Real-time analysis of amyloid fibril formation of alpha-synuclein using a fibrillation-state-specific fluorescent probe of JC-1.
alpha-Synuclein is a pathological component of PD (Parkinson's disease) by participating in Lewy body formation. JC-1 (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide) has been shown to interact with alpha-synuclein at the acidic C-terminal region with a K(d) of 2.6 microM. JC-1 can discriminated between the fibrillation states of alpha-synuclein (monomeric, oligomeric intermediate and fibrillar forms) by emitting the enhanced binding fluorescence of different colours at 590, 560 and 538 nm respectively with the common excitation at 490 nm. The fibrillation-state-specific interaction of JC-1 allowed us to perform real-time analyses of the alpha-synuclein fibrillation in the presence of iron as a fibrillation inducer, rifampicin as a fibrillation inhibitor, baicalein as a defibrillation agent and dequalinium as a protofibril inducer. In addition, various alpha-synuclein fibrils with different morphologies prepared with specific ligands such as metal ions, glutathione, eosin and lipids were monitored with their characteristic JC-1-binding fluorescence spectra. FRET (fluorescence resonance energy transfer) between thioflavin-T and JC-1 was also employed to specifically identify the amyloid fibrils of alpha-synuclein. Taken together, we have introduced JC-1 as a powerful and versatile probe to explore the molecular mechanism of the fibrillation process of alpha-synuclein in vitro. It could be also useful in high-throughput drug screening. The specific alpha-synuclein interaction of JC-1 would therefore contribute to our complete understanding of the molecular aetiology of PD and eventual development of diagnostic/therapeutic strategies for various alpha-synucleinopathies.
Lee
Jung-Ho
JH
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Gwanak-Ku, Seoul 151-744, South Korea.
Lee
In-Hwan
IH
Choe
Young-Jun
YJ
Kang
Sungsoo
S
Kim
Hui Young
HY
Gai
Wei-Ping
WP
Hahn
Ji-Sook
JS
Paik
Seung R
SR
D000682:Amyloid / Q000032:analysis* / Q000378:metabolism; D001562:Benzimidazoles / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D052160:Benzothiazoles; D002232:Carbocyanines / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology; D013379:Substrate Specificity; D013844:Thiazoles / Q000378:metabolism / Q000493:pharmacokinetics; D051844:alpha-Synuclein / Q000032:analysis / Q000378:metabolism*
D000682:Amyloid; D001562:Benzimidazoles; D052160:Benzothiazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D013844:Thiazoles; D051844:alpha-Synuclein; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C009462:thioflavin T