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Fichou, Y.

Publications and source records attributed to Fichou, Y..

7 recordsLinked to original sources
bioRxiv2025

DEER of Singly Labelled Proteins to Evaluate Supramolecular Packing of Amyloid Fibrils

1The formation of protein amyloid fibrils in the brain is a hallmark of various neurodegenerative diseases, including Alzheimer diseases and Parkinson diseases. Amyloid fibrils are highly ordered aggregates in which proteins folded in two-dimensional layers stack along one axis to form elongated linear assemblies. The specific conformations adopted by the proteins within each layer of the amyloid correlates with the pathology. Furthermore, their spreading and templating competency relies on strict in register packing of the folded proteins along the fibril growing axis. There is a need for tools to characterize not only the protein fold across the fibril cross section but also the spatial ordering of the proteins stacked along the amyloid fibril axis. We present an approach based on double electron electron resonance spectroscopy (DEER) using singly labelled tau protein assembled in amyloid fibrils that can deliver an apparent dimensionality of the supramolecular organization of tau fibrils. The parameters of the DEER background function can be used to assess the amyloid core location and packing order, and track time-resolved formation of aggregation intermediates. Showcasing the method on tau, we demonstrate that heparin-induced tau fibrils are mispacked while seeded aggregation can template amyloid fibrils with a higher packing order. This study benchmarks a new method that will provide critical structural insights into amyloid assemblies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/687719v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@8c42c9org.highwire.dtl.DTLVardef@74b0f8org.highwire.dtl.DTLVardef@10e8095org.highwire.dtl.DTLVardef@11bc727_HPS_FORMAT_FIGEXP M_FIG DEER-derived dimensionality parameter differentiates between spin-labelled tau protein in solution, in perfectly aligned fibril or in imperfectly aligned fibril (fibril structure edited from PDB 6qjh). C_FIG

biophysics↗
bioRxiv2025

Development of an EPR-based methodology to study protein-lipid interaction

The interaction of protein with other biomolecules is central to all cellular processes. In particular, protein-lipid interactions play an essential role in regulating soluble and membrane protein function, structure, and dynamics. However, probing these interactions remains challenging due to the complexity and heterogeneity of membranes. Various methods have been developed to characterize protein-membrane interaction, each presenting advantages and limitations. This study presents a robust methodology based on continuous-wave Electron Paramagnetic Resonance (CW-EPR) spectroscopy to characterize protein-membrane interactions. We focused on the protein Tau, an intrinsically disordered protein associated with neurodegenerative diseases. We show that the interaction of labelled Tau with lipids gives rise to a very distinct lineshape, which can be used to quantify the fraction of bound protein. This allows to obtain the apparent binding mode and affinity through titration experiments. In addition, we show that a single measurement provides the absolute concentration of free and bound protein. We argue that this information, which is rarely obtained by other methods providing relative signals, is very useful for mechanistic studies. Furthermore, using mathematical modeling, we developed a minimal-data approach and demonstrated that a single EPR measurement can be used to derive binding constants. The approach is applied to the Taumembrane interaction occurring in different conditions affecting the binding behavior. The presented methodology is expected to be applicable to other proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/672991v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@19cf0c5org.highwire.dtl.DTLVardef@833209org.highwire.dtl.DTLVardef@149fd8corg.highwire.dtl.DTLVardef@a4f50a_HPS_FORMAT_FIGEXP M_FIG C_FIG TOC Figure. Schematic representation of EPR-based analysis of Tau-membrane interactions using spin-labeled Tau and POPS vesicles

biophysics↗
bioRxiv2025

Localized Reconstruction of Multimodal Distance Distribution from DEER Data of Biopolymers

Pulsed Dipolar ESR Spectroscopy (PDS) is a uniquely powerful technique to characterize the structural property of intrinsically disordered proteins (IDPs) and polymers and the conformational evolution of IDPs and polymers, e.g. during assembly, by offering the probability distribution of segment end-to-end distances. However, it is challenging to determine distance distribution P(r) of IDPs by PDS because of the uncertain and broad shape information that is intrinsic to the distance distribution of IDPs. We demonstrate here that the Srivastava-Freed Singular Value Decomposition (SF-SVD) point-wise mathematical inversion method along with wavelet denoising (WavPDS) can aid in obtaining reliable shapes for the distance distribution, P(r), for IDPs. We show that broad regions of P(r) as well as mixed narrow and broad features within the captured distance distribution range can be effectively resolved and differentiated without a priori knowledge. The advantage of SF-SVD and WavPDS is that the methods are transparent, requiring no adjustable parameters, the processing of the magnitude for the probability distribution is performed separately for each distance increment, and the outcome of the analysis is independent of the users judgement. We demonstrate the performance and present the application of WavPDS and SF-SVD on model ruler molecules, model polyethylene glycol polymers with end-to-end spin labeling, and IDPs with pairwise labeling spanning different segments of the protein tau to generate the transparent solutions to the P(r)s including their uncertainties and error analysis.

