Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein
The aggregation of α-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-β-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current technique...
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2008
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00222836_v378_n5_p1064_Thirunavukkuarasu http://hdl.handle.net/20.500.12110/paper_00222836_v378_n5_p1064_Thirunavukkuarasu |
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paper:paper_00222836_v378_n5_p1064_Thirunavukkuarasu2025-07-30T17:30:47Z Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein Jares, Elizabeth Andrea fibrillization flourescence anisotropy neurodegenerative oligomers Parkinson's disease alpha synuclein pyrene anisotropy article fluorescence human in vivo study kinetics Parkinson disease point mutation priority journal protein aggregation protein analysis alpha-Synuclein Amino Acid Sequence Amyloid Anisotropy Fluorescent Dyes Humans Molecular Sequence Data Mutagenesis, Site-Directed Parkinson Disease Protein Folding Protein Structure, Quaternary Pyrenes Spectrometry, Fluorescence The aggregation of α-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-β-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of α-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of α-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 °C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (II/IIII), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0-160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinson's disease as well as in basic investigations. © 2008 Elsevier Ltd. All rights reserved. Fil:Jares-Erijman, E.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2008 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00222836_v378_n5_p1064_Thirunavukkuarasu http://hdl.handle.net/20.500.12110/paper_00222836_v378_n5_p1064_Thirunavukkuarasu |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
fibrillization flourescence anisotropy neurodegenerative oligomers Parkinson's disease alpha synuclein pyrene anisotropy article fluorescence human in vivo study kinetics Parkinson disease point mutation priority journal protein aggregation protein analysis alpha-Synuclein Amino Acid Sequence Amyloid Anisotropy Fluorescent Dyes Humans Molecular Sequence Data Mutagenesis, Site-Directed Parkinson Disease Protein Folding Protein Structure, Quaternary Pyrenes Spectrometry, Fluorescence |
| spellingShingle |
fibrillization flourescence anisotropy neurodegenerative oligomers Parkinson's disease alpha synuclein pyrene anisotropy article fluorescence human in vivo study kinetics Parkinson disease point mutation priority journal protein aggregation protein analysis alpha-Synuclein Amino Acid Sequence Amyloid Anisotropy Fluorescent Dyes Humans Molecular Sequence Data Mutagenesis, Site-Directed Parkinson Disease Protein Folding Protein Structure, Quaternary Pyrenes Spectrometry, Fluorescence Jares, Elizabeth Andrea Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein |
| topic_facet |
fibrillization flourescence anisotropy neurodegenerative oligomers Parkinson's disease alpha synuclein pyrene anisotropy article fluorescence human in vivo study kinetics Parkinson disease point mutation priority journal protein aggregation protein analysis alpha-Synuclein Amino Acid Sequence Amyloid Anisotropy Fluorescent Dyes Humans Molecular Sequence Data Mutagenesis, Site-Directed Parkinson Disease Protein Folding Protein Structure, Quaternary Pyrenes Spectrometry, Fluorescence |
| description |
The aggregation of α-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-β-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of α-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of α-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 °C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (II/IIII), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0-160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinson's disease as well as in basic investigations. © 2008 Elsevier Ltd. All rights reserved. |
| author |
Jares, Elizabeth Andrea |
| author_facet |
Jares, Elizabeth Andrea |
| author_sort |
Jares, Elizabeth Andrea |
| title |
Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein |
| title_short |
Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein |
| title_full |
Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein |
| title_fullStr |
Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein |
| title_full_unstemmed |
Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein |
| title_sort |
multiparametric fluorescence detection of early stages in the amyloid protein aggregation of pyrene-labeled α-synuclein |
| publishDate |
2008 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00222836_v378_n5_p1064_Thirunavukkuarasu http://hdl.handle.net/20.500.12110/paper_00222836_v378_n5_p1064_Thirunavukkuarasu |
| work_keys_str_mv |
AT jareselizabethandrea multiparametricfluorescencedetectionofearlystagesintheamyloidproteinaggregationofpyrenelabeledasynuclein |
| _version_ |
1840326801182687232 |