It is known that the humanin (HN) peptide binding to amyloid-β (Aβ) protects against its cytotoxic effects, while acetylcholinesterase (AChE) binding to Aβ increases its aggregation and cytotoxicity. HN is also known to bind the insulin-like growth factor binding protein-3 (IGFBP-3). Here, we examined the regulation of Aβ conformations by HN, AChE, and IGFBP-3 both in vitro and in the conditioned media from A549 and H1299 lung cancer cells. Our in vitro results showed the following: IGFBP-3 binds HN and blocks it from binding Aβ in the absence or presence of AChE; HN and AChE can simultaneously bind Aβ but not when in the presence of IGFBP-3; HN is unable to reduce the aggregation of Aβ in the presence of IGFBP-3; and HN abolishes the aggregation of Aβ induced by the addition of AChE in the absence of IGFBP-3. In the media, AChE and HN can simultaneously bind Aβ. While both AChE and HN are detected when using 6E10 Aβ antibodies, only AChE is detected when using the Aβ 17–24 antibody 4G8, the anti-oligomer A11, and the anti-amyloid fibril LOC antibodies. No signal was observed for IGFBP-3 with any of the anti-amyloid antibodies used. Exogenously added IGFBP-3 reduced the amount of HN found in a complex when using 6E10 antibodies and correlated with a concomitant increase in the amyloid oligomers. Immunodepletion of HN from the media of the A549 and H1299 cells increased the relative abundance of the oligomer vs the total amount of Aβ, the A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, and was also correlated with diminished cell viability and increased apoptosis.

Background: Amyloid fibrils in Alzheimer’s disease are composed of amyloid-β (Aβ) peptides of variant lengths. Humanin (HN), a 24 amino acid residue neuroprotective peptide, is known to interact with the predominant Aβ isoform in the brain, Aβ (1-40). Methods: Here, we constructed smaller segments of Aβ and HN and identified residues in HN important for both HN-HN and HN-Aβ interactions. Peptides corresponding to amino acid residues 5- 15 of HN, HN (5-15), HN (5-15, L11S), where Leu11 was replaced with Ser, and residues 17-28 of Aβ, Aβ (17-28), were synthesized and tested for their ability to block formation of the complex between HN and Aβ (1-40). Results: Co-immunoprecipitation and binding kinetics showed that HN (5-15) was more efficient at blocking the complex between HN and Aβ (1-40) than either HN (5-15, L11S) or Aβ (17-28). Binding kinetics of these smaller peptides with either full-length HN or Aβ (1-40) showed that HN (5- 15) was able to bind either Aβ (1-40) or HN more efficiently than HN (5-15, L11S) or Aβ (17-28). Compared to full-length HN, however, HN (5-15) bound Aβ (1-40) with a weaker affinity suggesting that while HN (5-15) binds Aβ, other residues in the full length HN peptide are necessary for maximum interactions. Conclusion: L11 was more important for interactions with Aβ (1-40) than with HN. Aβ (17-28) was relatively ineffective at binding to either Aβ (1-40) or HN. Moreover, HN, and the smaller HN (5-15), HN (5-15 L11S), and Aβ (17-28) peptides, had different effects on regulating Aβ (1-40) aggregation kinetics.