Biology subjects

Tsay, K.

Publications and source records attributed to Tsay, K..

6 recordsLinked to original sources

bioRxiv2026

A water-pinning hotspot drives templated tau aggregation

Tau assembles into fibrillar aggregates that are pathological hallmarks of a group of neurodegenerative diseases collectively called tauopathies. Templated aggregation of naive tau to seeding-competent fibrils that proceed from cell to cell is a key driver of prion-like progression of tauopathies. This study tests the hypothesis that tau, an intrinsically disordered protein (IDP), achieves in-register stacking to form seed-competent fibrils by a pinning action of tau to each other and/or the seed surface via a single dominant hotspot to avoid mismatch in tau stacking to fibrils. Structured solvation water has been proposed to be a signature of such hotspots at both the tau fibril-end surface and soluble tau monomers. Although jR2R3-P301L tau exhibits a heterogeneous hydration landscape in its intrinsically disordered state, with enhanced water structuring near the P301L mutation site, it is unclear whether a localized hotspot exists at the fibril end surface and surface water facilitates the initial contacts in templated aggregation. Using rapid 1H-15N SOFAST-HMQC NMR to track seed-induced aggregation of jR2R3-P301L in real time, complemented by molecular dynamics simulation of fibril surface hydration, we identify a residue-specific pinning hotspot that is prone to dewetting followed by sequential folding and incorporation of the remaining segment in a two-step dock-and-lock process. Site-specific spin labeling further demonstrates that blocking this pinning hotspot disrupts templated aggregation, leading to shorter fibrils. The identification of a dominant pinning site will facilitate the rational design of binders to effectively disrupt fibril extension or serve as diagnostic or therapeutic strategies.

biophysics↗

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

Structure-specific Mini-Prion Model for Alzheimer's Disease Tau Fibrils

A critical discovery of the past decade is that tau protein fibrils adopt disease-specific hallmark structures in each tauopathy. The faithful generation of synthetic fibrils adopting hallmark structures that can serve as targets for developing diagnostic and/or therapeutic strategies remains a grand challenge. We report on a rational design of synthetic fibrils built of a short peptide that adopts a critical structural motif in tauopathy fibrils found in Alzheimers Disease (AD) and Chronic Traumatic Encephalopathy (CTE). They serve as minimal prions with exquisite seeding competency, in vitro and in tau biosensor cells, for recruiting tau constructs ten times larger its size en route to AD or CTE fibril structures. We demonstrate that the generation of AD and CTE-like fibril structures is dramatically catalyzed in the presence of mini-AD prions and further influenced by salt composition in solution. Double Electron-Electron Resonance studies confirmed the preservation of AD-like folds across multi-generational seeding. Fibrils formed with the full AD/CTE-like core show strong seeding competency, with their templating effect dominating over the choice of salt composition that tunes the initial selection of AD- and CTE-like fibril populations. The mini-AD prions serve as a potent catalyst with templating capabilities that offer a novel strategy to design pathological tau fibril models.

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

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement

Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross {beta}-sheets. We solve a 3 [A] cryo-EM structure of jR2R3-P301L fibrils with a pseudo 21 screw symmetry in which each half of the fibrils cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion. Significance StatementThis study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L--among the widest used mutations in cell and animal models of Alzheimers Disease--destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimers Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

biophysics↗

bioRxiv2023

CBD and PSP cell-passaged Tau Seeds Generate Heterogeneous Fibrils with A Subpopulation Adopting Disease Folds

The recent discovery by cryo-electron microscopy (cryo-EM) that the neuropathological hallmarks of different tauopathies, including Alzheimers disease, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP), are caused by unique misfolded conformations of the protein tau is among the most profound developments in neurodegenerative disease research. To capitalize on these discoveries for therapeutic development, one must achieve in vitro replication of tau fibrils that adopt the representative tauopathy disease folds, which represents a grand challenge for the field. An widely used approach is seeded propagation using pathological tau fibrils derived from post-mortem patients in biosensor cells that expresses a fragment of the tau protein, known as K18 or Tau4RD, containing mainly the microtubule-binding repeat domain of tau as the substrate. The new insight from cryo-EM raised the question whether the Tau4RD fragment is capable of adopting the specific tau folds found in CBD and PSP patient fibrils, and whether cell-passaged and amplified tau fibrils can be used as seeds to achieve cell-free assembly of recombinant 4R tau into fibrils without the addition of any cofactors. Using Double Electron Electron Resonance (DEER) spectroscopy, we discovered that cell-passaged pathological seeds generate heterogeneous fibrils that are distinct between the CBD and PSP lysate-seeded fibrils. These fibrils are also distinct from heparin-induced tau fibril populations. Moreover, the lysate-seeded fibrils contain a characteristic sub-population that resembles the disease fold corresponding to the respective starting patient sample. These findings indicate that templated propagation using CBD and PSP patient-derived fibrils is possible with a tau fragment that does not contain the entire pathological fibril core, and that additional mechanisms must be tuned to converge to a homogeneous fibril population.

biophysics↗