Stabilization of Aβ42 oligomers through PICUP and Tyr “scanning” mutagenesis

Alzheimer’s disease (AD) is a neurodegenerative disorder causing irreversible damage to the brain that culminates in memory loss, cognitive dysfunction, and eventual death. Amyloid beta-protein (Abeta) forms deposits of fibrillar assemblies, which were long believed to be the key etiologic factors of AD. However, recent studies suggest that pre-fibrillar protein oligomers are the primary toxic effectors causing the disease, rather than fibrils. Abeta exists as either a 40- or 42-residue peptide, where the latter isoform appears to be more neurotoxic and clinically relevant. To study the folding dynamics of Abeta42, whose oligomers are intrinsically unstable, a method for quantitative oligomer frequency distribution was utilized termed PICUP (Photo-induced Cross-linking of Unmodified Proteins). PICUP allowed us to chemically stabilize oligomers of Abeta40. However, we found that cross-linking involving the Tyr¹⁰ residue of Abeta42 does not yield completely stable oligomers. To address this problem, we developed a technique termed “scanning PICUP” to determine regions of Abeta42 responsible for oligomerization. Tyr was substituted initially at four different positions along the peptide to produce [F10, Y1]Abeta42, [F10, Y20]Abeta42, [F10, Y30]Abeta42, and [F10, Y42]Abeta42. Based on SDS-PAGE and silver staining analysis, the [F10, Y42]Abeta42 substitution produces oligomers with substantial stability, whereas the other substitutions produce cross-linked peptides that are significantly less stable. Experiments are underway to purify [F10, Y42]Abeta42 oligomers of specific order and to use these pure oligomer populations for detailed biophysical and cell physiology analyses. These analyses will advance our understanding of Abeta42 assembly, a prerequisite for knowledge-based design of therapeutic compounds for AD.