Biology subjects

Linse, S.

Publications and source records attributed to Linse, S..

At least 19 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

DNAJB6b: a complete amyloid inhibitor

In Alzheimer's disease (AD), the aggregation of amyloid {beta} (A{beta}) peptides has been linked to disease pathology. Once aggregates have formed, existing fibrils catalyze the formation of new fibrils in a runaway process associated with neuro degeneration. One component of the cellular defense system against amyloid formation is the molecular chaperone DNAJB6b (JB6). JB6 is known to suppress both primary nucleation and fibril-catalyzed secondary nucleation. However, the extent of JB6 binding to A{beta} fibrils and whether this interaction leads to inhibition of fibril elongation also, have not been established. Here, we combine well-characterized aggregation kinetics of A{beta}42 and the knowledge of JB6 self-assembly equilibrium and kinetics, with high-sensitivity HPLC measurements of free JB6 at nanomolar concentrations to determine the affinity of JB6 for A{beta}42 fibrils. We show that JB6 binds to A{beta}42 fibrils with high affinity and that this binding can quantitatively explain the observed elongation inhibition. Diffusion-based techniques reveal that JB6 binds to amyloid intermediates, resulting in potent suppression of amyloid nucleation until the pool of free JB6 is depleted, after which rapid bulk aggregation ensues. Our results provide a mechanistic link between chaperone-fibril binding and amyloid suppression, offering a quantitative framework for understanding how molecular chaperones regulate amyloid formation.

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

Secondary nucleation of α-Synuclein drives Mitochondria dysfunctions and Lewy body formation in Parkinson's Disease

The seeding of -Synuclein (Syn) is a key driver of Lewy pathology propagation in Parkinsons disease (PD) and forms the basis for recent diagnostic advances. However, it remains unclear how the structural and biochemical features of Syn seeds dictate their propagation efficiency, capacity to induce Lewy body formation, and resulting cellular toxicity. Using genetic and idiopathic PD cell models, we map the pathogenic cascade beginning with the seed-driven conversion of endogenous Syn, followed by impaired degradation, mitochondrial dysfunction, and ultimately Lewy body formation. By coupling kinetic modelling of aggregation with functional readouts, we identify secondary nucleation as the predominant mechanism generating toxic Syn aggregation intermediates, identifying the critical process that links seeding to pathology. Extending this framework to PD brain, we quantitatively correlate seeding capacity with the spatiotemporal spread and severity of Lewy pathology, revealing a mechanistic connection between Syn aggregation dynamics and disease progression at molecular, cellular, and anatomical levels. By unifying molecular mechanism with clinicopathological progression, our work identifies catalytic Syn fibrillar seeds as tractable targets for both disease-modifying therapy and biomarker development in PD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/676873v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1eca299org.highwire.dtl.DTLVardef@a714d4org.highwire.dtl.DTLVardef@1486db5org.highwire.dtl.DTLVardef@1aa895_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISyn fibril-oligomer interplay drives mitochondrial abnormalities and Lewy pathology C_LIO_LIFibrillar Syn catalyse toxic aggregate formations via secondary nucleation C_LIO_LIPhosphorylated Syn evades lysosomal clearance and drives enhanced dysfunctions C_LIO_LISeeding capacity of Syn predicts Lewy pathology burden and disease progression C_LI

neuroscience↗

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↗

bioRxiv2025

Design of Tau Aggregation Inhibitors Using Iterative Machine Learning and a Polymorph-Specific Brain-Seeded Fibril Amplification Assay

