Abstract
Amyloid fibrils, which are caused by abnormal conformation and the mis-assembly of proteins, are responsible for several conformational diseases, including prion diseases. To develop methods to prevent amyloid formation, blocking peptides with hydrophilic substitutions covering the stem forming regions of barnase 1-24 were prepared and examined for their ability to block amyloid-forming fragments—prion, Amyloid β, Pmel 17—. When these fragments were mixed with the synthetic blocking peptides, the result was a decline in the intensity of fluorescence, suggesting that amyloid formation was inhibited. Therefore, amyloidogenesis appears to be specifically inhibited by disrupting the hydrophobic interactions between core amyloid regions.
Author Contributions
Copyright© 2024
Saiki Masatoshi, et al.
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
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Competing interests The authors have declared that no competing interests exist.
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Introduction
The formation of amyloid fibrils is responsible for several disorders including Alzheimer’s disease, prion disease, and dialysis amyloidosis, which are collectively known as “conformational diseases” Insight into mechanisms that inhibit amyloidogenesis is crucial for the therapeutic treatment and clinical analysis of amyloid-related disease To develop methods for preventing amyloid formation, it is necessary to inhibit the aggregation of β-structures
Materials And Methods
Peptides were prepared by solid-phase peptide synthesis using Fmoc strategy as previously described Each lyophilized synthetic peptide (SS-1, SS-2, and NC) was solubilized at 0.2 mM in 50 mM Tris chloride buffer (pH 7.5) in a microtube. Lyophilized peptides of BM1-24, prion180-193, Amyloid β, and serum amyloid A protein 1-27 (SAA1-27) were prepared in the same solution, whereas Pmel17 405-420 was solubilized in sodium acetate buffer (pH 6.0). These solutions were admixed with SS-1, SS-2, or NC, incubated for 7 d under static conditions at 4°C, and then analyzed by ThT assay and CD spectroscopy. The thioflavin T (ThT) assay was used for the detection of fibril formation by measuring ThT fluorescence enhancement that occurs in the presence of fibrils. Synthetic peptides were prepared by adding 20 mL of incubated peptide solution to 2 mL of aqueous ThT. The final concentrations of the peptide and ThT were 2 mM and 5 mM, respectively. The formation of amyloid fibrils was monitored by fluorescence enhancement of fibril-bound ThT in 50 mM Tris buffer (pH 7.5). Fluorescence emission spectra were collected in the range 460-600 nm with an excitation wavelength of 450 nm as previously described A circular dichroism (CD) spectrum was recorded in the far-UV region (200-260 nm) at 20°C with a JASCO J-725 spectropolarimeter, a quartz cuvette, and a 1.0 mm path length. The spectral data were recorded in terms of mean residue ellipticity, (θ), in degrees square centimeter per decimole.
Results
The ability of BM1-24 to form fibrils in the presence or absence of the synthetic blocking peptides (SS-1 and SS-2) and the negative control peptide (NC) was evaluated by ThT assay, as shown in To obtain structural information related to fibril formation of BM1-24 in the presence and absence of SS-1, we carried out CD measurements under the same biochemical conditions as the ThT assay ( Next, we investigated whether the SS-1 peptide might be applicable to the inhibition of other proteins that are known to form amyloid fibrils. We prepared peptides of the following amyloid-forming proteins, prion180-193
Conclusion
Herein, we have presented a structural model to design peptides for blocking amyloi-dogenesis via the formation of hydrophobic interactions. The SS-1 peptide, comprising eight amino acids, was designed to have hydrophilic residues (Gln, Glu, Asn, and Asp) on only one side of the β-sheet. In addition to inhibiting amyloid formation by BM1-24, SS-1 blocked amyloidogenesis by peptides of prion protein, amyloid-β, SSA, and Pmel17. In summary, amyloidogenesis seems to have been specifically inhibited by the disruption of hydrophobic interactions between core amyloid regions. Our newly designed peptide may be useful for analyzing amyloid β aggregate formation and for studying diseases associated with the formation of amyloid fibrils (amyloidosis). Further optimizing the design of inhibitory peptides and verifying their effectiveness in in vivo experiments will likely lead to the development of more effective amyloid fibril inhibitors.