biophysics↗
bioRxiv2023

Mutations in tau protein promote aggregation by favoring extended conformations

Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Mutations in the gene encoding tau are responsible for a class of inherited tauopathies called frontotemporal dementia and parkinsonism linked to chromosome 17Q (FTDP-17). These mutations are thought to trigger FTDP-17 by favoring the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the diseases-associated single point mutations promote amyloid formation. We combined biochemical characterization and small angle X-ray scattering (SAXS) to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radius of gyration. We show that mutations disfavor intramolecular protein interactions which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate aggregation propensity of tau variants.

biophysics↗
bioRxiv2020

Liquid-liquid phase separation of tau driven by hydrophobic interaction facilitates fibrillization of tau

Amyloid aggregation of tau protein is implicated in neurodegenerative diseases, yet its facilitating factors are poorly understood. Recently, tau has been shown to undergo liquid liquid phase separation (LLPS) both in vivo and in vitro. LLPS was shown to facilitate tau amyloid aggregation in certain cases, while independent of aggregation in other cases. It is therefore important to understand the differentiating properties that resolve this apparent conflict. We report on a model system of hydrophobically driven LLPS induced by high salt concentration (LLPS-HS), and compare it to electrostatically driven LLPS represented by tau-RNA/heparin complex coacervation (LLPS-ED). We show that LLPS-HS promotes tau protein dehydration, undergoes maturation and directly leads to canonical tau fibrils, while LLPS-ED is reversible, remains hydrated and does not promote amyloid aggregation. We show that the nature of the interaction driving tau condensation is the differentiating factor between aggregation-prone and aggregation-independent LLPS.

biophysics↗
bioRxiv2019

Liquid-liquid phase separation and fibrillization of tau are independent processes with overlapping conditions

Amyloid aggregation of the microtubule binding protein tau is a hallmark of Alzheimers disease and many other neurodegenerative diseases. Recently, tau has been found to undergo liquid-liquid phase separation (LLPS) near physiological conditions. Although LLPS and aggregation have been shown to simultaneously occur under certain common conditions, it remains to be seen whether tau LLPS promotes aggregation, or if they are two independent processes. In this study, we address this question by combining multiple biochemical and biophysical assays in vitro. We investigated the impacts of LLPS on tau aggregation at three stages: conformation of tau, kinetics of aggregation and fibril quantity. We showed that none of these properties are influenced directly by LLPS, while amyloid aggregation propensity of tau can be altered without affecting its LLPS behavior. LLPS and amyloid aggregation of tau occur under overlapping conditions of enhanced intermolecular interactions and localization, but are two independent processes.

biophysics↗
bioRxiv2018

First estimation of the scale of canonical 5’ splice site GT>GC mutations generating wild-type transcripts and their medical genetic implications

It has long been known that canonical 5 splice site (5SS) GT>GC mutations may be compatible with normal splicing. However, to date, the true scale of canonical 5SS GT>GC mutations generating wild-type transcripts, both in the context of the frequency of such mutations and the level of wild-type transcripts generated from the mutation alleles, remain unknown. Herein, combining data derived from a meta-analysis of 45 informative disease-causing 5SS GT>GC mutations (from 42 genes) and a cell culture-based full-length gene splicing assay of 103 5SS GT>GC mutations (from 30 genes), we estimate that [~]15-18% of the canonical GT 5SSs are capable of generating between 1 and 84% normal transcripts as a consequence of the substitution of GT by GC. We further demonstrate that the canonical 5SSs whose substitutions of GT by GC generated normal transcripts show stronger complementarity to the 5 end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were able to accurately predict the functional impact of 5SS GT>GC mutations. Our findings imply that 5SS GT>GC mutations may not invariably cause human disease but should also help to improve our understanding of the evolutionary processes that accompanied GT>GC subtype switching of U2-type introns in mammals.

genetics↗