The aggregation of tau into amyloid fibrils is associated with Alzheimers disease (AD) and related tauopathies. Since different tauopathies are characterised by the formation of distinct tau fibril morphologies, it is important to combine the search of tau aggregation inhibitors with the development of in vitro tau aggregation assays that recapitulate aggregation as it may occur in the brain. Here we address this problem by reporting an in vitro tau aggregation assay in which AD brain homogenates are used to seed the generation of first-generation tau fibrils in a polymorph-specific manner under quiescent conditions. These fibrils are then used to create amyloid seed libraries from which second-generation kinetic assays can be readily performed. Using this strategy, we illustrate an iterative machine learning method for the identification of small molecules for the polymorph-specific inhibition of the in vitro formation of tau fibrils. We further show that the small molecules selected by this procedure are potent inhibitors in a Drosophila tauopathy model.

biophysics↗

bioRxiv2025

On the transient interactions of α-synuclein in different dimensions

0.-Synuclein (Syn) is a neuronal protein predominantly found in the brain, whose native function seems to be associated with vesicle trafficking. While intrinsically disordered in solution, the first ca. 100 residues adopt an amphipathic -helical structure when the protein adsorbs onto membranes. Additionally, the aggregation of Syn into highly ordered {beta}-sheet rich amyloid fibrils is associated with Parkinso[n]s disease. The different regions of Syn and the interactions between them have been reported to play a key role in the behaviour of the protein in solution, its membrane binding, and its aggregation into fibrils. This study employs photo-induced cross-linking of unmodified proteins (PICUP) to capture and identify the transient contacts of Syn in different conformational states: free in solution, adsorbed to membranes, and aggregated into fibrils. By using tyrosine-to-phenylalanine mutations to block the reactivity of specific amino acid residues, we establish key cross-links in each state. In solution, we identify internal contacts between the N and C termini of monomers, as well as inter-monomer contacts between C termini in oligomers. When Syn is adsorbed to membranes, the internal cross-linking is blocked, while cross-linking between C-terminal regions persists. In fibrils, cross-linking is significantly reduced, primarily occurring between C-terminal residues of adjacent monomers. This work highlights the utility of PICUP for reporting on the transient contacts that occur on the pathways of self- and co-assembly of Syn.

biochemistry↗

bioRxiv2024

In situ generation of Aβ42 oligomers via secondary nucleation triggers neurite degeneration and synaptic dysfunction in human iPSC-derived glutamatergic neurons

The aggregation of A{beta}42 into misfolded oligomers is a central event in the pathogenesis of Alzheimers disease. In this study, we aimed to develop a robust experimental system that recapitulates A{beta}42 oligomerization in living cells to gain insight into their neurotoxicity and to provide a platform to characterize the effects of inhibitors of this process. Our strategy is based on the in situ generation of A{beta}42 oligomers via secondary nucleation by repeatedly treating the cells with A{beta}42 monomers in the presence of pre-formed A{beta}42 fibrils. This approach enables an accurate control over the levels of on-pathway soluble A{beta}42 oligomers and cell-associated aggregates, as well as the study of their neurotoxic effects. By implementing this approach in human glutamatergic neurons derived from induced pluripotent stem cells (iPSCs), we were able to replicate key aspects of Alzheimers disease, including neurite degeneration and synaptic dysfunction. Using BRICHOS, a molecular chaperone that specifically inhibits secondary nucleation, we confirmed that aggregation in this system occurs through secondary nucleation, and that quantitative parameters for comparing potential A{beta}42 aggregation inhibitors can be obtained. Overall, our results demonstrate that by in situ generation of on-pathway A{beta}42 oligomers, one can obtain translational cellular models of AD to bridge the gap between basic research and clinical applications.

cell biology↗

bioRxiv2024

Tipping point in α-synuclein-membrane interactions: stable protein-covered vesicles or amyloid aggregation

