Dataset Information

Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions.

ABSTRACT: Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid ? (A?) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to A? allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that A? fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration.

SUBMITTER: Nasir I

PROVIDER: S-EPMC4599436 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions.

Nasir Irem I Linse Sara S Cabaleiro-Lago Celia C

ACS chemical neuroscience 20150520 8

Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unsp ...[more]

PMID: 25946560

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Project description:Crespo2012 - Kinetics of Amyloid Fibril Formation This model is described in the article: A generic crystallization-like model that describes the kinetics of amyloid fibril formation. Crespo R, Rocha FA, Damas AM, Martins PM. J. Biol. Chem. 2012 Aug; 287(36): 30585-30594 Abstract: Associated with neurodegenerative disorders such as Alzheimer, Parkinson, or prion diseases, the conversion of soluble proteins into amyloid fibrils remains poorly understood. Extensive "in vitro" measurements of protein aggregation kinetics have been reported, but no consensus mechanism has emerged until now. This contribution aims at overcoming this gap by proposing a theoretically consistent crystallization-like model (CLM) that is able to describe the classic types of amyloid fibrillization kinetics identified in our literature survey. Amyloid conversion represented as a function of time is shown to follow different curve shapes, ranging from sigmoidal to hyperbolic, according to the relative importance of the nucleation and growth steps. Using the CLM, apparently unrelated data are deconvoluted into generic mechanistic information integrating the combined influence of seeding, nucleation, growth, and fibril breakage events. It is notable that this complex assembly of interdependent events is ultimately reduced to a mathematically simple model, whose two parameters can be determined by little more than visual inspection. The good fitting results obtained for all cases confirm the CLM as a good approximation to the generalized underlying principle governing amyloid fibrillization. A perspective is presented on possible applications of the CLM during the development of new targets for amyloid disease therapeutics. This model is hosted on BioModels Database and identified by: BIOMD0000000531. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Dataset Information

Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions.

Publications

Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions.

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