Molecular mechanisms of protein aggregation from global fitting of kinetic models

Abstract

The elucidation of the molecular mechanisms by which soluble proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity associated with aggregation reaction networks, the analysis of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quantitative kinetic assays and global fitting, to determine and to verify a molecular mechanism for aggregation reactions that is compatible with experimental kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic experiments that measure the concentration of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic experiment measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-separated text file. We also outline general experimental strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the analysis, we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global analysis of kinetic data without the need for extensive programming or detailed mathematical knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic analysis: determination of constraints to be placed on the aggregation mechanism based on the concentration dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate constants. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to determining mechanisms, can be completed in <1 d.

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

“…It has previously been shown that A␤ fibrils can adopt a pseudotransition state, termed the molten globule state, in which side chain packing at the fiber surface becomes perturbed (22). The molten globule state was defined by a reduction in ThT fluorescence and truncation of the fibrillar structure while the ␤-strand signal was maintained in CD spectroscopy, similar to what we have observed in the presence of DLPC (28). This molten globule state has previously only been observed following treatment of mature aggregates with ultrasonication.…”

Secondary nucleation of monomers on fibril surface dominatesα-synuclein aggregation and provides autocatalytic amyloid amplification

“…2b), for the different kinetic models tested are shown in Fig. 2d-f (Meisl et al 2016a). Two models were most consistent with the data: having either fragmentation or saturated secondary nucleation as the dominant processes of fibril multiplication (Fig.…”

Building reaction kinetic models for amiloid fibril growth

“…Recent intensive research into the physicochemical properties of amyloid and its formation into fibrils in the citoplasm points attention to growth models [3], [29], [38], [31]. In [39] the authors consider the growth of aminoid fibrils and look for a mechanistic explanation of the process in terms of a biochemical reaction network.…”

“…Let M be the total amount (concentration) of monomer, F be the fibril and C = M -F be the monomer-fibril complex at the time t of aggregation [3], [29], [38], [39]. After aggregation the complex C turns into fibril F , that is, the added monomer molecule M converts into (part of) the fibril F .…”