alpha-synuclein and pyrene

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Biofilms; Cell Survival; Fluorescent Dyes; HeLa Cells; Humans; Islet Amyloid Polypeptide; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Peptide Fragments; Pyrenes; Spectrophotometry, Ultraviolet; Staphylococcus aureus

The aggregation of alpha-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-beta-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of alpha-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 alpha-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 degrees C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (I(I)/I(III)), 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.

Topics: 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