Thioflavin T fluorescence to analyse amyloid formation kinetics: Measurement frequency as a factor explaining irreproducibility

Sebastiao, Mathew; Quittot, Noé; Bourgault, Steve

doi:10.1016/j.ab.2017.06.007

Cited by 54 publications

(54 citation statements)

References 22 publications

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“…The amyloid cross‐β‐sheet conformation was also evaluated by measuring thioflavin T (ThT) fluorescence. ThT is a small dye whose fluorescence emission increases sharply upon its binding to cross‐β‐sheet quaternary structure . Surprisingly, an increase of ThT fluorescence was only observed for uncapped I 10 , whereas the N‐capped assemblies were ThT‐negative (Figure c).…”

Section: Resultsmentioning

confidence: 97%

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods

Zottig

Al‐Halifa

Babych

et al. 2019

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Peptides that self‐assemble into cross‐β‐sheet amyloid structures constitute promising building blocks to construct highly ordered proteinaceous materials and nanoparticles. Nevertheless, the intrinsic polymorphism of amyloids and the difficulty of controlling self‐assembly currently limit their usage. In this study, the effect of electrostatic interactions on the supramolecular organization of peptide assemblies is investigated to gain insights into the structural basis of the morphological diversities of amyloids. Different charged capping units are introduced at the N‐terminus of a potent β‐sheet‐forming sequence derived from the 20–29 segment of islet amyloid polypeptide, known to self‐assemble into polymorphic fibrils. By tuning the charge and the electrostatic strength, different mesoscopic morphologies are obtained, including nanorods, rope‐like fibrils, and twisted ribbons. Particularly, the addition of positive capping units leads to the formation of uniform rod‐like assemblies, with lengths that can be modulated by the charge number. It is proposed that electrostatic repulsions between N‐terminal positive charges hinder β‐sheet tape twisting, leading to a unique control over the size of these cytocompatible nanorods by protofilament growth frustration. This study reveals the high susceptibility of amyloid formation to subtle chemical modifications and opens to promising strategies to control the final architecture of proteinaceous assemblies from the peptide sequence.

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Section: Resultsmentioning

confidence: 97%

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods

Zottig

Al‐Halifa

Babych

et al. 2019

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“…CD spectroscopy revealed a secondary conformational transition from a mixture of random coil and α-helix (two minima at 200 and 222 nm) to a β-sheet (one minimum at 217 nm) occurring between 96 to 168 h incubation ( Figure 2 c), in agreement with turbidity measurement and what has been reported for amyloid-related assemblies. ThT fluorescence, which reports the formation of cross-β-sheet quaternary structure [ 37 , 38 ], revealed that M2e-I 10 peptide assembled into ThT-positive structure after 48 h, and that a plateau of ThT fluorescence was reached between 96 and 168 h.…”

Section: Resultsmentioning

confidence: 99%

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-β Fibrils to Design Adjuvanted Nanovaccines

et al. 2020

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Protein fibrils characterized with a cross-β-sheet quaternary structure have gained interest as nanomaterials in biomedicine, including in the design of subunit vaccines. Recent studies have shown that by conjugating an antigenic determinant to a self-assembling β-peptide, the resulting supramolecular assemblies act as an antigen delivery system that potentiates the epitope-specific immune response. In this study, we used a ten-mer self-assembling sequence (I10) derived from an amyloidogenic peptide to biophysically and immunologically characterize a nanofibril-based vaccine against the influenza virus. The highly conserved epitope from the ectodomain of the matrix protein 2 (M2e) was elongated at the N-terminus of I10 by solid phase peptide synthesis. The chimeric M2e-I10 peptide readily self-assembled into unbranched, long, and twisted fibrils with a diameter between five and eight nm. These cross-β nanoassemblies were cytocompatible and activated the heterodimeric Toll-like receptor (TLR) 2/6. Upon mice subcutaneous immunization, M2e-fibrils triggered a robust anti-M2e specific immune response, which was dependent on self-assembly and did not require the use of an adjuvant. Overall, this study describes the efficacy of cross-β fibrils to activate the TLR 2/6 and to stimulate the epitope-specific immune response, supporting usage of these proteinaceous assemblies as a self-adjuvanted delivery system for antigens.

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“…We recently reported that, although the microplate kinetic assay is done under quiescent conditions (i.e. no agitation between each reading), the displacement of the microplate within the fluorimeter occurring during measurements is sufficient to accelerate amyloid formation (40). Thus, self-assembly of N21A, N21G, and N21Aib appears to be more dependent on agitation than the WT IAPP.…”

Section: Self-assembly-toxicity Relationships Of Iappmentioning

confidence: 99%

Identification of a hinge residue controlling islet amyloid polypeptide self-assembly and cytotoxicity

Godin

Nguyen

Zottig

et al. 2019

Journal of Biological Chemistry

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The islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone whose deposition as amyloid fibrils in the pancreatic islets is associated with type 2 diabetes. Previous studies have suggested that residue Asn-21 plays a critical role in the in vitro self-assembly of IAPP. Herein, we studied structure-self-assembly relationships focusing on position 21 to gain detailed insights into the molecular mechanisms of IAPP self-assembly and to probe the conformational nature of the toxic assemblies associated with ␤-cell death. Thioflavin T (ThT) fluorescence, CD spectroscopy, and transmission EM analysis revealed that the Asn-21 amide side chain is not required for IAPP nucleation and amyloid elongation, as N21A and N21G variants assembled into prototypical fibrils. In contrast, Asn-21 substitution with the conformationally constrained and turn-inducing residue Pro accelerated IAPP self-assembly. Successive substitutions with hydrophobic residues led to the formation of ThT-negative ␤-sheet-rich aggregates having high surface hydrophobicity. Cell-based assays revealed no direct correlation between the in vitro amyloidogenicity of these variants and their toxicity. In contrast, leakage of anionic lipid vesicles disclosed that membrane disruption is closely associated with cytotoxicity. We observed that the N21F variant self-assembles into worm-like aggregates, causing loss of lipid membrane structural integrity and inducing ␤-cell apoptosis. These results indicate that specific intra-and intermolecular interactions involving Asn-21 promote IAPP primary nucleation events by modulating the conformational conversion of the oligomeric intermediates into amyloid fibrils. Our study identifies position 21 as a hinge residue that modulates IAPP amyloidogenicity and cytotoxicity.

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Thioflavin T fluorescence to analyse amyloid formation kinetics: Measurement frequency as a factor explaining irreproducibility

Cited by 54 publications

References 22 publications

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-β Fibrils to Design Adjuvanted Nanovaccines

Identification of a hinge residue controlling islet amyloid polypeptide self-assembly and cytotoxicity

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