Biology subjects

Thacker, D.

Publications and source records attributed to Thacker, D..

6 recordsLinked to original sources

bioRxiv2026

Heterogeneous primary nucleation determines Aβ42 fibrilstructure.

The current developments in the field of amyloid fibril structure determination has revealed multiple structures for peptides of identical sequence. This may reflect variations in sample environment or similar nucleation barriers for multiple folds. Here we investigate the influence of aggregating surface and during fibril formation from monomeric amyloid {beta} peptide (A{beta}42). The fibril structures formed in identical solutions in glass vials / bottles and PEGylated polystyrene 96-well plates display very different monomer fold within the fibrils. This is a clear indication of extremely slow homogeneous primary nucleation. Instead, heterogeneous primary nucleation at vessel walls or at the three-line interface between air, water and vessel wall dictate the outcome in each solution, where the initially formed structure is propagated through elongation and secondary nucleation. The results further imply that the conversion towards the thermodynamically stable fibril structure one is slow for A{beta}42.

biochemistry↗

bioRxiv2026

Errors in peptide synthesis are a source of discrepancies in Aβ42 studies

Amyloid-{beta}42 (A{beta}42) aggregation is highly sensitive to experimental conditions, making reproducibility a persistent challenge in Alzheimers disease research. Among the many variables that influence aggregation, the impact of peptide production remains poorly understood. Direct comparison of recombinant and chemically synthesised A{beta}42 prepared under carefully controlled conditions reveals that, despite following similar aggregation mechanism and forming the same predominant fibril structures, synthetic A{beta}42 aggregates more slowly and exhibits reduced seeding efficiency. Consequently, synthetic A{beta}42 produces fewer oligomeric species and displays lower cellular toxicity. Mass spectrometric analyses identify low-abundance sequence imperfections introduced during peptide synthesis as the origin of these differences. By linking synthesis-derived imperfections to variations in A{beta}42 behaviour, this work reveals a previously underappreciated source of discrepancies in amyloid studies. In addition, we provide a framework for evaluating the impact of sequence impurities on biophysical studies that are sensitive to peptide composition.

biochemistry↗

bioRxiv2025

Aducanumab Binding to Aβ1-42 Fibrils Alters Dynamics of the N-Terminal Tail While Preserving the Fibril Core

Aducanumab, a human IgG1 antibody with plaque-clearing effects and modest clinical benefit, binds selectively to aggregated A{beta} via the N-terminal region. Yet, the molecular details of how the antibody engages A{beta}1-42 fibrils remain unresolved. Using magic-angle spinning nuclear magnetic resonance, we show that binding of aducanumab preserves the overall architecture of the A{beta}1-42 fibril core while inducing significant structural and dynamic perturbations in the N-terminal region. Antibody binding markedly reduces flexibility in this domain, with the appearance of sidechain resonances from residues D1, E3, and histidine (likely H6) in dipolar-based experiments. These sidechains--previously observed only in scalar-coupling spectra of the unbound state--indicate rigidification of residues that were dynamic. The interaction extends to S8 and Y10, indicating broader fibril engagement than the minimal epitope (residues 3-7) defined in fragment-based studies. Perturbations in the C-terminal segment (G37-A42) are consistent with its spatial proximity to the antibody-bound N-termini of neighboring monomers. Cryo-TEM images reveal fibrils bundling in the presence of aducanumab, consistent with lateral association via antibody cross-linking, supporting a model where surface coating and steric hindrance suppress secondary nucleation. This mode of action restricts monomer access to catalytic sites on fibril surface, resulting in partial inhibition ([~]three-fold reduction) of secondary nucleation. The effect depends on high avidity and relatively high stoichiometry, but is ultimately limited by antibody size relative to N-terminal spacing along the fibril. These findings provide atomic-level insights into aducanumabs binding mode and supply a structural framework for understanding antibody-mediated fibril recognition and for guiding next-generation therapies targeting A{beta} aggregates in Alzheimers disease. Significance StatementUnderstanding how therapeutic antibodies interact with amyloid-{beta} (A{beta}) fibrils is crucial for developing effective Alzheimers disease treatments. Magic angle spinning NMR provides atomic-level insights into the binding of aducanumab to mature A{beta}1-42 fibrils. Aducanumab binding preserves the fibrils core structure but slows the dynamics of the N-terminal domain of A{beta}. This interaction, which spans D1 to S8 and extends to Y10 on the fibril surface, is consistent with a mechanism in which N-terminal binding by the antibody interferes with aggregation steps like secondary nucleation. These findings detail how aducanumab engages its target fibril and provides insights relevant to other clinically approved antibodies and next-generation therapies that recognize the A{beta} N-terminal region.

