Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases.
Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases.
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MazF 与 MazE 片段融合的 MazF 序列特异性 mRNA 干扰酶活性的分子内调节，该片段具有可被特定蛋白酶切割的接头。
DOI：
10.1128/aem.00364-12
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发表时间：
2012
影响因子：
4.4
通讯作者：
Inouye,Masayori
中科院分区：
文献类型：
--
作者：
Park,Jung-Ho;Yamaguchi,Yoshihiro;Inouye,Masayori
人类免疫缺陷病毒1型（HIV-1）和丙型肝炎病毒（HCV）的基因组由编码多蛋白的单链RNA组成，这些多蛋白通过病毒编码的特异性蛋白酶加工成单个功能蛋白。这些蛋白酶已被用作药物开发的靶点。在这里，我们不是靶向这些蛋白酶来抑制病毒感染，而是利用蛋白酶活性来激活毒性蛋白质以防止病毒感染。我们设计了大肠杆菌的MazE-MazF抗毒素-毒素系统，以将抗毒素MazE的C-末端41-残基片段与毒素MazF的N-末端融合，其中接头具有针对HIV PR（HIV-1蛋白酶）、NS 3蛋白酶（HCV蛋白酶）或因子Xa的特异性蛋白酶切割位点。这些融合蛋白形成了一个稳定的二聚体（而不是MazF 2-MazE 2-MazF 2异源六聚体），以抑制MazF的ACA（序列）特异性mRNA干扰酶活性。当融合蛋白与相应的蛋白酶一起孵育时，迷宫片段从融合蛋白上切割下来，释放出活性MazF，然后MazF作为ACA特异性mRNA干扰酶切割单链MS 2噬菌体RNA。所证明的蛋白酶对MazF毒性的分子内调节可能为HIV-1、HCV和其他单链RNA病毒感染的预防性和治疗性治疗提供新的方法。
The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF2-MazE2-MazF2heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.