Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching |
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Authors: | Anne S Wagner Antonio Z Politi Anne Ast Kenny Bravo-Rodriguez Katharina Baum Alexander Buntru Nadine U Strempel Lydia Brusendorf Christian Hänig Annett Boeddrich Stephanie Plassmann Konrad Klockmeier Juan M Ramirez-Anguita Elsa Sanchez-Garcia Jana Wolf Erich E Wanker |
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Institution: | 1. Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany;2. Mathematical Modelling of Cellular Processes, Max Delbrueck Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany;3. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 2, 45470 Mülheim an der Ruhr, Germany;4. Computational Biochemistry, University Duisburg-Essen, Universitätsstr. 2, 45141 Essen, Germany |
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Abstract: | Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends. |
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Keywords: | Huntingtin fibrillogenesis aggregation mechanism nucleation amyloidogenesis nucleated fibril branching HTT Huntingtin polyQ polyglutamine GST Ex1Q49 exon-1 fragment with 49 glutamines PP PreScission protease FRA filter retardation assay ThT thioflavin T DLS dynamic light scattering AFM atomic force microscopy TEM transmission electron microscopy DBAs dot blot assays QM/MM MD quantum mechanics/molecular mechanics molecular dynamics SD standard deviation AICc corrected Akaike information criterion |
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