Abstract
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.
Highlights
Lies with autosomal dominant inheritance of the disease
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 ␣-syn. These results suggest that inappropriate C-terminal cleavage of ␣-syn, which is known to occur in vivo in Parkinson’s disease (PD) brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated ␣-syn inclusion formation and dopaminergic neurodegeneration
To further explore the effect of covalent modification by The discovery of insoluble, ␣-syn fibrils in Lewy body inclucatechols on ␣-syn fibril formation, recombinant purified sions of PD brain has suggested that aberrant conformational
Summary
Generation of ␣-Syn-containing Plasmids—The bacterial expression vector prk172 encoding ␣-syn Y125F,E126A, M127A,P128F,S129A (␣-syn 125m) was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described previously [20]. For incubations of ␣-syn with catechols, L-DOPA, DA, and dihydroxyphenylacetic acid (DOPAC) (Sigma) were dissolved at a stock concentration of 10 mM in D-PBS and immediately diluted in the reaction mixture to a final concentration of 0.5 M (1:1.5 protein/catechol ratio). Several cell lines were generated and analyzed for A53T-125m expression levels by Western blot analysis (Fig. 1, a and b). To determine the Diagnostica, Inc., Greenwich, CT) aggregation was initiated by effect of increasing catechol levels on ␣-syn aggregation in the addition of 0.1 units/ml human thrombin (American Diag- A53T-125m cells, TH was expressed by transduction with a nostica) in Tris-buffered saline Commensurate to increasing as the mean Ϯ S.E. One-way analysis of variance followed by catechol levels, the number of cells containing ␣-syn aggregates. The statistically different, with p values of Ͻ0.05 considered same treatment in A53T-125m cells had no effect on ␣-syn significant
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