Alpha-synuclein-decorated nanoconjugates targeting alpha-synuclein assembly

Alpha-synuclein (alpha S) is a neuronal protein implicated in synaptic regulation, whose pathological aggregation is a hallmark of synucleinopathies, including Parkinson's disease. Despite its physiological importance, its intrinsically disordered nature and conformational plasticity complicate both mechanistic understanding and therapeutic targeting. Here, we investigate the use of protein-functionalized nanoparticles (NPs) as tools to modulate and probe alpha S assembly pathways, including aggregation and liquid-liquid phase separation. We designed three nanoconjugates by covalently attaching single-cysteine alpha S variants at positions 18, 76, and 140, spanning the protein's three functional regions, onto PEG-coated silica NPs. These conjugates preserved the disordered character of alpha S, exhibited low cytotoxicity, and were internalized into neuronal cells. Functional assays revealed site-dependent effects on aggregation: NP-alpha SA76C significantly delayed alpha S fibril formation and further inhibited aggregation of the amyloidogenic tau protein, suggesting interference with nucleation mechanisms, while NP alpha SA18C modestly accelerated aggregation and showed strong affinity for mature fibrils. All nanoconjugates partitioned into alpha S condensates, though without marked site-specific differences, and interacted with model lipid membranes. Overall, alpha S-functionalized NPs influence protein aggregation and condensate interactions, with context-dependent site specificity. These findings highlight the importance of conjugation position in tuning NP-protein interactions and underscore the potential of these nanoconjugates for investigating and modulating pathological protein assembly in neurodegenerative diseases.