Amyloid-like aggregation or fibrillization of alpha-synuclein (alpha-Syn) and the filamentous deposits in Lewy bodies are believed to be closely associated with several fatal neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Here, we report the importance of a nine-residue peptide motif, (66)VGGAVVTGV(74), in the fibrillization and cytotoxicity of human alpha-Syn. Mutagenesis combined with thioflavin T fluorescence detection, atomic force microscopic imaging, and cytotoxicity assays reveal that deletion of this sequence completely eliminates alpha-Syn fibrillization and cell toxicity. However, deletion of the (71)VTGV(74) sequence decreases the fibrillization rate while the cytotoxicity remains unchanged. Incorporation of charged residues within this region slows aggregation and even impedes filament formation. In addition, substitution of Gly68 with Ala or C-terminal truncations of alpha-Syn accelerate the fibrillization processes. Circular dichroism studies suggest that beta-sheet formation is often concomitant with filament formation. Thus, this segment, namely, the GAV motif, is responsible for aggregation or fibrillization of alpha-Syn and perhaps other amyloidogenic proteins. The oligomers formed during fibrillogenesis might be associated with the cytotoxicities of various alpha-Syn species. This finding may provide further insight into the understanding of the molecular mechanism underlying the fibrillogenesis implicated in neurodegeneration as well as aid in drug design and development of transgenic models.

Parkinson's disease (PD) is a common movement disorder characterized by degeneration of dopaminergic neurons and deposition of fibrillar Lewy bodies comprising primarily a-synuclein (a-Syn) in the substantia nigra [1][2][3][4]. A growing body of evidence strongly supports the theory that formation of a-Syn fibrils and dopamine (DA) metabolism are closely associated with the pathogenesis of this fatal disease [5][6][7]. These findings imply an intrinsic link between the presynaptic a-Syn protein and the DA molecule [8], a synaptic neurotransmitter that functions in signal transmission. Recent research has focused on oxidative stress in brain, DA metabolism and dysfunction of a-Syn in synapses, in an attempt to elucidate an overview linking these important biological processes. Conway et al. [9] reported that DA stabilized the a-Syn protofibrils by forming a DA-a-Syn adduct. They proposed that DA could react with the phenol Fibrillization of a-synuclein (a-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit a-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of a-Syn chain, generating a-Syn-quinone adducts. It is likely that the amino groups of a-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked a-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact a-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of a-Syn with DA is relevant to the selective dopaminergic loss in PD.Abbreviations Ab, amyloid b-protein; AFM, atomic force microscopy; CA, catechol; DA, dopamine; DAQ, dopamine-quinone; EGCG, (-)-epigallocatechin gallate; GAV, a peptide motif with a homologous sequence of VGGAVVAGV; HQ, hydroquinone; HSQC, heteronuclear single quantum coherence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, nonamyloid component; NBT, nitroblue tetrazolium chloride; pNP, p-nitrophenol; PrP, prion protein; pXG, p-xylylene glycol; Q, p-benzoquinone; a-Syn, human a-synuclein; ThT, thioflavin T.