靶向识别新型冠状病毒上刺突蛋白S1亚基的嵌合型E3泛素连接酶的设计与功能验证
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

天津市合成生物技术创新能力提升行动(TSBICIP-CXRC-048)


Design and functional validation of a chimeric E3 ubiquitin ligase targeting the spike protein S1 subunit of SARS-CoV-2
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    刺突(spike, S)蛋白在新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)侵染宿主的过程中起着至关重要的作用,其中S1亚基的受体结合域(receptor binding domain, RBD)结构域与宿主的血管紧张素转换酶2 (angiotensin-converting enzyme 2, ACE2)受体结合,介导病毒侵入宿主细胞。因此,降解S1是预防SARS-CoV-2侵染的可行策略之一。本研究旨在开发一种针对S1的靶向降解工具。首先利用三质粒慢病毒系统构建了稳定表达S1的HEK 293细胞系,发现在该细胞系中过表达线粒体E3泛素连接酶1 (mitochondrial E3 ubiquitin protein ligase 1, MUL1)可以有效促进S1的泛素化修饰,并加速其通过蛋白酶体降解过程。进一步研究显示,MUL1催化的S1的多泛素修饰主要是以K48的侧链加成方式进行。此外,将特异性靶向识别S1的小肽LCB1与MUL1偶联,构建了嵌合型E3泛素连接酶LCB1-MUL1。研究发现,该嵌合酶活性依赖于MUL1,并且相比于MUL1,LCB1-MUL1表现出更高的催化效率,将胞内S1的蛋白质半衰期从12 h缩短至9 h。本研究阐明了MUL1通过催化S1的泛素化修饰并促进其通过蛋白酶体降解的机制,初步验证了针对S1蛋白设计的靶向降解嵌合酶LCB1-MUL1的有效性。

    Abstract:

    The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1’s half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.

    参考文献
    相似文献
    引证文献
引用本文

代艳,林佳雨,张肖雅,逯浩睿,饶朗. 靶向识别新型冠状病毒上刺突蛋白S1亚基的嵌合型E3泛素连接酶的设计与功能验证[J]. 生物工程学报, 2024, 40(11): 4071-4083

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2024-03-05
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2024-11-07
  • 出版日期: 2024-11-25
文章二维码
您是第位访问者
生物工程学报 ® 2024 版权所有

通信地址:中国科学院微生物研究所    邮编:100101

电话:010-64807509   E-mail:cjb@im.ac.cn

技术支持:北京勤云科技发展有限公司