Biology subjects

Hoyer, W.

Publications and source records attributed to Hoyer, W..

9 recordsLinked to original sources

bioRxiv2026

Direct NMR evidence for transient binding of the vesicular SNARE protein synaptobrevin-2/VAMP2 to the α-synuclein C-terminus

The protein -synuclein (-syn) plays a central role in several neurodegenerative diseases, including Parkinson's disease; however, its physiological function remains enigmatic. We provide direct evidence by nuclear magnetic resonance (NMR) spectroscopy, microscale thermophoresis (MST), and biolayer interferometry (BLI) measurements that the -synuclein C-terminus binds to the vesicular SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein synaptobrevin-2 (syb-2), also known as VAMP2 (vesicle-associated membrane protein). The observed dissociation constant (Kd) in the 10 - 50 micromolar range falls within the estimated concentration range of -synuclein in neurons, while local concentrations may be substantially higher. NMR chemical shift perturbation (CSP) and paramagnetic relaxation enhancement (PRE) experiments mapped the binding site with residue-specific resolution to the -synuclein C-terminus (V118 - E137) and to the synaptobrevin-2 SNARE motif/linker domain interface (region A82 - K87), and also affected the synaptobrevin-2 N-terminus (M1 - S2). NMR CSPs and PREs indicate a transient interaction in the fast exchange (s-ms) regime. These transient weak binding interactions are characteristic of intrinsically disordered proteins (IDPs) and allow both proteins to engage in multiple binding interactions, thereby enabling -synuclein to play a regulatory role upstream of or during neuronal exocytosis. While the exact physiological role of -synuclein remains debated, the involvement of its pathological form in several neurodegenerative diseases (dubbed synucleinopathies) is well established. Our data suggest a direct link between proteins involved in neurodegeneration and neuronal exocytosis.

biophysics↗

bioRxiv2024

Phase Separation and Aggregation of α-Synuclein Diverge at Different Salt Conditions

The coacervation and structural rearrangement of the protein alpha-synuclein (Syn) into cytotoxic oligomers and amyloid fibrils are considered pathological hallmarks of Parkinsons disease. While aggregation is recognized as the key element of amyloid diseases, liquid-liquid phase separation (LLPS) and its interplay with aggregation have gained increasing interest. Previous work showed that factors promoting or inhibiting amyloid formation have similar effects on phase separation. Here, we provide a detailed scanning of a wide range of parameters including protein, salt and crowding concentrations at multiple pH values, revealing different salt dependencies of aggregation and phase separation. The influence of salt on aggregation under crowded conditions follows a non-monotonic pattern, showing increased effects at medium salt concentrations. This behavior can be elucidated through a combination of electrostatic screening and salting-out effects on the intramolecular interactions between the N-terminal and C-terminal regions of Syn. By contrast, we find a monotonic salt dependence of phase separation due to the intermolecular interaction. Furthermore, we observe the time evolution of the two distinct assembly states, with macroscopic fibrillar-like bundles initially forming at medium salt concentration but subsequently converting into droplets after prolonged incubation. The droplet state is therefore capable of inhibiting aggregation or even dissolving the aggregates through a variety of heterotypic interactions, thus preventing Syn from its dynamically arrested state.

biophysics↗

bioRxiv2023

Direct disassembly of alpha-syn preformed fibrils into native alpha-syn monomers by an all-D-peptide

Parkinsons disease (PD) is the most common neurodegenerative movement disorder worldwide. One of its central features is the neurodegeneration that starts in the substantia nigra and progressively tends to involve other brain regions. -Synuclein (-syn) and its aggregation during pathogenesis have been drawn into the center of attention, where especially soluble oligomeric and fibrillar structures are thought to play a key role in cell-to-cell transmission and induction of toxic effects. Here, we report the development of all-D-enantiomeric peptide ligands that bind monomeric -syn with high affinity, thereby stabilizing the physiological intrinsically disordered structure and preventing initiation of aggregation, and more important, disassembling already existing aggregates. This "anti prionic" mode of action (MoA) has the advantage over other MoAs that it eliminates the particles responsible for disease propagation directly and independently of the immune system, thereby restoring the physiological monomer. Based on mirror image phage display on the D-enantiomeric full-length -syn target, we identified SVD-1 and SVD-1a by next generation sequencing, Thioflavin-T screens and rational design. The compounds were analyzed with regard to their anti-aggregation potential and both compounds showed aggregation delaying as well as seed capacity reducing effects in de novo and seeded environments, respectively. High affinity towards the monomeric -syn, in the low nano- to picomolar KD range was identified by surface plasmon resonance (SPR). SVD-1a reduced toxic effects as well as intracellular seeding capacity of -syn pre-fromed fibrils (PFF) in cell culture. SVD-1a disassembled -syn PFF into monomers as identified by atomic force microscopy (AFM), time dependent dynamic light scattering (DLS) and size exclusion chromatography (SEC) analysis. The present work provides promising results on the development of lead compounds with this anti-prionic mode of action for treatment of Parkinsons disease and other synucleinopathies.