biophysics↗

bioRxiv2025

Structural defects in amyloid-β fibrils drive secondary nucleation

The nucleation of amyloid fibrils from monomeric protein, catalyzed by the surface of existing fibrils, is an important driver of many disorders such as Alzheimers and Parkinsons diseases. The structural basis of this secondary nucleation process, however, is poorly understood. Here, we ask whether secondary nucleation sites are found predominantly at rare growth defects: defects in the fibril core structure generated during their original assembly. We first demonstrate using the specific inhibitor of secondary nucleation, Brichos, that secondary nucleation sites on Alzheimers disease-associated fibrils composed of A{beta}40 and A{beta}42 peptides are rare compared to the number of protein molecules they contain. We then grow A{beta}40 fibrils under conditions designed to eliminate most growth defects while leaving the regular fibril morphology unchanged, and confirm the latter using cryo-electron microscopy. We measure both the ability of these annealed fibrils to promote secondary nucleation and the stoichiometry of their secondary nucleation sites, finding that both are greatly reduced as predicted. Re-analysis of published data for other proteins suggests that fibril growth defects that expose monomer planes or other structural units may also drive secondary nucleation generally, across most or all amyloids. These findings could unlock structure-based drug design of therapeutics that aim to halt amyloid disorders by inhibiting secondary nucleation sites.

biophysics↗

bioRxiv2024

Residues 2-7 of alpha-synuclein regulate amyloid formation via lipid-dependent and -independent pathways

Amyloid formation by -synuclein (Syn) occurs in Parkinsons disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate Syn amyloid fibril formation will not only provide mechanistic insight, but may also reveal new targets to prevent and treat disease. Previous investigations have identified several regions of Syn to be important in the regulation of amyloid formation, including the non-amyloid-{beta} component (NAC), P1 region (residues 36-42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of Syn for both its physiological and pathological roles. Here, the role of residues 2-7 in the N-terminal region of Syn are investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (Syn{Delta}N7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (SynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length Syn. Strikingly, fibril formation of Syn{Delta}N7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to SynWT. A Caenorhabditis elegans model also showed that Syn{Delta}N7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of SynWT::YFP. Together, the results demonstrate the involvement of residues 2-7 of Syn in amyloid formation, revealing a new target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed. Significance StatementAmyloid formation of -synuclein (Syn) is associated with Parkinsons disease. Attempts to target Syn aggregation to treat synucleinopathies, thus far, have been unsuccessful. A better understanding of residues that regulate amyloid formation may reveal new targets for therapeutics. Here, six residues at the N-terminus of Syn are identified as regulators of amyloid formation. Deletion of these residues slows lipid-independent assembly, ablates lipid-dependent amyloid formation in vitro, and prevents aggregation and its associated cellular toxicity in vivo. Importantly, these residues are not necessary for binding to synthetic membranes. The work reveals a new target for the prevention of synucleinopathies by disfavouring aggregation without perturbing membrane binding, a property considered to be essential for the physiological function of Syn at the synapse.

biophysics↗

bioRxiv2022

A Kinetic Map of the Influence of Biomimetic Lipid Membrane Models on Aβ42 Aggregation

The aggregation of the amyloid {beta} peptide (A{beta}) is one of the major molecular hallmarks of Alzheimers disease. Although A{beta} deposits have been mostly observed extracellularly, various studies have reported the presence of also intracellular A{beta} assemblies. Because these intracellular A{beta} aggregates might play a role in the onset and progression of Alzheimers disease, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein (APP), from which A{beta} is derived by proteolytic cleavage. Since lipid bilayers have been shown to promote the aggregation of A{beta}, in this study we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of the 42-residue form of A{beta} (A{beta}42). By using small unilamellar vesicles modelling cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the endoplasmic reticulum (ER), and the Golgi apparatus, we show that A{beta}42 aggregation is inhibited by the ER and Golgi membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on A{beta} aggregation, and suggest the presence of an evolutionary optimization of lipid composition to prevent the intracellular aggregation of A{beta}.

biophysics↗