双定位信号增强工程化蛋白线粒体靶向性呈递

doi:10.16288/j.yczz.24-171

遗传 ›› 2024, Vol. 46 ›› Issue (11): 937-946.doi: 10.16288/j.yczz.24-171

双定位信号增强工程化蛋白线粒体靶向性呈递

周冰倩¹(), 李尚朴¹, 王旭¹, 孟祥宇¹, 邓竞荣¹, 邢金良²(), 王建刚¹, 徐坤¹()

1.西北农林科技大学动物科技学院，杨凌 712100
2.空军军医大学肿瘤生物学国家重点实验室，西安 710000

收稿日期:2024-06-12 修回日期:2024-09-01 出版日期:2024-11-20 发布日期:2024-09-04
通讯作者: 邢金良，博士，教授，研究方向：线粒体生物学与肿瘤防治。E-mail: xingjl@fmmu.edu.cn;
徐坤，博士，副教授，研究方向：基因编辑工具的开发及研究应用。E-mail: xukun@nwafu.edu.cn
作者简介:周冰倩，本科，专业方向：动物科学。E-mail: 2770998747@qq.com
基金资助:
农业生物育种重大专项(2023ZD04074);农业生物育种重大专项(2023ZD04051);肿瘤生物学国家重点实验室开放基金(CBSKL2022ZDKF11)

Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins

Bingqian Zhou¹(), Shangpu Li¹, Xu Wang¹, Xiangyu Meng¹, Jingrong Deng¹, Jinliang Xing²(), Jiangang Wang¹, Kun Xu¹()

1. College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China
2. State Key Laboratory of Tumour Biology, Fourth Military Medical University, Xi'an 710000, China

Received:2024-06-12 Revised:2024-09-01 Published:2024-11-20 Online:2024-09-04
Supported by:
Biological Breeding-Major Projects(2023ZD04074);Biological Breeding-Major Projects(2023ZD04051);Open Fund Project from State Key Laboratory of Tumor Biology-China(CBSKL2022ZDKF11)

摘要/Abstract

摘要：

有效传递工程化改造的蛋白进入线粒体对开发高效的线粒体DNA编辑工具、实现线粒体疾病精准治疗具有重要意义。本研究选取eGFP和Cas9基因，在其上游或/和下游引入不同的线粒体定位信号(mitochondrial localization signal，MLS)序列，分别构建了相应的工程化蛋白表达载体。将不同表达载体转染HEK293T细胞后，利用荧光共定位实验和免疫印迹实验分析不同工程化蛋白的线粒体靶向性呈递效果。结果显示，相比单端添加MLS的eGFP和Cas9蛋白，双端MLS改造均显著提高了工程化蛋白的线粒体靶向性呈递效率。推测双MLS策略可增强工程化蛋白的线粒体靶向性，为以后开发高效的线粒体DNA编辑工具奠定了理论基础。

关键词: 线粒体疾病, 线粒体DNA编辑, 工程化蛋白, 线粒体定位信号, 线粒体靶向

Abstract:

Effective delivery of engineered proteins into mitochondria is of great significance for developing efficient mitochondrial DNA editing tools and realizing accurate treatment of mitochondrial diseases. Here, the candidate genes, eGFP and Cas9, were engineered with different mitochondrial localization signal (MLS) sequences introduced at their up- or/and down-streams. The corresponding expression vectors for the engineered proteins were constructed respectively, and HEK293T cells were transfected with these vectors. The fluorescence colocalization and Western blotting assays were used to analyze the mitochondrial targeting presentation effect of different engineered proteins. The results demonstrated that the daul-MLS modification of the eGFP and Cas9 proteins significantly improved the efficiency of mitochondrial targeted presentation, compared with the engineered proteins with single MLS added. Hence, it is speculated that dual MLS strategy can enhance the mitochondrial targeting of engineered proteins, which lays a theoretical foundation for the future development of efficient mitochondrial DNA editing tools.

Key words: mitochondrial diseases, mtDNA editing, protein engineering, mitochondrial localization signal, mitochondrial targeting

周冰倩, 李尚朴, 王旭, 孟祥宇, 邓竞荣, 邢金良, 王建刚, 徐坤. 双定位信号增强工程化蛋白线粒体靶向性呈递[J]. 遗传, 2024, 46(11): 937-946.

Bingqian Zhou, Shangpu Li, Xu Wang, Xiangyu Meng, Jingrong Deng, Jinliang Xing, Jiangang Wang, Kun Xu. Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins[J]. Hereditas(Beijing), 2024, 46(11): 937-946.

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线粒体编辑器	组成元件	编辑类型	偏好性	编辑效率	脱靶	参考文献
CRISPR Cas9	Cas9, gRNA	Indel	碱基识别偏好	极小	严重的脱靶效应	[26,27]
DdCBEs	DddA, TALE	C·G-T·A	偏好TC序列	4.9%~49%	严重的脱靶效应	[18]
DdCBEs(V11)	DddA11, TALE	C·G-T·A	HC	15%~30%	严重的脱靶效应	[22]
DdCBE-Ss	Ddd-Ss, TALE	C·G-T·A	DC	6%~51%	严重的脱靶效应	[23]
Mito-TALED	DddA, TadA8e, TALE	A·T-G·C	编辑位点偏好	大约40%	严重的脱靶效应	[19]
Mito-BEs	TALE, Chymotrypsin, Deaminase	A·T-G·C或 C·G-T·A	链选择偏好	大约40%	暂未发现明确的脱靶效应	[28]

双定位信号增强工程化蛋白线粒体靶向性呈递

Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins

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