在活细胞和活体中基于酸酶的近距离标记
Hao Zhu1, Jae Hoon Oh2, Yuna Matsuda1
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Journal of the American Chemical Society
|March 6, 2024
概括
研究人员开发了一种用于近距离标记的新型细菌酸酶 (BmTyr),可在活细胞和体内进行快速的低背景蛋白标记. 这种酶克服了现有方法的局限性,推进了空间蛋白质组学和细胞网络的发现.
科学领域:
- 蛋白质组学
- 分子和细胞生物学
- 生物化学
背景情况:
- 了解有机体蛋白质成分和蛋白质邻居对于阐明有机体功能和蛋白质网络至关重要.
- 近距离标记 (PL) 是一个强大的空间蛋白质组技术,但现有的酶如APEX,BioID和TurboID具有包括细胞毒性,缓慢动力学和高背景的限制.
- 在体内应用中需要改进的PL酶,具有更好的生物相容性,速度和特异性.
研究的目的:
- 介绍和描述一种新型的细菌酸酶 (BmTyr) 作为近距离标记酶.
- 证明BmTyr在活细胞中无H2O2,快速和低背景蛋白质标记的实用性.
- 开发一种在体内使用联结BmTyr的PL策略,并将其应用于研究神经递质受体蛋白质.
主要方法:
- 细菌酸酶 (BmTyr) 的遗传编码用于近距离标记.
- 在活细胞中应用BmTyr用于亚细胞溶解的PL和蛋白质组.
- 在活小鼠大脑模型中设计BmTyr连接策略.
主要成果:
- BmTyr 作为一种高效的 PL 酶,可在活细胞中在 10 分钟内进行无 H2O2 的标记.
- 与现有方法相比,BmTyr具有快速标记动力学和低背景生物化.
- 在小鼠大脑突触中,BmTyr成功识别了神经递质受体 (Grm1和Drd2) 的周围蛋白质组.
结论:
- 细菌酸酶 (BmTyr) 是一种用于近距离标记的新型多功能酶,与当前的PL工具相比,具有优势.
- 在细胞和体内环境中,BmTyr促进了快速,特定和生物相容的空间蛋白质组.
- 这项工作扩大了近距离标签的工具包,并有望加强器官功能和蛋白质相互作用网络的发现.
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