Dequalinium-induced Protofibril Formation of α-Synuclein

Abstract

␣-Synuclein is the major constituent of Lewy bodies, a pathological signature of Parkinson disease, found in the degenerating dopaminergic neurons of the substantia nigra pars compacta. Amyloidosis generating the insoluble fibrillar protein deposition has been considered to be responsible for the cell death observed in the neurodegenerative disorder. In order to develop a controlling strategy toward the amyloid formation, 1,1-(1,10-decanediyl)-bis-[4-amino-2-methylquinolinium] (dequalinium), was selected and examined in terms of its specific molecular interaction with ␣-synuclein. The protein was self-oligomerized by dequalinium, which gave rise to the ladder formation on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-PAGE in the presence of a coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. The double-headed structure of dequalinium with the two cationic 4-aminoquinaldinium rings was demonstrated to be critical for the protein self-oligomerization. The dequaliniumbinding site was located on the acidic C-terminal region of the protein with an approximate dissociation constant of 5.5 M. The protein self-oligomerization induced by the compound has resulted in the protofibril formation of ␣-synuclein before it has developed into amyloids. The protofibrils were demonstrated to affect the membrane intactness of liposomes, and they have also been shown to influence cell viability of human neuroblastoma cells. In addition, dequalinium treatment of the ␣-synuclein-overexpressing cells exerted a significant cell death. Therefore, it is pertinent to consider that dequalinium could be used as a molecular probe to assess toxic mechanisms related to the amyloid formation of ␣-synuclein. Ultimately, the compound could be employed to develop therapeutic and preventive strategies toward ␣-synucleinopathies including Parkinson disease.

Resveratrol Selectively Remodels Soluble Oligomers and Fibrils of Amyloid Aβ into Off-pathway Conformers

“…That resveratrol promotes high molecular weight aggregation in- stead of disassembling soluble and insoluble A␤ conformers into low molecular weight species is contrary to conventional wisdom. However, this observation is supported by a few disparate reports for resveratrol and other polyphenols (57,58,60,63,91,92), as well as the broader literature demonstrating that many small molecules and peptides redirect the aggregation cascade instead of inhibiting it completely (40,56,62,(93)(94)(95)(96). For example, the aggregation of monomeric A␤ and ␣-synuclein was studied in the presence of EGCG.…”

Dequalinium-Induced Cell Death of Yeast Expressing α-Synuclein-GFP Fusion Protein

“…Although lactacystin itself exerted cytotoxicity by inducing abnormal protein accumulation due to the suppressed proteasome activity [23,24], the lactacystin-induced cell death of SH-SY5Y cells was further augmented as a-synuclein was overexpressed [14]. DQ was also demonstrated to affect the cells overexpressing a-synuclein [19]. DQ facilitated the protein self-interaction of a-synuclein and led to the protofibril formation which has been suggested to be a toxic species for the cytotoxicity related to amyloidosis [9,10].…”

“…They were previously shown to cause the cell death of mammalian dopaminergic SH-SY5Y cells in a-synuclein-dependent manner [14,19]. Although lactacystin itself exerted cytotoxicity by inducing abnormal protein accumulation due to the suppressed proteasome activity [23,24], the lactacystin-induced cell death of SH-SY5Y cells was further augmented as a-synuclein was overexpressed [14].…”

“…DQ actually facilitated the protofibril formation of a-synuclein prior to the final amyloid fibril formation [19]. By binding to the acidic C-terminus of a-synuclein, the compound was shown to remove the membrane-bound a-synuclein and lead to the oligomeric protofibrillar state [19]. Although the DQ-induced protofibrils were suggested to exert their cytotoxicity by influencing membrane stability, actual intracellular effects of the protofibrils have not been examined.…”

“…The compound has been also demonstrated to interact with several enzymes such as mitochondrial F 1 -ATPase, protein kinase C, calmodulin-dependent phosphodiesterase, and calcium-activated K + channel [16][17][18]. DQ actually facilitated the protofibril formation of a-synuclein prior to the final amyloid fibril formation [19]. By binding to the acidic C-terminus of a-synuclein, the compound was shown to remove the membrane-bound a-synuclein and lead to the oligomeric protofibrillar state [19].…”