| Autor: |
Silva A; IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal. a.silva@ibmc.up.pt.; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. a.silva@ibmc.up.pt., Sárkány Z; IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal. zsarkany@ibmc.up.pt.; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. zsarkany@ibmc.up.pt., Fraga JS; IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal. Joana.Fraga@ibmc.up.pt.; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. Joana.Fraga@ibmc.up.pt.; ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal. Joana.Fraga@ibmc.up.pt., Taboada P; Área de Física de la Materia Condensada, Facultad de Física, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain. pablo.taboada@usc.es.; Instituto de Investigación Sanitaria (IDIS), 15706 de Santiago de Compostela, Spain. pablo.taboada@usc.es., Macedo-Ribeiro S; IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal. sribeiro@ibmc.up.pt.; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. sribeiro@ibmc.up.pt., Martins PM; IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal. pmartins@ibmc.up.pt.; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. pmartins@ibmc.up.pt.; ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal. pmartins@ibmc.up.pt. |
| Abstrakt: |
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. |
