Biology subjects

Gremer, L.

Publications and source records attributed to Gremer, L..

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↗

bioRxiv2025

High-Level Expression, Purification and Biophysical Characterization of GPI-anchored native-like human Prion Protein using Leishmania tarentolae

The human prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-linked membrane-bound glycoprotein, containing two glycosylation sites. Human PrP is associated with a number of neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Pathogenesis involves a structural conversion of the cellular form (PrPC), rich in -helical and random coil structure, into the scrapie form (PrPSc) characterized by parallel in register intermolecular {beta}-sheet conformation. To get a better understanding of this structural conversion, it is crucial to first characterize the non-pathogenic cellular isoform including all posttranslational modifications, like GPI-anchoring and native-like human glycosylation pattern. So far, studies on PrPC or PrPSc as well as the transition from one state to the other rely on non-native constructs of PrP studied far away from physiological conditions. We, therefore, established the expression of GPI-linked human PrP with close to native glycosylation pattern (native-like human PrP) using the eukaryotic LEXSY expression system in Leishmania tarentolae. This expression system has the added advantage that it allows for large-scale production of the native-like human PrP, which results in [~]1 mg purified protein per liter culture. Sedimentation velocity analysis and far-UV circular dichroism spectroscopy confirm the high structural homogeneity and monomeric native-like conformation of the purified GPI-anchored human PrP.

biophysics↗

bioRxiv2025

Lecanemab binds to transgenic mouse model-derived amyloid-β fibril structures resembling Alzheimer 's disease type-I, type-II and Arctic folds

Lecanemab, an Alzheimers disease US Food and Drug Administration approved monoclonal antibody was previously reported to have a high affinity against intermediately sized amyloid-{beta} aggregates. Subsequently, it was observed by immunogold labelling that lecanemab can also bind to human type-I amyloid-{beta} fibrils. Therefore, to determine whether lecanemab binds to amyloid-{beta} fibril structures other than type-I, we performed immunogold labelling on extracted amyloid-{beta} fibril preparations from six different Alzheimers disease mouse models whose structures were previously solved by cryo-EM. Our results show that lecanemab exhibits high binding affinity to amyloid-{beta} fibril structures that have a flexible N-terminus in common, as it is the case for type-I, type-II and murine type-III amyloid-{beta} fibril polymorphs which resemble or are identical to human structures observed in sporadic and familial cases of Alzheimers disease, including a case with the Arctic (E22G) mutation. In contrast, only weak, if any, lecanemab binding was observed for amyloid-{beta} fibril folds with a fixed and ordered N-terminus. Key points- Lecanemab binds to A{beta} fibrils from several Alzheimers disease tg-mice whose structures resemble the type-I, type-II and Arctic folds found in Alzheimers patients, all of which share a flexible, unstructured N-terminus. - Lecanemab is therefore expected to be active against all common familial and sporadic Alzheimers cases containing these folds. - Lecanemab binding ability is unaffected by and tolerates the Arctic E22G mutation, at least in type-I or Arctic folds. - Weak, if any, lecanemab binding was observed to A{beta} fibrils derived from tg-SwDI mice, whose structures DI1, DI2 and DI3 all share structured and fixed N-termini. - Since the fixed N-termini of tg-SwDI DI1 fibrils and human meningeal A{beta}40 fibrils derived from CAA-affected brain are identical, most likely preventing lecanemab binding, treatment with lecanemab may be less or ineffective against CAA, but may explain the reported beneficial low ARIA-E frequency with this antibody. Table of Contents Graphic (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/633637v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b1e636org.highwire.dtl.DTLVardef@180efe4org.highwire.dtl.DTLVardef@58668org.highwire.dtl.DTLVardef@bb481e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗

bioRxiv2023

Cryo-EM Structures of Amyloid-β Fibrils from Alzheimer's Disease Mouse Models

The development of novel drugs for Alzheimers disease has proven difficult, with a high failure rate in clinical trials. Typically, transgenic mice displaying amyloid-{beta} peptide brain pathology are used to develop therapeutic options and to test their efficacy in preclinical studies. However, the properties of A{beta} in such mice have not been systematically compared to A{beta} from the patient brains. Here, we determined the structures of nine ex vivo A{beta} fibrils from six different mouse models by cryo-EM. We found novel A{beta} fibril structures in the APP/PS1, ARTE10, and tg-SwDI models, whereas the human familial type II fibril fold was found in the ARTE10, tg-APPSwe, and APP23 models. The tg-APPArcSwe mice showed an A{beta} fibril whose structure resembles the human sporadic type I fibril. These structural elucidations are key to the selection of adequate mouse models for the development of novel plaque-targeting therapeutics and PET imaging tracers. One Sentence SummaryCryo-EM structures of A{beta} fibrils extracted from brains of mouse models used for Alzheimers disease preclinical research are presented.

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

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↗

bioRxiv2018

An atomic-scale view at the composition ofamyloid-beta fibrils by atom probe tomography

Amyloid-beta (A{beta}) proteins play an important role in a number of neurodegenerative diseases. A{beta} is found in senile plaques in brains of Alzeimers disease patients. The 42 residues of the monomer form dimers which stack to fibrils gaining several micrometers in length. Using A{beta} fibrils with 13C and 15N marker substitution, we developed an innovative approach to obtain insights to structural and chemical information of the protein. We deposited the modified protein fibrils to pre-sharped aluminium needles with >100-nm apex diameters and, using the position-sensitive mass-to-charge spectrometry technique of atom probe tomography, we acquired the chemically-resolved three dimensional information for every detected ion evaporated in small fragments from the protein. We also discuss the influence of experimental parameters such as pulse energy and pulse frequency of the used Laser beam which lead to differences in the size of the gained fragments, developing the capability of localising metal atom within A{beta} plaques.

biochemistry↗