-synuclein is a neuronal protein implicated in neurotransmitter release. Its function is thought to critically depend on the dynamic equilibrium between free and membrane-bound protein. -synuclein amyloid formation implicated in Parkinsons Disease was also shown to be modulated by lipid membranes. However, it remains elusive whether -synuclein-related pathology is due to loss-of-function or gain-of-toxic-function. To help address this question, we studied the coupling of the equilibrium between free and membrane-bound -synuclein and membrane-induced amyloid formation - phenomena that are usually treated separately. We present a description of the system on a wide range of length scales and timescales for lipid-to-protein ratio conditions where amyloid formation is either accelerated or inhibited by lipid membranes. We find a clear difference between the dynamics and heterogeneity of the protein-covered membrane interface in the two sets of conditions. In aggregation-accelerating conditions, the membrane interface is dynamic and heterogeneous with rapid exchange between free and membrane-bound protein, and disordered protein segments of varying lengths exposed to solution. All these characteristics of the membrane interface are likely to decrease the free energy barrier for amyloid formation. Conversely, the membrane interface is homogeneous and less dynamic in conditions where amyloid formation is inhibited. Importantly, any factors affecting the equilibrium between free and membrane-bound -synuclein may trigger a change from non-aggregating to aggregating conditions. Altogether, our results highlight a strong coupling of the dynamic equilibrium between the free and membrane-bound -synuclein and membrane-modulated amyloid formation and thus of the physiological function of -synuclein and its aberrant aggregation.

biochemistry↗

bioRxiv2024

The Alzheimer's Aβ peptide forms biomolecular condensates that trigger amyloid aggregation

The onset and development of Alzheimers disease (AD) is linked to the accumulation of pathological aggregates formed from the normally monomeric amyloid-{beta} peptide within the central nervous system. These A{beta} aggregates are increasingly successfully targeted with clinical therapies, but the fundamental molecular steps that trigger the initial nucleation event leading to the conversion of monomeric A{beta} peptide into pathological aggregates remain unknown. Here we show that the A{beta} peptide can form biomolecular condensates on lipid bilayers both in molecular assays and in living cells. Our results reveal that these A{beta} condensates can significantly accelerate the primary nucleation step in the amyloid conversion cascade that leads to the formation of amyloid aggregates and plaque. We show that A{beta} condensates contain phospholipids, are intrinsically heterogenous, and are prone to undergo a liquid-to-solid transition leading to the formation amyloid fibrils. These findings uncover the liquid-liquid phase separation behaviour of the A{beta} peptide, and reveal a new molecular step very early in the amyloid-{beta} aggregation cascade that can form the basis for novel therapeutic intervention strategies. Significance statementThe hallmark of Alzheimers disease is the abnormal buildup of the normally soluble amyloid {beta} protein aggregates in the central nervous system. While the molecular mechanisms at the late stages of the amyloid {beta} aggregation cascade are well understood, the initial steps remained elusive until now. Our current study demonstrates that amyloid {beta} undergoes liquid-liquid phase separation on lipid surfaces, which triggers primary nucleation and initiates the amyloid {beta} aggregation cascade. This newly identified step in the molecular mechanism of Alzheimers disease represents a promising target for the development of alternative innovative therapeutic strategies.

biophysics↗

bioRxiv2023

Molecular mechanism of α-synuclein aggregation on lipid membranes revealed.

The central hallmark of Parkinsons disease pathology is the aggregation of the -synuclein protein, which, in its healthy form, is associated with lipid membranes. Purified monomeric -synuclein is relatively stable in vitro, but its aggregation can be triggered by the presence of lipid vesicles. Despite this central importance of lipids in the context of -synuclein aggregation, their mechanistic role in this process has not been established to date. Here, we use chemical kinetics to develop a detailed mechanistic model that is able to globally describe the aggregation behaviour of -synuclein in the presence of DMPS lipid vesicles, across a range of lipid and protein concentrations. Through the application of our kinetic model to experimental data, we find that the reaction is a co-aggregation process involving both protein and lipids and that lipids promote aggregation predominantly by enabling the elongation process. Moreover, we find that the initial formation of aggregates, via primary nucleation, takes place not on the surface of lipid vesicles but at the interfaces present in vitro. Our model will enable mechanistic insights, also in other lipid-protein co-aggregation systems, which will be crucial in the rational design of drugs that inhibit aggregate formation and act at the key points in the -synuclein aggregation cascade.

