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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\u2014a starting point for further studies with cell and animal models of SCA3.<\/jats:p>","DOI":"10.3390\/cells11121969","type":"journal-article","created":{"date-parts":[[2022,6,19]],"date-time":"2022-06-19T10:14:52Z","timestamp":1655633692000},"page":"1969","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["A Robust Assay to Monitor Ataxin-3 Amyloid Fibril Assembly"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5933-9875","authenticated-orcid":false,"given":"Francisco","family":"Figueiredo","sequence":"first","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"International Iberian Nanotechnology Laboratory (INL), 4715-330 Braga, Portugal"},{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar (ICBAS), Universidade do Porto, 4050-313 Porto, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8597-4730","authenticated-orcid":false,"given":"M\u00f3nica","family":"Lopes-Marques","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Department of Biology, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9757-4240","authenticated-orcid":false,"given":"Bruno","family":"Almeida","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal"},{"name":"ICVS\/3B\u2019s\u2014PT Government Associate Laboratory, 4710-057 Braga, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1309-3640","authenticated-orcid":false,"given":"Nena","family":"Matscheko","sequence":"additional","affiliation":[{"name":"Dynamic Biosensors GmbH, 82152 Martinsried, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6908-1828","authenticated-orcid":false,"given":"Pedro M.","family":"Martins","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7604-792X","authenticated-orcid":false,"given":"Alexandra","family":"Silva","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7698-1170","authenticated-orcid":false,"given":"Sandra","family":"Macedo-Ribeiro","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1016\/S0306-4522(00)00391-2","article-title":"Amyloid-like inclusions in Huntington\u2019s disease","volume":"100","author":"McGowan","year":"2000","journal-title":"Neuroscience"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/S0896-6273(00)80943-5","article-title":"Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3","volume":"19","author":"Paulson","year":"1997","journal-title":"Neuron"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1111\/j.1365-2990.2011.01220.x","article-title":"Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3)","volume":"38","author":"Seidel","year":"2012","journal-title":"Neuropatho. 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