Probing the Influence of Single-Site Mutations in the Central Cross-β Region of Amyloid β (1–40) Peptides

Abstract

Amyloid β (Aβ) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer’s disease. A complete mechanistic understanding how Aβ peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aβ40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aβ assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aβ40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aβ fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aβ fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aβ40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aβ40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aβ fibrillation pathway.

How Single Site Mutations Can Help Understanding Structure Formation of Amyloid β1−40

“…To this end, several in vitro assays have been established: i) the classical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene sulfonate) (WST-1) conversion assays representing the proportion of metabolically active cells, ii) the LDH release assay is indicative for plasma membrane disruption (dead cells), iii) caspase-3 staining which is used to investigate one of the possible pathways toward cell apoptosis, and iv) neurite length measurements to assess the effect of A𝛽 peptides on the outgrowth of neurites and thus information on the wellbeing under the applied conditions can be obtained. [84,[91][92][93]…”

“…To this end, the positions F20 or G33 were mutated using a small library of four new mutants, namely, F20K , F20Y , F20Cha , and G33A . [ 91 ] The mutants F20K and F20Y showed a slightly shorter lag time, while F20Cha prolongs it. This could be attributed to the lack of a flat aromatic ring in the side chain at position 20, which might be important for the initiation of the fibril growth phase.…”

“…Aliphatic ring [91] G33A Increased sterical demand [91] pS26-A𝜷 1−40 Increased sterical demand and negative charge [69] S26BMAA Increased sterical demand while maintaining hydrogen bonding [92]…”

Does Inflammation Play a Major Role in the Pathogenesis of Alzheimer's Disease?

“…(Kageyama et al, 2021). Aβ42 oligomers cause synaptotoxicity and memory loss (Fritzsch et al, 2021). It is therefore warranted to develop agents that can effectively inhibit the formation of Aβ oligomers or block their toxicity.…”

Amyloidogenesis: What Do We Know So Far?

“…Almost a year later Tycko et al showed that the hepta residue Aβ16-22: N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2, formed full length and highly ordered amyloid fibrils in vitro [155]. Substitutions for positions that stabilize the α-helical conformation (for instance, substituting Val18 by Ala) have been shown to reduce the amyloidogenic propensity of the peptides [156]. It is indeed intriguing that even single point substitutions can act as a veto against fibrillation.…”