Abstract
The pathogenesis of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is highly heterogeneous and still unclear. Additional novel variants have been recently detected in the population. The molecular and cellular effects of these previously unreported variants are still poorly understood and require further characterization. To address this problem, we have evaluated the various functions and biochemical consequences of six novel missense variants that lead to mild VLCAD deficiency. Marked deficiencies in fatty acid oxidation (FAO) and other mitochondrial defects were observed in cells carrying one of these six variants (c.541C>T, c.863T>G, c.895A>G, c.1238T>C, c.1276G>A, and c.1505T>A), including reductions in mitochondrial respiratory-chain function and adenosine triphosphate (ATP) production, and increased levels of mitochondrial reactive oxygen species (ROS). Intriguingly, higher apoptosis levels were found in cells carrying the mutant VLCAD under glucose-limited stress. Moreover, the stability of the mutant homodimer was disturbed, and major conformational changes in each mutant VLCAD structure were predicted by molecular dynamics (MD) simulation. The data presented here may provide valuable information for improving management of diagnosis and treatment of VLCAD deficiency and for a better understanding of the general molecular bases of disease variability.
摘要
目的
鉴定并阐述6 个ACADVL 新发突变的对细胞和分 子功能的影响, 为后期分子诊断和临床病例管理 提供科学依据.
创新点
本文从分子功能和结构的角度阐述6 个新位点对 蛋白和细胞功能的不同影响.
方法
通过体外构建突变载体建立各突变的过表达细胞 系, 检测各细胞系脂肪酸代谢能力、线粒体呼吸 链功能、线粒体产三磷酸腺苷(ATP)能力、活 性氧(ROS)和凋亡水平.同时检测了新发突变 对蛋白二聚体稳定性的影响并用分子动力学模 拟了突变蛋白构像的改变.
结论
突变(c.541C>T、c.863T>G、c.895A>G、c.1238T>C、 c.1276G>A 和c.1505T>A)过表达细胞系均表现 出脂肪酸代谢障碍, 线粒体呼吸链功能障碍, ATP 产生水平下降和线粒体ROS 水平上升.在无糖条 件下, 各细胞系的凋亡水平上升.各突变影响了 极长链酰基辅酶A 脱氢酶(VLCAD)蛋白二聚 体的稳定性, 分子动力学模拟预测每个突变均会 导致VLCAD 蛋白结构的发生重要构象变化.
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We thank all patients and their family members for their participation.
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Project supported by the National Natural Science Foundation of China (No. 81741090), the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, and the National Key R&D Program of China (Nos. 2017YFC1001703 and 2018YFC1002700)
Contributors
Ping-ping JIANG and Qiang SHU designed the research, wrote the manuscript, and had primary responsibility for final content. Ting CHEN and Fan TONG conducted the biochemical experiments and drafted the manuscript. Xiao-yu WU performed the cell culture and mitochondrial function analysis. Ling ZHU and Jing ZHENG supported the interpretation of the molecular genetics data. Ting CHEN, Xiao-hui CANG, and Qiu-zi YI were responsible for molecular dynamics analysis. Fan TONG, Ru-lai YANG, and Zheng-yan ZHAO carried out the clinical evaluation.
Compliance with ethics guidelines
Ting CHEN, Fan TONG, Xiao-yu WU, Ling ZHU, Qiu-zi YI, Jing ZHENG, Ru-lai YANG, Zheng-yan ZHAO, Xiao-hui CANG, Qiang SHU, and Ping-ping JIANG declare that they have no conflicts of interest.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study.
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Chen, T., Tong, F., Wu, Xy. et al. Novel ACADVL variants resulting in mitochondrial defects in long-chain acyl-CoA dehydrogenase deficiency. J. Zhejiang Univ. Sci. B 21, 885–896 (2020). https://doi.org/10.1631/jzus.B2000339
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DOI: https://doi.org/10.1631/jzus.B2000339
Key words
- Mitochondrial dysfunction
- Very-long-chain acyl-CoA dehydrogenase (VLCAD)
- β-Oxidation
- Molecular dynamics (MD) simulation