Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid <i>β</i>-protein fibrillation and cytotoxicity

doi:10.1007/s11705-017-1687-2

Front. Chem. Sci. Eng.

2018, Vol. 12

Issue (2) : 283-295 https://doi.org/10.1007/s11705-017-1687-2

RESEARCH ARTICLE

Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity

Shuai Ma¹, Huan Zhang¹, Xiaoyan Dong¹(

), Linling Yu¹, Jie Zheng², Yan Sun¹

¹. Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China
². Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325, USA

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Abstract

Amyloid-β (Aβ) protein aggregation is the main hallmark of Alzheimer’s disease (AD). Inhibition of Aβ fibrillation is thus a promising therapeutic approach to the prevention and treatment of AD. Recently, we designed a heptapeptide inhibitor, LVFFARK (LK7). LK7 shows a promising inhibitory capability on Aβ fibrillation, but is prone to self-assembling and displays high cytotoxicity, which would hinder its practical application. Herein, we modified LK7 by a head-to-tail cyclization and obtained a cyclic LK7 (cLK7). cLK7 exhibits a different self-assembly behavior from LK7, and has higher stability against proteolysis than LK7 and little cytotoxicity to SH-SY5Y cells. Thermodynamic analysis revealed that both LK7 and cLK7 could bind to Aβ₄₀ by electrostatic interactions, hydrogen bonding and hydrophobic interactions, but the binding affinity of cLK7 for Aβ₄₀ (K_D = 4.96 µmol/L) is six times higher than that of LK7 (K_D = 32.2 µmol/L). The strong binding enables cLK7 to stabilize the secondary structure of Aβ₄₀ and potently inhibit its nucleation, fibrillation and cytotoxicity at extensive concentration range, whereas LK7 could only moderately inhibit Aβ₄₀ fibrillation and cytotoxicity at low concentrations. The findings indicate that the peptide cyclization is a promising approach to enhance the performance of peptide-based amyloid inhibitors.

Keywords Alzheimer’s disease amyloid β-protein cyclic peptide inhibition protein aggregation

Corresponding Author(s): Xiaoyan Dong

Just Accepted Date: 25 September 2017 Online First Date: 11 January 2018 Issue Date: 09 May 2018

Cite this article:

Shuai Ma,Huan Zhang,Xiaoyan Dong, et al. Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity[J]. Front. Chem. Sci. Eng., 2018, 12(2): 283-295.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-017-1687-2
https://academic.hep.com.cn/fcse/EN/Y2018/V12/I2/283

Fig.1 Scheme 1 Chemical structures of (a) LK7 and (b) cLK7

Fig.2 Characterization of LK7 and cLK7. TEM images of (a) LK7 and (b) cLK7 (200 µmol/L for both) after incubation at 37 °C for 72 h. The scale bar of 200 nm is for the two images. (c) The time course of intact peptides by incubating with α-chymotrypsin. Error bars represent the standard deviation of the measurement (n = 3). (d) Cytotoxicity induced by LK7 and cLK7 aggregates detected using SH-SY5Y cells. In the MTT assay, the cell viability treated with PBS buffer alone was set to 100%

Fig.3 Aggregation kinetics of Aβ₄₀ incubated without and with different concentrations of (a) LK7, and (b) cLK7. Aβ₄₀ concentration was 25 µmol/L

Tab.1 Lag time (T_lag) and transition time (T_trans) for aggregation kinetics of 25 µmol/L Aβ₄₀ in the presence of different concentrations of LK7 or cLK7

Fig.4 TEM images of Aβ₄₀ (25 µmol/L) in the absence and presence of LK7 and cLK7. (a) Aβ₄₀ alone; (b to e) Aβ₄₀ with LK7 at concentrations of (b) 25 µmol/L, (c) 50 µmol/L, (d) 100 µmol/L and (e) 200 µmol/L; (f to i) Aβ₄₀ with cLK7 at concentrations of (f) 25 µmol/L, (g) 50 µmol/L, (h) 100 µmol/L and (i) 200 µmol/L. The scale bar of 200 nm is for all the images. All samples were taken after incubation at 37 °C for 72 h

Fig.5 Far-UV circular dichroism spectra of Aβ₄₀ (25 µmol/L) incubated in the absence and presence of different concentrations of (a) LK7 and (b) cLK7. The co-incubation of Aβ₄₀ was conducted at 37 °C

Fig.6 Inhibitory effects of LK7 and cLK7 on Aβ₄₀-induced cytotoxicity for SH-SY5Y cell lines: ***p <0.001, compared to control groups; #p<0.05, ##p<0.01, ###p<0.001, compared to the Aβ₄₀ treated group (Aβ₄₀ only)

Tab.2 Thermodynamic parameters for the interactions between the inhibitors and Aβ₄₀ at 37 °C

Fig.7 Schematic representations of Aβ₄₀ aggregation and cytotoxicity: (a) on-pathway aggregation of Aβ₄₀ monomers, (b) Aβ₄₀ aggregation in the presence of LK7, and (c) Aβ₄₀ aggregation in the presence of cLK7

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