Singlet oxygen-mediated oxidation of methionine residues induces the formation of “donut”-shaped aggregates of a-synuclein

C. D. BORSARELLI; V. OSTATNA; J. FAUERBACH; E. PALECEK; E.A. JARES-ERIJMAN; T. M. JOVIN

San Javier, Tucumán

Congreso; XLI REUNION ANUAL DE LA SOCIEDAD ARGENTINA DE BIOFISICA; 2012

Sociedad Argentina de Biofísica

Alpha-synuclein (aS), a 140 aminoacid presynaptic protein, is the mayor component of the fibrillar aggregates (Lewy bodies, LBs) observed in dopaminergic neurons of Parkinson’s disease patients.It is currently believed that non-covalent oligomeric forms of αS, arising as early 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. There is plenty evidence of the role of oxidative stress in the generation of neuropathies, due to the action of several reactive oxygen species (ROS), e.g. HO•, H2O2, O2•-, etc. In this work we focused the effect of singlet molecular oxygen (1O2), a neutral and non-radical ROS that can be generated intra- and intercellular by chemical, photochemical or enzymatic reaction, on the aggregation fate of aS. Specific and local generation of 1O2 was performed by UVB excitation of pyrene residues covalently linked to aS in several positions of Cys-modified protein, e.g. A18C, A90C and A140C. System characterization by fiber aggregation kinetic studies, fluorescence and mass spectroscopy, electrochemistry, and atomic force microscopy indicated that the direct effect of 1O2 on aS is the total oxidation of Met residues to Met sulfoxide, inducing changes in both the kinetics of fiber formation and the morphology of the non-covalent aggregates. AFM analysis revealed the formation of normal amyloid fibrils with non-oxidized aS. However, in the case of 1O2-oxidized aS, smaller and rounded (donut-shaped) aggregates were observed. The biological consequences of these new non-covalent aggregates of aS are under study. Acknowledgements: CDB thanks Alexander von Humboldt Foundation for a Georg Förster fellowship.