A robust assay to monitor ataxin-3 amyloid fibril assembly
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | https://hdl.handle.net/1822/80014 |
Resumo: | Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3. |
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A robust assay to monitor ataxin-3 amyloid fibril assemblyThioflavin-TPolyglutamine expansionReproducibilityUbiquitinSelf-association ratesEquilibrium dissociation constantSwitchSENSEScience & TechnologySpinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3.This study was supported by FEDER funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; Portuguese funds through FCT in the framework of the projects “PQTools: Molecular tools for Machado-Joseph Disease” (POCI-01-0145-FEDER-031173), “NAPPIT-MJD:Nuclear ataxin-3 protein-protein interactions as therapeutic targets in Machado-Joseph disease” (POCI-01-0145-FEDER-029056), “AggreGATE: Targeting diffusible oligomers of alpha-synuclein and ataxin-3: a drug repurposing opportunity for the treatment of neurodegenerative diseases” (POCI-01-0145-FEDER-031323), and “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274); the European Union’s Horizon 2020 Research and Innovation programme under grant agreement ID 952334 “PhasAGE”. The work was also supported by a research grant from National Ataxia Foundation to A.S. F.F. is the recipient of an FCT PhD fellowship (SFRH/BD/133009/2017).Multidisciplinary Digital Publishing Institute (MDPI)Universidade do MinhoFigueiredo, FranciscoLopes-Marques, MónicaAlmeida, BrunoMatscheko, NenaMartins, Pedro M.Silva, AlexandraMacedo-Ribeiro, Sandra2022-06-192022-06-19T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/1822/80014engFigueiredo, F.; Lopes-Marques, M.; Almeida, B.; Matscheko, N.; Martins, P.M.; Silva, A.; Macedo-Ribeiro, S. A Robust Assay to Monitor Ataxin-3 Amyloid Fibril Assembly. Cells 2022, 11, 1969. https://doi.org/10.3390/cells111219692073-440910.3390/cells11121969357410991969https://www.mdpi.com/2073-4409/11/12/1969info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:50:43Zoai:repositorium.sdum.uminho.pt:1822/80014Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:49:27.364834Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
title |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
spellingShingle |
A robust assay to monitor ataxin-3 amyloid fibril assembly Figueiredo, Francisco Thioflavin-T Polyglutamine expansion Reproducibility Ubiquitin Self-association rates Equilibrium dissociation constant SwitchSENSE Science & Technology |
title_short |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
title_full |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
title_fullStr |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
title_full_unstemmed |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
title_sort |
A robust assay to monitor ataxin-3 amyloid fibril assembly |
author |
Figueiredo, Francisco |
author_facet |
Figueiredo, Francisco Lopes-Marques, Mónica Almeida, Bruno Matscheko, Nena Martins, Pedro M. Silva, Alexandra Macedo-Ribeiro, Sandra |
author_role |
author |
author2 |
Lopes-Marques, Mónica Almeida, Bruno Matscheko, Nena Martins, Pedro M. Silva, Alexandra Macedo-Ribeiro, Sandra |
author2_role |
author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Figueiredo, Francisco Lopes-Marques, Mónica Almeida, Bruno Matscheko, Nena Martins, Pedro M. Silva, Alexandra Macedo-Ribeiro, Sandra |
dc.subject.por.fl_str_mv |
Thioflavin-T Polyglutamine expansion Reproducibility Ubiquitin Self-association rates Equilibrium dissociation constant SwitchSENSE Science & Technology |
topic |
Thioflavin-T Polyglutamine expansion Reproducibility Ubiquitin Self-association rates Equilibrium dissociation constant SwitchSENSE Science & Technology |
description |
Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-06-19 2022-06-19T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://hdl.handle.net/1822/80014 |
url |
https://hdl.handle.net/1822/80014 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Figueiredo, F.; Lopes-Marques, M.; Almeida, B.; Matscheko, N.; Martins, P.M.; Silva, A.; Macedo-Ribeiro, S. A Robust Assay to Monitor Ataxin-3 Amyloid Fibril Assembly. Cells 2022, 11, 1969. https://doi.org/10.3390/cells11121969 2073-4409 10.3390/cells11121969 35741099 1969 https://www.mdpi.com/2073-4409/11/12/1969 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799133076341653504 |