biophysics↗

bioRxiv2023

Self-replication of Abeta42 aggregates occurs on small and isolated fibril sites

Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyse the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modelling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behaviour in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimers A{beta}42 amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A{beta}42 fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.

biophysics↗

bioRxiv2023

α-Synuclein oligomers form by secondary nucleation

Oligomeric species arising during aggregation of -synuclein are proposed to be a major source of toxicity in Parkinsons disease, and thus a major potential drug target. However, their mechanism of formation and role in aggregation are largely unresolved. Here we first show that, at physiological pH, -synuclein aggregates by secondary nucleation, rather than fragmentation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under physiologically relevant conditions.

biophysics↗

bioRxiv2022

Uncovering the universality of self-replication in protein aggregation and its link to disease.

Fibrillar protein aggregates are a hallmark of the pathology of a range of human disorders, from prion diseases to dementias. Yet, the same aggregated structures that are formed in disease are also encountered in several functional contexts. The fundamental properties that determine whether these protein assembly processes are functional or, by contrast, pathological, have remained elusive. Here, we address this question by analysing the aggregation kinetics of a large set of self-assembling proteins, from those associated with disease, over those whose aggregates fulfil functional roles in biology, to those that aggregate only under artificial conditions. Remarkably, we find that essentially all systems that assemble by a nucleated-growth mechanism are capable of significant self-replication on experimentally accessible timescales. However, comparing the intrinsic timescales of self-replication with the timescales over which the corresponding aggregates form in a biological context yields a clear distinction; for aggregates which have evolved to fulfil a structural role, the rate of self-replication is too low to be significant on the biologically relevant timescale. By contrast, all analysed proteins that aggregate in the context of disease are able to self-replicate quickly compared to the timescale of the associated disease. Our findings establish the ability to self-replicate as both a ubiquitous property of protein aggregates and one that has the potential to be a key process across aggregation-related disorders.

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↗

bioRxiv2021

Structure-based discovery of small molecule inhibitors of the autocatalytic proliferation of alpha-synuclein aggregates

The presence of amyloid fibrils of -synuclein is closely associated with Parkinsons disease and related synucleinopathies. It is still very challenging, however, to systematically discover small molecules that prevent the formation of these aberrant aggregates. Here, we describe a structure-based approach to identify small molecules that specifically inhibit the surface-catalyzed secondary nucleation step in the aggregation of -synuclein by binding to the surface of the amyloid fibrils. The resulting small molecules are screened using a combination of kinetic and thermodynamic assays for their ability to bind -synuclein fibrils and prevent the further generation of toxic oligomers. This study demonstrates that the combination of structure-based and kinetic-based drug discovery methods can lead to the identification of small molecules that selectively inhibit the autocatalytic proliferation of -synuclein aggregates.

biophysics↗

bioRxiv2021

The bacterial amyloids phenol soluble modulins from Staphylococcus aureus catalyze alpha-synuclein aggregation

Aggregated -synuclein (-syn) is the main constituent of Lewy bodies, the main pathological hallmark of Parkinsons disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric -syn. Braaks seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role that the microbiome might play in the pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called Phenol Soluble Modulins (PSMs). Here, we studied the kinetics of -syn aggregation under quiescent conditions in the presence or absence of four different PSM peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure -syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSM peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSM peptides displayed distinct effects on the kinetics of -syn aggregation. Kinetic analyses of the data suggest that while all four peptides catalyze -syn aggregation, the underlying mechanisms might differ with a model of nucleation and elongation fitting the -syn aggregation induced by PSM2 but not PSM1. The results of immunogold TEM imply that the aggregates are fibrillar and composed of -syn. Addition of the co-aggregated materials to HEK cells expressing the A53T -syn variant fused to GFP was found to catalyze -syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.

biochemistry↗