amyloid-beta-peptides and 5-5--bis(8-(phenylamino)-1-naphthalenesulfonate)

Although abundant evidence suggests that amyloid accumulation plays a significant role in the pathogenesis of degenerative disease, the mechanism of amyloid formation and toxicity remains elusive. Early hypotheses for disease pathogenesis proposed that large amyloid deposits, which are composed primarily of 6-10-nm mature amyloid fibrils, were the primary causative agent in pathogenesis, but this hypothesis required modification to consider the central role of oligomers or aggregation intermediates, because the accumulation of these large aggregates does not correlate well with pathogenesis. Recent evidence supports the hypothesis that small soluble aggregates representing intermediates in the fibril assembly process may represent the primary culprits in a variety of amyloid-related degenerative diseases. Investigating the role of soluble amyloid oligomers in pathogenesis presents a problem for distinguishing these aggregates from the mature fibrils, soluble monomer, and natively folded precursor proteins, especially in vivo and in complex mixtures. Recently, we generated a conformation-specific antibody that recognizes soluble oligomers from many types of amyloid proteins, regardless of sequence. These results indicate that soluble oligomers have a common, generic structure that is distinct from both fibrils and low-molecular-weight soluble monomer/dimer. Conformation-dependent, oligomer-specific antibodies represent powerful tools for understanding the role of oligomers in pathogenesis. The purpose of this chapter is to review the methods for the production, characterization, and application of this antibody to understanding the contribution of amyloid oligomers to the disease process.

Topics: Amino Acid Sequence; Amyloid; Amyloid beta-Peptides; Anilino Naphthalenesulfonates; Animals; Antibodies; Benzothiazoles; Circular Dichroism; Epitopes; Humans; Molecular Mimicry; Molecular Sequence Data; Neurodegenerative Diseases; Peptide Fragments; Protein Conformation; Protein Structure, Quaternary; Rabbits; Thiazoles

The venerable fluorescent probe of protein hydrophobic regions, 4,4(')-dianilino-1,1(')-binaphthyl-5,5(')-disulfonate (bis-ANS), unexpectedly increases in fluorescence with soluble beta(1-40) in acidic buffer solutions but reacts weakly with amyloid fibrils while other hydrophobic probes react with the fibrils. CD analysis correlates reaction with the probe with random coil/mixed conformations and alpha-helical forms of beta(1-40) in buffer solutions but less so with soluble beta-sheet forms or amyloid fibrils. The kinetics of the fluoroalcohol-induced interconversion of conformers can be followed by changes in bis-ANS fluorescence. Formation of the beta-sheet form in aqueous buffer is limited by a slow component (minutes) while fluoroalcohol-promoted changes between beta-sheet and alpha-helix occur over seconds. Variants of beta(1-40) such as beta(1-42) or the Dutch E22Q mutation of beta(1-40) and fragments beta(1-28), beta(12-28), beta(10-20 amide), and beta(10-35 amide) react with bis-ANS under conditions that do not support fibril formation. Primary amino acid sequence is important as beta(1-11) does not cause bis-ANS fluorescence while beta(1-16) does, but hydrophobicity is not as beta(25-35) and beta(15-20 amide) are unreactive. bis-ANS is a useful biophysical tool for characterizing particular, but not all, soluble Abeta conformations distinct from the fibrillar form of amyloid peptides detected by Thioflavin T.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Anilino Naphthalenesulfonates; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Protein Structure, Secondary; Solubility; Spectrometry, Fluorescence