Alpha-synuclein-decorated nanoconjugates targeting alpha-synuclein assembly.

Abstract

Alpha-synuclein (αS) 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 αS assembly pathways, including aggregation and liquid-liquid phase separation. We designed three nanoconjugates by covalently attaching single-cysteine αS 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 αS, exhibited low cytotoxicity, and were internalized into neuronal cells. Functional assays revealed site-dependent effects on aggregation: NP-αSA76C significantly delayed αS fibril formation and further inhibited aggregation of the amyloidogenic tau protein, suggesting interference with nucleation mechanisms, while NP-αSA18C modestly accelerated aggregation and showed strong affinity for mature fibrils. All nanoconjugates partitioned into αS condensates, though without marked site-specific differences, and interacted with model lipid membranes. Overall, αS-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.