Biophysical properties and cellular toxicity of covalent cross-linked oligomers of a-synuclein formed by photoinduced side-chain tyrosyl radicals

C. D. BORSARELLI

Córdoba

Encuentro; XI ENCUENTRO LATINOAMERICANO DE FOTOQUIMICA Y FOTOBIOLOGIA.; 2012

UNRC

Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson’s disease. It is currently believed that non-covalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by cross-linking via tyrosyl radicals generated by blue-light photosensitization of the cationic metal coordination complex ruthenium (II) tris-bipyridine, Ru(bpy)32+, in the presence of persulfate anion, S2O82-. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state and dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by –C–C– bonds between Tyr residues to form dityrosine (diTyr) as covalent bridges, with a predominant involvement of Y39 residue. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson´s disease.