Lakomek, N.-A. | Search bioRxiv

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↗

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↗