蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞的毒性

doi:10.11843/j.issn.0366-6964.2022.08.029

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (8): 2721-2728.doi: 10.11843/j.issn.0366-6964.2022.08.029

蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞的毒性

白瑜*, 王武, 王碧, 李颖, 冯自立

陕西理工大学生物科学与工程学院, 陕西省资源生物重点实验室, 汉中 723000

收稿日期:2021-11-15 出版日期:2022-08-23 发布日期:2022-08-23
通讯作者: 白瑜,E-mail:baiu@snut.edu.cn
作者简介:白瑜(1980-),女,山西忻州人,副教授,博士,主要从事兽医病理学研究
基金资助:
国家自然科学基金项目（31702206）；陕西省科技厅项目（2022JM-115）

The Cytotoxicity of Amyloid Fibrils Formed by Hen Egg-White Lysozyme on SH-SY5Y Cells

BAI Yu*, WANG Wu, WANG Bi, LI Ying, FENG Zili

Shaanxi Province Key Laboratory of Bio-resources, College of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong 723000, China

Received:2021-11-15 Online:2022-08-23 Published:2022-08-23

摘要/Abstract

摘要： 本研究旨在探讨一定理化条件下形成的蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞（SH-SY5Y）的毒性作用。采用高温（57±0.1）℃、pH 2.0甘氨酸溶液、振荡条件下孵育100 μmol·L^-1的蛋清溶菌酶蛋白，使其聚集成为蛋清溶菌酶淀粉样纤维；透射电子显微镜观察蛋清溶菌酶淀粉样纤维的超微结构，ANS荧光法测定纤维的疏水性，圆二色谱法测定并计算纤维的二级结构含量；将此纤维作用于培养的SH-SY5Y神经细胞，MTT法检测细胞活力，核染色法检测细胞凋亡状况。透射电镜结果显示，蛋清溶菌酶在孵育4 d后形成了呈短杆状超微结构的淀粉样纤维；与天然蛋清溶菌酶蛋白相比，纤维的疏水性和二级结构β折叠含量均显著提高；且1~3 μmol·L^-1纤维作用于SH-SY5Y细胞12、24和48 h均产生了显著的毒性作用（P<0.01），1、2和3 μmol·L^-1纤维作用细胞12 h时的细胞活力分别为75.16%±15.51%、67.54%±12.13%和67.89%±10.26%，作用24 h的细胞活力分别为75.78±13.01%、58.41%±5.55%和61.90%±8.94%，作用48 h的细胞活力分别为71.59%±14.75%、55.65%±5.78%和46.45%±6.23%，细胞活力降低呈现明显的浓度依赖性和时间依赖性，且细胞核染色呈现典型的核凋亡变化。结果提示，蛋清溶菌酶在一定理化条件下可形成具有神经细胞毒性的淀粉样纤维，为解释朊蛋白或其他蛋白质形成淀粉样纤维的机制提供参考。

