alpha-synuclein and nitroblue-formazan

The mechanisms that govern the formation of alpha-synuclein (alpha-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of alpha-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of alpha-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter alpha-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-alpha-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of alpha-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues (125)YEMPS(129) in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent protein-ortho-quinone adducts showed that although covalent modification of alpha-syn occurs, this does not affect alpha-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for alpha-syn. These results suggest that inappropriate C-terminal cleavage of alpha-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated alpha-syn inclusion formation and dopaminergic neurodegeneration.

Topics: alpha-Synuclein; Amino Acid Sequence; Catechols; Cell Line, Tumor; Cell-Free System; Fluorescent Antibody Technique, Indirect; Formazans; Genetic Vectors; Humans; Lentivirus; Light; Mass Spectrometry; Neuroblastoma; Oxidation-Reduction; Plasmids; Recombinant Proteins; Scattering, Radiation; Spectrometry, Fluorescence; Transduction, Genetic; Transfection