRÉSUMÉ
Amyloid diseases represent a growing social and economic burden in the developed world. Understanding the assembly pathway and the inhibition of amyloid formation is key to developing therapies to treat these diseases. The neurodegenerative condition Machado-Joseph disease is characterised by the self-aggregation of the protein ataxin-3. Ataxin-3 consists of a globular N-terminal Josephin domain, which can aggregate into curvilinear protofibrils, and an unstructured, dynamically disordered C-terminal domain containing three ubiquitin interacting motifs separated by a polyglutamine stretch. Upon expansion of the polyglutamine region above 50 residues, ataxin-3 undergoes a second stage of aggregation in which long, straight amyloid fibrils form. A peptide inhibitor of polyglutamine aggregation, known as polyQ binding peptide 1, has been shown previously to prevent the maturation of ataxin-3 fibrils. However, the mechanism of this inhibition remains unclear. Using nanoelectrospray ionisation-mass spectrometry, we demonstrate that polyQ binding peptide 1 binds to monomeric ataxin-3. By investigating the ability of polyQ binding peptide 1 to bind to truncated ataxin-3 constructs lacking one or more domains, we localise the site of this interaction to a 39-residue sequence immediately C-terminal to the Josephin domain. The results suggest a new mechanism for the inhibition of polyglutamine aggregation by polyQ binding peptide 1 in which binding to a region outside of the polyglutamine tract can prevent fibril formation, highlighting the importance of polyglutamine flanking regions in controlling aggregation and disease.
Sujet(s)
Amyloïde/composition chimique , Amyloïde/métabolisme , Ataxine-3/composition chimique , Ataxine-3/métabolisme , Peptides/métabolisme , Sites de fixation , Humains , Maladie de Machado-Joseph/diagnostic , Maladie de Machado-Joseph/métabolisme , Spectrométrie de masse , Peptides/composition chimique , Liaison aux protéinesRÉSUMÉ
Polyglutamine (polyQ) repeat-containing proteins are widespread in the human proteome but only nine of them are associated with highly incapacitating neurodegenerative disorders. The genetic expansion of the polyQ tract in disease-related proteins triggers a series of events resulting in neurodegeneration. The polyQ tract plays the leading role in the aggregation mechanism, but other elements modulate the aggregation propensity in the context of the full-length proteins, as implied by variations in the length of the polyQ tract required to trigger the onset of a given polyQ disease. Intrinsic features such as the presence of aggregation-prone regions (APRs) outside the polyQ segments and polyQ-flanking sequences, which synergistically participate in the aggregation process, are emerging for several disease-related proteins. The inherent polymorphic structure of polyQ stretches places the polyQ proteins in a central position in protein-protein interaction networks, where interacting partners may additionally shield APRs or reshape the aggregation course. Expansion of the polyQ tract perturbs the cellular homeostasis and contributes to neuronal failure by modulating protein-protein interactions and enhancing toxic oligomerization. Post-translational modifications further regulate self-assembly either by directly altering the intrinsic aggregation propensity of polyQ proteins, by modulating their interaction with different macromolecules or by modifying their withdrawal by the cell quality control machinery. Here we review the recent data on the multifaceted aggregation pathways of disease-related polyQ proteins, focusing on ataxin-3, the protein mutated in Machado-Joseph disease. Further mechanistic understanding of this network of events is crucial for the development of effective therapies for polyQ diseases.
Sujet(s)
Ataxine-3/métabolisme , Protéine huntingtine/métabolisme , Peptides/composition chimique , Peptides/métabolisme , Protéines de répression/métabolisme , Animaux , Ataxine-3/composition chimique , Ataxine-3/génétique , Humains , Protéine huntingtine/composition chimique , Protéine huntingtine/génétique , Maladie de Machado-Joseph/génétique , Maladie de Machado-Joseph/anatomopathologie , Peptides/génétique , Phosphorylation , Maturation post-traductionnelle des protéines , Protéines de répression/composition chimique , Protéines de répression/génétique , Répétitions de trinucléotidesRÉSUMÉ
The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation.
Sujet(s)
Ataxine-3/génétique , Protéines fongiques/génétique , Réaction de choc thermique , Pichia/métabolisme , Adénosine triphosphate/métabolisme , Ataxine-3/composition chimique , Ataxine-3/métabolisme , Métabolisme énergétique , Protéines fongiques/composition chimique , Protéines fongiques/métabolisme , Régulation de l'expression des gènes fongiques , Pichia/génétiqueRÉSUMÉ
p97 is an essential ATPase associated with various cellular activities (AAA+) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum-associated degradation. However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants.
Sujet(s)
Ataxine-3/métabolisme , Coenzymes/métabolisme , Myopathies distales/métabolisme , Modèles moléculaires , Troubles de l'homéostasie des protéines/métabolisme , Protéines de répression/métabolisme , Protéine contenant la valosine/métabolisme , ADP/composition chimique , ADP/métabolisme , Adénosine triphosphate/composition chimique , Adénosine triphosphate/métabolisme , Substitution d'acide aminé , Ataxine-3/composition chimique , Ataxine-3/génétique , Sites de fixation , Fixation compétitive , Coenzymes/composition chimique , Coenzymes/génétique , Cristallographie aux rayons X , Bases de données de protéines , Myopathies distales/enzymologie , Myopathies distales/génétique , Humains , Microscopie électronique à transmission , Mutation , Fragments peptidiques/composition chimique , Fragments peptidiques/génétique , Fragments peptidiques/métabolisme , Conformation des protéines , Motifs et domaines d'intéraction protéique , Cartographie d'interactions entre protéines , Multimérisation de protéines , Troubles de l'homéostasie des protéines/enzymologie , Troubles de l'homéostasie des protéines/génétique , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/métabolisme , Protéines de répression/composition chimique , Protéines de répression/génétique , Protéine contenant la valosine/composition chimique , Protéine contenant la valosine/génétiqueRÉSUMÉ
Amyloid fibrils and soluble oligomers are two types of protein aggregates associated with neurodegeneration. Classic therapeutic strategies try to prevent the nucleation and spread of amyloid fibrils, whilst diffusible oligomers have emerged as promising drug targets affecting downstream pathogenic processes. We developed a generic protein aggregation model and validate it against measured compositions of fibrillar and non-fibrillar assemblies of ataxin-3, a protein implicated in Machado-Joseph disease. The derived analytic rate-law equations can be used to 1)â identify the presence of parallel aggregation pathways and 2)â estimate the critical sizes of amyloid fibrils. The discretized population balance supporting our model is the first to quantitatively fit time-resolved measurements of size and composition of both amyloid-like and oligomeric species. The new theoretical framework can be used to screen a new class of drugs specifically targeting toxic oligomers.
Sujet(s)
Amyloïde/composition chimique , Protéines/composition chimique , Ataxine-3/composition chimique , Biopolymères/composition chimique , Chromatographie sur gel , Cinétique , Microscopie électronique à transmission , Liaison aux protéines , Protéines de répression/composition chimiqueRÉSUMÉ
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.