Abstract
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.
Highlights
The deposition of amyloid fibrils in the brain is a pathological hallmark of several different neurodegenerative disorders, yet the pathogenic role of these insoluble aggregates is not fully understood [1]
For the experimental conditions adopted in each model protein, human insulin and ataxin-3 produce fibrillar species with distinct morphologies (Figure 1A,B) and at markedly different aggregation rates (Figure 1C)
Chemical kinetic analysis pinpoints these differences, and reveals how the presence of soluble oligomers influences each type of protein aggregation curves
Summary
The deposition of amyloid fibrils in the brain is a pathological hallmark of several different neurodegenerative disorders, yet the pathogenic role of these insoluble aggregates is not fully understood [1]. A classical cornerstone for drug discovery [3], is hardly applicable to the study of this new and pre-eminent target [4,5,6], in part due to the lack of straightforward methods to monitor the formation of a highly heterogeneous group of Biomolecules 2018, 8, 108; doi:10.3390/biom8040108 www.mdpi.com/journal/biomolecules. Extensive research has been devoted to protein aggregation kinetics based on the characteristic tinctorial properties of amyloid fibrils [9]. An important step towards the kinetic quantification of off-pathway aggregation was taken after the observation of protein precipitation occurring in parallel with the formation of amyloid fibrils of lysozyme [10]. Kinetic analysis of amyloid aggregation of the islet amyloid polypeptide (IAPP)
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