neuroscience↗

bioRxiv2021

Structural Basis for the Inhibition of IAPP Fibril Formation by the Hsp60 Co-Chaperonin Prefoldin

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin (PFD) inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal PFD interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined NMR spectroscopy with EM to establish that the inhibition of fibril formation is mediated by the binding of prefoldins coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. AFM demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens new perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.

biophysics↗

bioRxiv2020

Endo-lysosomal Aβ concentration and pH enable formation of Aβ oligomers that potently induce Tau missorting

Amyloid-β peptide (Aβ) forms metastable oligomers >50 kD, termed AβOs or protofibrils, that are more effective than Aβ amyloid fibrils at triggering Alzheimer’s disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aβ accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AβO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AβO formation and the high endo-lysosomal Aβ concentration together enable extensive AβO formation of Aβ42 under physiological conditions. Exploiting the enhanced AβO formation of the dimeric Aβ variant dimAβ we furthermore demonstrate targeting of AβOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AβOs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Structural details of amyloid beta oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy

Human PrP (huPrP) is a high-affinity receptor for oligomeric A{beta}. Synthetic oligomeric A{beta} species are known to be heterogeneous, dynamic and transient, rendering their structural investigation particularly challenging. Here, we used huPrP to preserve A{beta} oligomers by co-precipitating them into large hetero-assemblies to investigate the conformation of A{beta}(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the last two -helices. For A{beta}(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal a substantial {beta}-strand content. Importantly, not all A{beta}(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic A{beta} fibrils suggests that the A{beta} oligomer preparation represents a heterogeneous mixture of {beta}-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of A{beta} to adopt variable {beta}-structure conformers.

molecular biology↗

bioRxiv2020

Amyloid fibril structure of islet amyloid polypeptide by cryo-electron microscopy reveals similarities with amyloid beta

A critical role of the hormone islet amyloid polypeptide (IAPP) is vividly discussed for Type 2 Diabetes (T2D), where amyloid deposits in pancreatic islets consisting of fibrillar IAPP have been associated with beta cell loss. Here, we applied cryo-electron microscopy to elucidate the structure of IAPP fibrils prepared at physiological pH and reconstructed densities of three dominant polymorphs. An atomic model of the main polymorph comprising residues 13 - 37 in a density map of 4.2 [A] resolution reveals two S-shaped, intertwined protofilaments. The segment 21-NNFGAIL-27, which is essential for IAPP amyloidogenicity, forms the protofilament interface together with tyrosine 37 and the amidated C-terminus. The main IAPP fibril polymorph resembles polymorphs of the Alzheimer disease (AD)-associated amyloid-{beta} peptide (A{beta}), which is striking in light of the epidemiological link between T2D and AD and reports on IAPP-A{beta} cross-seeding in vivo. The results structurally link the early-onset T2D IAPP genetic polymorphism S20G with the early-onset AD Arctic mutation E22G of A{beta}, rationalize previous data on IAPP fibrils, help to elucidate mechanisms of amyloid formation and toxicity, and support the design of fibril growth inhibitors as well as imaging probes for early detection of IAPP fibrils.

biochemistry↗

bioRxiv2019

An engineered monomer binding-protein for α-synucleinefficiently inhibits the proliferation of amyloid fibrils

Removing or preventing the formation of -synuclein aggregates is a plausible strategy against Parkinsons disease. To this end we have engineered the {beta}-wrapin AS69 to bind monomeric -synuclein with high affinity. In cultured cells, AS69 reduced the occurrence of -synuclein oligomers and of visible -synuclein aggregates. In flies, AS69 reduced -synuclein aggregates and the locomotor deficit resulting from -synuclein expression in neuronal cells. In a mouse model based on the intracerebral injection of pre-formed -synuclein seed fibrills (PFFs), AS69 co-injection reduced the density of dystrophic neurites observed three months later. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited auto-catalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69--synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition. These results represent a new paradigm that high affinity monomer binders can be strongly sub-stoichiometric inhibitors of nucleation processes.

bioengineering↗

bioRxiv2017

A structural and kinetic link between membrane association and amyloid fibril formation of α-Synuclein

The protein -Synuclein (S) is linked to Parkinsons disease through its abnormal aggregation, which is thought to involve an interplay between cytosolic and membrane-bound forms of S. Therefore, better insights into the molecular determinants of membrane association and their implications for protein aggregation may help deciphering the pathogenesis of Parkinsons disease. Following previous studies using micelles and vesicles, we present a comprehensive study of S interaction with phospholipid bilayer nanodiscs. Using a combination of NMR - spectroscopic and complementary biophysical as well as computational methods we structurally and kinetically characterize S interaction with defined stable planar membranes in a quantitative and site-resolved way. We probe the role of S acetylation as well as membrane charge, plasticity and available surface area in modulating S membrane binding modes and directly link these findings to their consequences for S amyloid fibril formation.

biophysics↗