Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid beta Peptide Derivatives

Abstract

The self-assembly of two derivatives of KLVFF, a fragment Aβ(16–20) of the amyloid beta (Aβ) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Aβ (1–42) is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by the β-sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above the cac. Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of β-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF-CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and shows their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Aβ-induced toxicity.

Seeding and Growth of β-Amyloid Aggregates upon Interaction with Neuronal Cell Membranes

“…Our results show that the interaction of aggregates with the cellular membrane plays a role in the enhanced cellular stress leading to toxicity. We found that aggregates formed in the initial 0.5-1 h of incubation cause the most cellular stress (Figure 6), which correlates well with our cell viability and toxicity experiments ( Figure 7) as well as with previous toxicity studies [40,42,43] suggesting that aggregates formed at early stages of the aggregation process are the most powerful for inducing membrane permeability [8]. However, importantly, during the very early stages, the large majority of aggregates are nonfibrillar, but a dynamic population usually returns to monomers [54].…”

“…These values are in good agreement with previous toxicity studies of the Aβ peptide interacting with N2a cells, which reported viability values of almost 50% at a much higher peptide concentration (10 µM) than that used in our assay [42]. Regarding toxicity with other cell lines, it has also been shown Aβ42 aggregates at 10 µM that were previously incubated for 24 h at 37 • C, provoked a reduction in the cell viability of SH-SY5Y neuroblastoma cells by almost 20% [40], but the effect was even larger in primary neurons from E16 rat embryos in which Aβ42 aggregates at 20 µM caused a 65% reduction in cell viability [43]. In order to obtain additional information regarding the induction effect of different formed Aβ-647 aggregates on N2a neuronal cells, we took advantage of the fact that GG-DNBS is a dual sensor that is capable of not only simultaneously measuring levels of biothiols generated inside the cell but also detecting changes in the global levels of phosphate anions through the analysis of the fluorescence lifetime of the released fluorophore from the fluorogenic reaction, probe [41,44].…”

Pep‐Lipid Cubosomes and Vesicles Compartmentalized by Micelles from Self‐Assembly of Multiple Neuroprotective Building Blocks Including a Large Peptide Hormone PACAP‐DHA

“…Despite of the reported variety of nanoparticulate assemblies of liquid crystalline order and hierarchical complexity, the investigations in which the structural organization of the lipid nanocarriers has been suggested as beneficial for curing of neurodegenerative disorders are scarce . The modifications of GPCR ligands and other neuropeptides by lipophilic anchors is suggested as a possibility for enhancement of the neuropeptide drug bioavailability.…”

Hybrid Conjugates Formed between Gold Nanoparticles and an Amyloidogenic Diphenylalanine‐Cysteine Peptide

“…To evaluate the phenomena of self‐organization and study the aggregation mechanism of CFF in aqueous media, seven CFF solutions in a range of 0.3‐2.0 mmol⋅L −1 peptide concentrations were analyzed by steady‐state fluorescence spectroscopy. The critical aggregation concentration ( cac ) was assessed by the normalized intensity of the phenylalanine fluorescence emission at λ = 289 nm . In this way, the CFF cac (∼0.8 mmol⋅L −1 at 25 °C) was estimated from the crossover between linear fits corresponding to different emission regimes (Figure A).…”