Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

Sidhu, Arshdeep; Vaneyck, Jonathan; Blum, Christian; Segers-Nolten, Ine; Subramaniam, Vinod

doi:10.1080/13506129.2018.1517736

Cited by 61 publications

(56 citation statements)

References 35 publications

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“…More specifically, stronger H-bonds and/or longer -chains would probably add more constraints and less flexibility to the ThT binding site with the result of inhibiting more efficiently the non-radiative ThT rotation and increasing the fluorophore's lifetime 68 . This picture is supported by Sidhu and co-workers, which employ the analysis of ThT fluorescence decays in terms of two components (both in the ns range) to discriminate among different fibril polymorphs 78 . Shorter lifetime components were attributed to ThT binding sites characterized by larger flexibility, while longer lifetime components to tighter binding sites.…”

Section: Data and Interpretation Presented Inmentioning

confidence: 73%

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Luca

Galparsoro

Sancataldo

et al. 2020

Journal of Colloid and Interface Science

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Ensembles of protein aggregates are characterized by a nano-and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable -structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also on the position within a single spherulite at submicron scale. We unambiguously reveal that the surface of the spherulites are characterized by -structures with an enhanced H-bond coupling compared to the core. This information, inaccessible via bulk methods, allows us to relate the aggregate structure at molecular level to the overall morphology of the aggregates. Our findings robustly solve the problem of probing the ensemble and single particle heterogeneity of amyloid samples. Furthermore, they offer a unique, scalable and ready-to-use screening methodology for in-depth characterization of self-assembled structures, being this translatable to material sciences, drug quality control and clinical imaging of amyloid-affected tissues.

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Section: Data and Interpretation Presented Inmentioning

confidence: 73%

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Luca

Galparsoro

Sancataldo

et al. 2020

Journal of Colloid and Interface Science

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“…The same approach using NAS probes was performed to sense changes upon alpha-synuclein aggregation [ 106 ]. Additionally, the study of the τ and correlation time of the pathogenic alpha-synuclein mutants, A30P and A53T, showed that they had distinct types of binding sites, which was in concordance with the difference in fibril morphology [ 120 ]. Additionally, the anisotropy decay profiles of fluorescein labelled-alpha synuclein have recently been used to study its liquid–liquid phase separation into droplets, an event that occurs before protein fibrillation [ 121 ].…”

Section: Fluorescence Spectroscopy Is a Versatile Tool For Evaluatmentioning

confidence: 89%

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

2020

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The self-assembly of proteins is an essential process for a variety of cellular functions including cell respiration, mobility and division. On the other hand, protein or peptide misfolding and aggregation is related to the development of Parkinson’s disease and Alzheimer’s disease, among other aggregopathies. As a consequence, significant research efforts are directed towards the understanding of this process. In this review, we are focused on the use of UV-Visible Absorption Spectroscopy, Fluorescence Spectroscopy and Circular Dichroism to evaluate the self-organization of proteins and peptides in solution. These spectroscopic techniques are commonly available in most chemistry and biochemistry research laboratories, and together they are a powerful approach for initial as well as routine evaluation of protein and peptide self-assembly and aggregation under different environmental stimulus. Furthermore, these spectroscopic techniques are even suitable for studying complex systems like those in the food industry or pharmaceutical formulations, providing an overall idea of the folding, self-assembly, and aggregation processes, which is challenging to obtain with high-resolution methods. Here, we compiled and discussed selected examples, together with our results and those that helped us better to understand the process of protein and peptide aggregation. We put particular emphasis on the basic description of the methods as well as on the experimental considerations needed to obtain meaningful information, to help those who are just getting into this exciting area of research. Moreover, this review is particularly useful to those out of the field who would like to improve reproducibility in their cellular and biomedical experiments, especially while working with peptide and protein systems as an external stimulus. Our final aim is to show the power of these low-resolution techniques to improve our understanding of the self-assembly of peptides and proteins and translate this fundamental knowledge in biomedical research or food applications.

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“…1 ), factors other than the difference of length between HP- and DS-induced tau filaments may affect the level of fluorescence. The latter may indicate qualitatively different thioflavin binding sites as the result of structural differences between tau filaments formed by HP and the more sulphated DS, as has been reported for α-synuclein filaments ( Sidhu et al , 2018 ). This is also supported by the findings that murine tau in the presence of HP exhibited greater sarkosyl-insolubility than that in the presence of DS ( Fig.…”

Section: Discussionmentioning

confidence: 61%

Dextran sulphate-induced tau assemblies cause endogenous tau aggregation and propagation in wild-type mice

Masuda-Suzukake

Suzuki

Hosokawa

et al. 2020

Brain Communications

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Abstract Accumulation of assembled tau protein in the central nervous system is characteristic of Alzheimer’s disease and several other neurodegenerative diseases, called tauopathies. Recent studies have revealed that propagation of assembled tau is key to understanding the pathological mechanisms of these diseases. Mouse models of tau propagation are established by injecting human-derived tau seeds intracerebrally; nevertheless, these have a limitation in terms of regulation of availability. To date, no study has shown that synthetic assembled tau induce tau propagation in non-transgenic mice. Here we confirm that dextran sulphate, a sulphated glycosaminoglycan, induces the assembly of recombinant tau protein into filaments in vitro. As compared to tau filaments induced by heparin, those induced by dextran sulphate showed higher thioflavin T fluorescence and lower resistance to guanidine hydrochloride, which suggests that the two types of filaments have distinct conformational features. Unlike other synthetic filament seeds, intracerebral injection of dextran sulphate-induced assemblies of recombinant tau caused aggregation of endogenous murine tau in wild-type mice. AT8-positive tau was present at the injection site one month after injection, from where it spread to anatomically connected regions. Induced tau assemblies were also stained by anti-tau antibodies AT100, AT180, 12E8, PHF1, anti-pS396 and anti-pS422. They were thioflavin- and Gallyas-Braak silver-positive, indicative of amyloid. In biochemical analyses, accumulated sarkosyl-insoluble and hyperphosphorylated tau was observed in the injected mice. In conclusion, we revealed that intracerebral injection of synthetic full-length wild-type tau seeds prepared in the presence of dextran sulphate caused tau propagation in non-transgenic mice. These findings establish that propagation of tau assemblies does not require tau to be either mutant and/or overexpressed.

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Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

Cited by 61 publications

References 35 publications

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

Dextran sulphate-induced tau assemblies cause endogenous tau aggregation and propagation in wild-type mice

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