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Author(s): Rosa Crespo
Eva Villar-Alvarez
Pablo Taboada
Fernando A. Rocha
Ana M. Damas
Pedro M. Martins
Title: Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation
Issue Date: 2017
Abstract: : The study of drug candidates for the treatmentof amyloidosis and neurodegenerative diseases frequentlyinvolves in vitro measurements of amyloid fibril formation.Macromolecular crowding and off-pathway aggregation (OPA)are, by different reasons, two important phenomena affectingthe scalability of amyloid inhibitors and their successfulapplication in vivo. On the one hand, the cellular milieu iscrowded with macromolecules that drastically increase theeffective (thermodynamic) concentration of the amyloidogenicprotein. On the other hand, off-pathway aggregates, ratherthan amyloid fibrils, are increasingly appointed as causativeagents of toxicity. The present contribution reveals thatinsoluble off-pathway aggregates of hen egg-white lysozyme (HEWL) are a peculiar type of crowding agents that, unlike classicalmacromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resumeafter lowering the fraction of insoluble aggregates at a constant soluble HEWL concentration. Protein depletion and thioflavin-Tfluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. Thecrystallization-like model extended to account for OPA and time-dependent activity coefficients is able to fit multiple kineticresults using a single set of three parameters describing amyloid nucleation, autocatalytic growth, and off-pathway nucleation. Thelist of fitted results notably includes the cases of aggregation rebirth and all types of progress curves measured for differentHEWL concentrations. The quantitative challenges posed by macromolecular crowding and OPA find here a unified responsewith broader implications for the development of on- and off-pathway inhibitors
Related Information: info:eu-repo/grantAgreement/FCT - Fundação para a Ciência e Tecnologia/Projetos Estratégicos/UID/EQU/00511/2013 - POCI-01-0145-FEDER-006939/Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia/LEPABE
Document Type: Artigo em Revista Científica Internacional
Rights: restrictedAccess
Appears in Collections:FEUP - Artigo em Revista Científica Internacional

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