关键词: 淀粉样纤维, 蛋清溶菌酶, β折叠, 人神经母细胞瘤细胞

Abstract: The aim of this study was to investigate the cytotoxicity effect of amyloid fibrils of hen egg-white lysozyme (HEWL) on SH-SY5Y cells. The 100 μmol·L^-1 HEWL protein was incubated with agitation at 57℃ in glycine solution of pH 2.0. The ultrastructure of HEWL amyloid fibrils was observed under transmission electron microscopy, the hydrophobicity was detected by ANS fluorescence, the secondary structural contents were measured and analyzed by circular dichroism. After SH-SY5Y cells were treated by the formed HEWL fibrils, the cell viability was determined by MTT assay and the apoptosis features were detected by nuclear staining. The transmission electron microscopy results showed that HEWL protein formed short and rod-shaped amyloid fibrils after 4 days of incubation. Meanwhile, compared with the native HEWL protein, the surface hydrophobicity and β-sheet structure of aggregates were both greatly increased. Furthermore, 1-3 μmol·L^-1 HEWL fibrils all produced significant cytotoxicity in SH-SY5Y cells after 12, 24 and 48 h treatments(P<0.01). For different concentrations of 1, 2 and 3 μmol·L^-1 HEWL fibrils, the cell viability were 75.16%±15.51%, 67.54%±12.13% and 67.89%±10.26% for 12 h treatment,respectively; 75.78±13.01%, 58.41%±5.55% and 61.90%±8.94% for 24 h treatment,respectively; 71.59%±14.75%, 55.65%±5.78% and 46.45%±6.23% for 48 h treatment,and the decreased cell viability displayed time-dependent and concentration-dependent manner. Nuclear staining results further confirmed the apoptosis features. These results suggest that HEWL protein could form into toxic amyloid fibrils under certain physicochemical conditions, which could provide basis for understanding the mechanisms of amyloid fibrils forming of prion protein and other amyloid proteins.

Key words: amyloid fibrils, hen egg-white lysozyme, β-sheet, SH-SY5Y cells

中图分类号:

S852.34

白瑜, 王武, 王碧, 李颖, 冯自立. 蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞的毒性[J]. 畜牧兽医学报, 2022, 53(8): 2721-2728.

BAI Yu, WANG Wu, WANG Bi, LI Ying, FENG Zili. The Cytotoxicity of Amyloid Fibrils Formed by Hen Egg-White Lysozyme on SH-SY5Y Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2721-2728.

