Formation of Covalent Linked Oligomers of alpha-Synuclein using Photoinduced Generation of Tyrosyl Radicals

C. D. BORSARELLI; V. OSTATNA; J. FAUEBACH; L. FALOMIR; E. PALEčEK; E. A. JARES-ERIJMAN; T.M. JOVIN

Salta

Encuentro; 3rd LATIN AMERICAN PROTEIN SOCIETY MEETING; 2010

SAB-CeBEM-LAPSM

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 believed that oligomeric (dimer, trimer, …n-mer) aS arise as intermediates in the aggregation of aS and may constitute the major neurotoxic species. However, attempts to isolate such noncovalent oligomers have not been very successful, presumably due to their transient nature, low concentration, and inherent instability.  In this work, we prepared different covalent oligomeric species of aS by action of protein tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine (Rubpy) in the presence of ammonium persulfate (APS). The reaction products were fractionated by ion-exchange chromatography.Numerous biochemical (SDS-PAGE), spectroscopic (UV/Vis absorption, stationary and dynamic fluorescence, dynamic light scattering), mass spectrometric (MS) and electrochemical (peak H) techniques were used to characterize the aS oligomers. The formation of intra- and inter-molecular di-tyrosine –C–C– bonds was confirmed. Finally, in vitro aggregation assays showed that the diverse oligomeric species induce a differential effect on the fibrillization of native aS, implying the existence of inherent complexity in the pathologic response to intracellular oxidative stress.