Identification of on- and off-pathway oligomers in amyloid fibril formation

Dear, Alexander J.; Meisl, Georg; Šarić, Anđela; Michaels, Thomas C. T.; Kjærgaard, Magnus; Linse, Sara; Knowles, Tuomas P. J.

doi:10.1039/c9sc06501f

Cited by 68 publications

(82 citation statements)

References 65 publications

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“… Because monomers and oligomers reach pre-equilibrium on a timescale significantly faster than that of the measurement intervals, altering this model to make oligomers off-pathway does not affect the fit quality. As such, the oligomers cannot be resolved as on- or off-pathway and must instead be considered part of the reactant ensemble at this experimental time resolution [ 42 ]. Left: Oligomer formation ( k o ) and dissociation (k d ) interconvert monomers (m) and oligomers (O).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, monomeric and oligomeric species should be considered part of the same ensemble of reactants for the purposes of a kinetic description. In other words, whether new fibrils are formed directly from monomers or by conversion of oligomeric intermediates is thus not distinguishable based on these kinetic measurements alone [ 42 ]. We verify this observation also by showing that fits to a model where oligomers cannot convert to fibrils, and fibrils are instead formed directly from monomer, describe the data equally well (see S5 Fig of S1 File ).…”

Section: Discussionmentioning

confidence: 99%

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In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers

et al. 2021

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Knowledge of the mechanisms of assembly of amyloid proteins into aggregates is of central importance in building an understanding of neurodegenerative disease. Given that oligomeric intermediates formed during the aggregation reaction are believed to be the major toxic species, methods to track such intermediates are clearly needed. Here we present a method, electron paramagnetic resonance (EPR), by which the amount of intermediates can be measured over the course of the aggregation, directly in the reacting solution, without the need for separation. We use this approach to investigate the aggregation of α-synuclein (αS), a synaptic protein implicated in Parkinson’s disease and find a large population of oligomeric species. Our results show that these are primary oligomers, formed directly from monomeric species, rather than oligomers formed by secondary nucleation processes, and that they are short-lived, the majority of them dissociates rather than converts to fibrils. As demonstrated here, EPR offers the means to detect such short-lived intermediate species directly in situ. As it relies only on the change in size of the detected species, it will be applicable to a wide range of self-assembling systems, making accessible the kinetics of intermediates and thus allowing the determination of their rates of formation and conversion, key processes in the self-assembly reaction.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers

et al. 2021

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“…The minimal model for the amyloid-forming proteins is based on a coarse-grained model which has proven useful for studying both primary (29) and secondary amyloid nucleation (12) and has been cross-validated with experiments (12,(30)(31)(32). Proteins are modeled as hard, patchy spherocylinders and can exist in two distinct conformational states: a soluble (s) and a β-sheet-forming (β) conformation, both of which are equipped with different arrangements of interaction patches (Fig.…”

Section: Resultsmentioning

confidence: 99%

Physical mechanisms of amyloid nucleation on fluid membranes

Krausser

Knowles

Šarić

2020

Proc. Natl. Acad. Sci. U.S.A.

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Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein–membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways—protein-rich and lipid-rich—and quantify how the membrane fluidity and protein–membrane affinity control the relative importance of those molecular pathways. We find that the membrane’s susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson’s disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context.

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“…Several lines of evidences have confirmed the importance of pre-fibril aggregates, such as oligomers, in the pathogenesis of prion-based diseases, although the way these aggregates express their harmful potential is still widely debated [ 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

Hampering the early aggregation of PrP-E200K protein by charge-based inhibitors: a computational study

Agamennone

Storchi

Marrone

et al. 2021

J Comput Aided Mol Des

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A multilayered computational workflow was designed to identify a druggable binding site on the surface of the E200K pathogenic mutant of the human prion protein, and to investigate the effect of the binding of small molecules in the inhibition of the early aggregation of this protein. At this purpose, we developed an efficient computational tool to scan the molecular interaction properties of a whole MD trajectory, thus leading to the characterization of plausible binding regions on the surface of PrP-E200K. These structural data were then employed to drive structure-based virtual screening and fragment-based approaches to the seeking of small molecular binders of the PrP-E200K. Six promising compounds were identified, and their binding stabilities were assessed by MD simulations. Therefore, analyses of the molecular electrostatic potential similarity between the bound complexes and unbound protein evidenced their potential activity as charged-based inhibitors of the PrP-E200K early aggregation.

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Identification of on- and off-pathway oligomers in amyloid fibril formation

Abstract: A general non-binary definition for on- and off-pathway intermediates is developed, enabling comparison of amyloid oligomers' contributions to fibril formation.

Cited by 68 publications

References 65 publications

In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers

In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers

Physical mechanisms of amyloid nucleation on fluid membranes

Hampering the early aggregation of PrP-E200K protein by charge-based inhibitors: a computational study

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