参考文献 28

[1]	YU K H, LEE C I. Quercetin disaggregates prion fibrils and decreases fibril-induced cytotoxicity and oxidative stress[J]. Pharmaceutics, 2020, 12(11):1081.
[2]	NASR S H, DASARI S, MILLS J R, et al. Hereditary lysozyme amyloidosis variant p. Leu102Ser associates with unique phenotype[J]. J Am Soc Nephrol, 2017, 28(2):431-438.
[3]	HE L, WANG X S, ZHU D S, et al. Methionine oxidation of amyloid peptides by peroxovanadium complexes:inhibition of fibril formation through a distinct mechanism[J]. Metallomics, 2015, 7(12):1562-1572.
[4]	DE FELICE F G, VIEIRA M N N, MEIRELLES M N L, et al. Formation of amyloid aggregates from human lysozyme and its disease-associated variants using hydrostatic pressure[J]. FASEB J, 2004, 18(10):1099-1101.
[5]	KREBS M R H, WILKINS D K, CHUNG E W, et al. Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the β-domain[J]. J Mol Biol, 2000, 300(3):541-549.
[6]	SIVALINGAM V, PRASANNA N L, SHARMA N, et al. Wild-type hen egg white lysozyme aggregation in vitro can form self-seeding amyloid conformational variants[J]. Biophys Chem, 2016, 219:28-37.
[7]	FENG Z L, LI Y, BAI Y. Elevated temperatures accelerate the formation of toxic amyloid fibrils of hen egg-white lysozyme[J]. Vet Med Sci, 2021, 7(5):1938-1947.
[8]	WANG L Q, ZHAO K, YUAN H Y, et al. Cryo-EM structure of an amyloid fibril formed by full-length human prion protein[J]. Nat Struct Mol Biol, 2020, 27(6):598-602.
[9]	NORLIN N, HELLBERG M, FILIPPOV A, et al. Aggregation and fibril morphology of the Arctic mutation of Alzheimer's Aβ peptide by CD, TEM, STEM and in situ AFM[J]. J Struct Biol, 2012, 180(1):174-189.
[10]	SETHURAMAN A, BELFORT G. Protein structural perturbation and aggregation on homogeneous surfaces[J]. Biophys J, 2005, 88(2):1322-1333.
[11]	OW S Y, DUNSTAN D E. The effect of concentration, temperature and stirring on hen egg white lysozyme amyloid formation[J]. Soft Matter, 2013, 9(40):9692-9701.
[12]	MAHDAVIMEHR M, MERATAN A A, GHOBEH M, et al. Inhibition of HEWL fibril formation by taxifolin:Mechanism of action[J]. PLoS One, 2017, 12(11):e0187841.
[13]	PAN K M, BALDWIN M, NGUYEN J, et al. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins[J]. Proc Natl Acad Sci U S A, 1993, 90(23):10962-10966.
[14]	YAMAGUCHI K I, KUWATA K. Formation and properties of amyloid fibrils of prion protein[J]. Biophys Rev, 2018, 10(2):517-525.
[15]	MARIÑO L, PAUWELS K, CASASNOVAS R, et al. Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis[J]. Sci Rep, 2015, 5:12052.
[16]	李颖, 冯自立, 白瑜, 等. 蛋清溶菌酶的改造及其抑菌活性[J]. 畜牧兽医学报, 2021, 52(4):1094-1102.LI Y, FENG Z L, BAI Y, et al. The modification of hen egg-white lysozyme and its antibacterial activity[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4):1094-1102. (in Chinese)
[17]	SHARMA P, VERMA N, SINGH P K, et al. Characterization of heat induced spherulites of lysozyme reveals new insight on amyloid initiation[J]. Sci Rep, 2016, 6:22475.
[18]	GLYNN C, SAWAYA M R, GE P, et al. Cryo-EM structure of a human prion fibril with a hydrophobic, protease-resistant core[J]. Nat Struct Mol Biol, 2020, 27(5):417-423.
[19]	GREMER L, SCHÖLZEL D, SCHENK C, et al. Fibril structure of amyloid-β(1-42) by cryo-electron microscopy[J]. Science, 2017, 358(6359):116-119.
[20]	SWAMINATHAN R, RAVI V K, KUMAR S, et al. Lysozyme:a model protein for amyloid research[J]. Adv Protein Chem Struct Biol, 2011, 84:63-111.
[21]	SERAJ Z, GROVES M R, SEYEDARABI A. Cinnamaldehyde and Phenyl Ethyl Alcohol promote the entrapment of intermediate species of HEWL, as revealed by structural, kinetics and thermal stability studies[J]. Sci Rep, 2019, 9(1):18615.
[22]	GU W, WANG T T, ZHU J, et al. Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high temperature conditions[J]. Biophys Chem, 2003, 104(1):79-94.
[23]	SINGH J, UDGAONKAR J B. Unraveling the molecular mechanism of pH-induced misfolding and oligomerization of the prion protein[J]. J Mol Biol, 2016, 428(6):1345-1355.
[24]	YAMAGUCHI K I, KAMATARI Y O, FUKUOKA M, et al. Nearly reversible conformational change of amyloid fibrils as revealed by pH-jump experiments[J]. Biochemistry, 2013, 52(39):6797-6806.
[25]	BRUDAR S, HRIBAR-LEE B. The role of buffers in wild-type HEWL amyloid fibril formation mechanism[J]. Biomolecules, 2019, 9(2):65.
[26]	WANG W, ROBERTS C J. Protein aggregation-Mechanisms, detection, and control[J]. Int J Pharm, 2018, 550(1-2):251-268.
[27]	PELLARIN R, CAFLISCH A. Interpreting the aggregation kinetics of amyloid peptides[J]. J Mol Biol, 2006, 360(4):882-892.
[28]	CERDÀ-COSTA N, ESTERAS-CHOPO A, AVILÉS F X, et al. Early kinetics of amyloid fibril formation reveals conformational reorganisation of initial aggregates[J]. J Mol Biol, 2007, 366(4):1351-1363.

蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞的毒性

The Cytotoxicity of Amyloid Fibrils Formed by Hen Egg-White Lysozyme on SH-SY5Y Cells

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 28

相关文章 1

编辑推荐

Metrics

本文评价