Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation.
Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation.
复制标题
DOI：
10.1128/mbio.00328-17
复制
发表时间：
2017-03-28
期刊：
影响因子：
6.4
通讯作者：
Dudley JP
中科院分区：
文献类型：
--
作者：
Byun H;Das P;Yu H;Aleman A;Lozano MM;Matouschek A;Dudley JP
多种病原体，包括病毒和细菌，操纵内质网相关降解（ERAD），以避免宿主的免疫反应，并促进其复制。β逆转录病毒小鼠乳腺肿瘤病毒（MMTV）编码Rem，Rem是切割成98个氨基酸的信号肽（SP）和C末端蛋白（Rem-CT）的前体蛋白。SP利用逆向转位提取ER膜，但通过未知机制避免ERAD进入细胞核并作为Rev样蛋白发挥功能。为了确定SP如何逃脱ERAD，我们使用了泛素激活的相互作用陷阱（UBAIT）屏幕捕捉和识别与SP的瞬时蛋白质相互作用，包括ERAD相关的p97 ATP酶，但不E3连接酶或Derlin蛋白连接到retrotranslocation，polyubiquitylation和蛋白酶体降解提取的蛋白质。显性负性p97 ATP酶抑制Rem和SP功能。免疫沉淀实验表明，雷姆，而不是SP，是聚泛素化。使用酵母和哺乳动物表达系统，连接的泛素样结构域（UbL）SP或雷姆诱导降解的蛋白酶体，而SP是稳定的UbL的情况下。ERAD相关的Derlin蛋白不需要SP活性。总之，这些结果表明，雷姆使用一种新的p97依赖性，Derlin独立的逆转录机制不同于其他病原体，以避免SP泛素化和蛋白酶体降解。细菌和病毒感染产生病原体特异性蛋白质，干扰宿主功能，包括免疫反应。小鼠乳腺肿瘤病毒（MMTV）是研究人类复杂逆转录病毒（如HIV-1）以及癌症诱导的模型系统。我们已经表明，MMTV编码的调节蛋白，Rem，这是切割成N-末端信号肽（SP）和C-末端蛋白（Rem-CT）内的内质网（ER）膜。SP的功能需要通过逆向易位提取ER膜，这是称为ER相关降解（ERAD）的蛋白质质量控制系统的一部分，对细胞健康至关重要。通过不太清楚的机制，某些病原体衍生的蛋白质被逆转录，但不降解。我们在这里证明，MMTV SP retrotranslocation从ER膜避免降解通过一个独特的过程，涉及与细胞p97 ATP酶的相互作用和未能获得细胞蛋白酶体靶向序列。
Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation. Bacterial and viral infections produce pathogen-specific proteins that interfere with host functions, including the immune response. Mouse mammary tumor virus (MMTV) is a model system for studies of human complex retroviruses, such as HIV-1, as well as cancer induction. We have shown that MMTV encodes a regulatory protein, Rem, which is cleaved into an N-terminal signal peptide (SP) and a C-terminal protein (Rem-CT) within the endoplasmic reticulum (ER) membrane. SP function requires ER membrane extraction by retrotranslocation, which is part of a protein quality control system known as ER-associated degradation (ERAD) that is essential to cellular health. Through poorly understood mechanisms, certain pathogen-derived proteins are retrotranslocated but not degraded. We demonstrate here that MMTV SP retrotranslocation from the ER membrane avoids degradation through a unique process involving interaction with cellular p97 ATPase and failure to acquire cellular proteasome-targeting sequences.