Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

• Autores: Borsarelli, Claudio Darío; Falomir Lockhart, Lisandro Jorge; Ostatná, Veronika; Fauerbach, Jonathan Arturo; Hsiao, He Hsuan; Urlaub, Henning; Palecek, Emil; Jares Erijman, Elizabeth A.; Jovin, Thomas M.
• Año de publicación: 2012
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: NEURODEGENERATION; OXIDATIVE STRESS; PARKINSON'S DISEASE; PHOTOCROSSLINKING; PROTEIN AGGREGATION; PROTEIN PHOTOOXIDATION; SH-SY5Y
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

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 noncovalent 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 crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. 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, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr?Tyr (dityrosine) dimers through ?C?C? bonds acting as covalent bridges, with a predominant involvement of residue Y39. 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.
Fil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina
Fil: Falomir Lockhart, Lisandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Ostatná, Veronika. Max Planck Institut Für Biophysikalische Chemie; Alemania
Fil: Fauerbach, Jonathan Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina
Fil: Hsiao, He Hsuan. Institute Of Biophysics, Academy Of Sciences; República Checa
Fil: Urlaub, Henning. Institute Of Biophysics, Academy Of Sciences; República Checa
Fil: Palecek, Emil. No especifíca;
Fil: Jares Erijman, Elizabeth A.. Max Planck Institut Für Biophysikalische Chemie; Alemania
Fil: Jovin, Thomas M.. Max Planck Institut Für Biophysikalische Chemie; Alemania