The role of shear forces in primary and secondary nucleation of amyloid fibrils
Emil AxellJing HuMax LindbergAlexander J. DearLei Ortigosa-PascualEwa AndrzejewskaGreta ŠneiderienėDev ThackerTuomas P. J. KnowlesEmma SparrSara Linse
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Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A Crystal (programming language)
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Huntingtin (HTT) fragments with extended polyglutamine (polyQ) tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington9s disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). We demonstrate that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent monomers, and (2) a fast secondary fibril-dependent nucleation process, which involves branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs primary nucleation and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modelling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates. All results indicate that in vitro, HTT exon-1 fibrillar aggregates are formed by a branching mechanism.
Fibrillogenesis
Branching (polymer chemistry)
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Abstract Amyloid fibrils are self‐associating filamentous structures deposited in extracellular tissue in various neurodegenerative and protein misfolding disorders. It has been shown that beta‐sheet‐breaker (BSB) peptides may interfere with amyloid fibril assembly. Although BSB peptides are prospective therapeutic agents in amyloidosis, there is ambiguity about the mechanisms and generality of their action. In the present work we analyzed the effect of the BSB peptide LPFFD on the growth kinetics, morphologic, and mechanical properties of amyloid β25‐35 (Aβ25‐35) fibrils assembled in an oriented array on mica surface. Aβ25‐35 is thought to represent the biologically active, toxic fragment of the full‐length Aβ peptide. Growth kinetics and morphologic features were analyzed using in situ atomic force microscopy in the presence of various concentrations of LPFFD. We found that the addition of LPFFD only slightly altered the assembly kinetics of Aβ25‐35 fibrils. Already formed fibrils did not disassemble in the presence of high concentrations of LPFFD. The mechanical stability of the fibrils was explored with force spectroscopy methods. The nanomechanical behavior of Aβ25‐35 fibrils is characterized by the appearance of force staircases which correspond to the force‐driven unzipping and dissociation of several protofilaments. In the presence of LPFFD single‐plateau force traces dominated. The effects of LPFFD on Aβ25‐35 fibril assembly and stability suggest that inter‐protofilament interactions were slightly weakened. Complete disassembly of fibrils, however, was not observed. Thus, under the conditions explored here, LPFFD may not be considered as a BSB peptide with generalized beta‐sheet breaking properties. Copyright © 2011 John Wiley & Sons, Ltd.
Amyloid (mycology)
Beta sheet
Force Spectroscopy
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Collagen fibril
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An experimental investigation to explore the interaction between bubbles forming at adjacent nucleation sites is presented. The results obtained are consistent with the results of Calka’s and Knowles’ experimental investigations and confirm that nucleation site activation/deactivation, whereby a bubble growing at a nucleation site is able to promote/hinder the formation of a bubble at an adjacent nucleation site by depositing/displacing a vapor nucleus in the nucleation cavity, is instrumental in determining how a bubble forming at a nucleation site influences the nucleation of the subsequent bubble at an adjacent nucleation site.
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The induction times for electrodeposition of individual Ag nanoparticles on Pt nanodisk electrodes in acetonitrile were used to determine the critical nucleus size and activation energy barrier associated with the formation of Ag nuclei. Induction times for the nucleation and growth of a single Ag nanoparticle were determined following the application of a potential step to reduce Ag+ at overpotentials, η, ranging from −130 to −70 mV. Sufficiently small Pt electrodes (5.1 × 10–10–2.6 × 10–11 cm2) were used to ensure that the detection of a single Ag nucleation event occurred during the experimental observation time (150 ms–1000 s). Multiple measurements of Ag nucleation induction times were recorded to determine nucleation rates as a function of η using cumulative probability theory. Both classical nucleation theory (CNT) and the atomistic theory of electrochemical nucleation were employed to analyze experimental nucleation rates, without a requisite knowledge of the nucleus geometry or surface free energy. Using the CNT, the number of atoms comprising the critical size nucleus, Nc, was estimated to be 1–9 atoms for η ranging from −130 to −70 mV, in good agreement with 1–5 atoms obtained using atomistic theory. The experimental nucleation rates were also used to determine the activation energy barriers for nucleation from the CNT, which varied from 3.31 ± 0.05 to 13 ± 1 kT over the same range of η.
Classical nucleation theory
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Fibril formation is an obligatory process in amyloid diseases and is characterized by nucleation and elongation phases that result in the formation of long filaments with cross-β sheet structure. The kinetics of this process, as well as that of secondary nucleation, is controlled by a variety of factors, including nucleus (seed) structure, monomer conformation, and biochemical milieu. Some fibrillar amyloid assemblies act as prions, replicating themselves from protein monomers templated by existing prion seeds. Prion strains, which are characterized by distinct physicochemical and pathologic properties, may also form due to perturbation of the templating process within the susceptible organism. Understanding the types and effects of perturbations occurring during the development and progression of Parkinson's disease is an area requiring more study. Here, we used high-speed atomic force microscopy to determine the kinetics and structural dynamics of α-synuclein fibril elongation initiated by self-seeding or cross-seeding of wild-type (WT) or mutant α-synuclein with WT or mutant α-synuclein seeds. We found that cross-seeding modulated not only elongation rates but also the structures of the growing fibrils. Some fibrils produced in this manner had structures distinct from their "parent" seeds. In other cases, cross-seeding was not observed at all. These findings suggest that α-synuclein sequence variants can produce different types of strains by self- or cross-seeding. Perpetuation of specific strains then would depend on the relative rates of fibril growth and the relative stabilities of the fibrils formed by each strain.
Elongation
Morphology
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While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are examined. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ≈1.2 labels per collagen molecule preserves these characteristics. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labeled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development at low concentration, the labeled collagen is incorporated into a fine extracellular matrix (ECM) network associated with the cells within 24 h.
Fibrillogenesis
Type I collagen
Collagen fibril
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