Molecular mechanisms of protein aggregation from global fitting of kinetic models

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.

[1]  Thomas C T Michaels,et al.  Role of filament annealing in the kinetics and thermodynamics of nucleated polymerization. , 2014, The Journal of chemical physics.

[2]  Sara Linse,et al.  A facile method for expression and purification of the Alzheimer’s disease-associated amyloid β-peptide , 2009, The FEBS journal.

[3]  Michele Vendruscolo,et al.  Nucleated polymerization with secondary pathways. II. Determination of self-consistent solutions to growth processes described by non-linear master equations. , 2011, The Journal of chemical physics.

[4]  Thomas Scheibel,et al.  The elongation of yeast prion fibers involves separable steps of association and conversion. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Atanas V Koulov,et al.  Functional amyloid--from bacteria to humans. , 2007, Trends in biochemical sciences.

[6]  Michele Vendruscolo,et al.  Nucleated polymerization with secondary pathways. I. Time evolution of the principal moments. , 2011, The Journal of chemical physics.

[7]  Georg Meisl,et al.  Preventing peptide and protein misbehavior , 2015, Proceedings of the National Academy of Sciences.

[8]  Christopher M. Dobson,et al.  Direct Observation of the Interconversion of Normal and Toxic Forms of α-Synuclein , 2012, Cell.

[9]  Sara Linse,et al.  Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides , 2014, Proceedings of the National Academy of Sciences.

[10]  Sara Linse,et al.  Quantification of the concentration of Aβ42 propagons during the lag phase by an amyloid chain reaction assay. , 2014, Journal of the American Chemical Society.

[11]  Sara Linse,et al.  Role of aromatic side chains in amyloid β-protein aggregation. , 2012, ACS chemical neuroscience.

[12]  J. Doye,et al.  Global Optimization by Basin-Hopping and the Lowest Energy Structures of Lennard-Jones Clusters Containing up to 110 Atoms , 1997, cond-mat/9803344.

[13]  J. Hofrichter,et al.  Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism. , 1985, Journal of molecular biology.

[14]  Michele Vendruscolo,et al.  Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism , 2013, Proceedings of the National Academy of Sciences.

[15]  J R Ghilardi,et al.  Alzheimer's disease amyloid propagation by a template-dependent dock-lock mechanism. , 2000, Biochemistry.

[16]  C. Dobson,et al.  Protein misfolding, functional amyloid, and human disease. , 2006, Annual review of biochemistry.

[17]  Tuomas P. J. Knowles,et al.  The physical basis of protein misfolding disorders , 2015 .

[18]  F. Oosawa,et al.  A theory of linear and helical aggregations of macromolecules. , 1962, Journal of molecular biology.

[19]  Tuomas P. J. Knowles,et al.  An Analytical Solution to the Kinetics of Breakable Filament Assembly , 2009, Science.

[20]  A. Miranker,et al.  Fiber-dependent amyloid formation as catalysis of an existing reaction pathway , 2007, Proceedings of the National Academy of Sciences.

[21]  C. Dobson.  Protein folding and misfolding , 2003, Nature.

[22]  Astrid Gräslund,et al.  Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation , 2015, Proceedings of the National Academy of Sciences.

[23]  Jin-Moo Lee,et al.  Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide , 2009, Proceedings of the National Academy of Sciences.

[24]  Michele Vendruscolo,et al.  Chemical kinetics for drug discovery to combat protein aggregation diseases. , 2014, Trends in pharmacological sciences.

[25]  Michele Vendruscolo,et al.  The molecular chaperone Brichos breaks the catalytic cycle that generates toxic Aβ oligomers , 2015, Nature Structural &Molecular Biology.

[26]  Roger M. Nitsch,et al.  The recombinant amyloid-beta peptide Abeta1-42 aggregates faster and is more neurotoxic than synthetic Abeta1-42. , 2010, Journal of molecular biology.

[27]  Sara Linse,et al.  Amyloid β-protein aggregation produces highly reproducible kinetic data and occurs by a two-phase process. , 2010, ACS chemical neuroscience.

[28]  Christian Haass,et al.  Games Played by Rogue Proteins in Prion Disorders and Alzheimer's Disease , 2003, Science.

[29]  Michele Vendruscolo,et al.  From macroscopic measurements to microscopic mechanisms of protein aggregation. , 2012, Journal of molecular biology.

[30]  Tracy O'Connor,et al.  Protein aggregation diseases: pathogenicity and therapeutic perspectives , 2010, Nature Reviews Drug Discovery.

[31]  J. Hardy,et al.  The Amyloid Hypothesis of Alzheimer ’ s Disease : Progress and Problems on the Road to Therapeutics , 2009 .