Mechanism of HIV-1 Env Degradation by the ERAD pathway

ERAD 途径降解 HIV-1 Env 的机制

基本信息

批准号：
9324121
负责人：
YONG-HUI ZHENG
金额：
$ 19.38万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9324121
关键词：
Alpha-glucosidase Alpha-mannosidase Amino Acids Anti-Retroviral Agents Biochemical CRISPR/Cas technology Calnexin Carbohydrates Carrier Proteins Cells Cleaved cell Client Complex Consensus Cytoplasm Degradation Pathway Destinations Development Drug Targeting Ectopic Expression Endoplasmic Reticulum Enzymes Excision Family Glucose Glycoproteins Glycoside Hydrolases Golgi Apparatus HIV-1 Infection Knock-out Lead Libraries Link Manic Mannose Mannosidase Mediating Membrane Molecular Molecular Chaperones Oligosaccharides Pathway interactions Pharmacology Play Polysaccharides Process Production Proteins Proteolysis Public Health Quality Control Research Role Signaling Protein Small Interfering RNA System Technology Testing Therapeutic Thiol Disulfide Oxidoreductase Ubiquitin calreticulin chaperone machinery env Glycoproteins glycoprotein biosynthesis glycosylation knock-down man member misfolded protein multicatalytic endopeptidase complex novel protein degradation protein misfolding protein transport public health relevance sugar ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): The endoplasmic reticulum-associated protein degradation (ERAD) pathway serves as quality control for cellular glycoprotein folding in the ER by disposing of misfolded glycoproteins through the ubiquitin/proteasome system. HIV-1 Env is heavily N-glycosylated, which intrinsically accelerates its folding and stability, and is required or its functions. N-glycosylation involves a number of enzymes and chaperones in the ER. Noticeably, the glycoprotein folding in the ER is error-prone, resulting in production of misfolded proteins that are toxic to cells, so cells have evolved the ERAD pathway to specifically target misfolded proteins for degradation. GH47 enzymes are class I mannosidases that include ERManI, EDEM1, EDEM2, EDEM3, Golgi ManIA, Golgi ManIB, and Golgi ManIC. They cleave 1,2-lnked mannose residues from N-linked high- mannose glycan precursors during N-glycosylation, and importantly, they also play an indispensable role in ERAD. In general, ERAD is divided into three steps: substrate recognition, cytoplasmic retrotranslocation, and proteolysis GH47 enzymes are engaged in the recognition step and initiate the degradation. It has been suggested that EDEM1 extracts misfolded proteins from the calnexin/calreticulin cycle, and misfolded proteins are targeted to the ER-derived quality control compartment (ERQC) where ERManI is enriched. ERManI and possibly the EDEM proteins then catalyze extensive demannosylation, which constitutes a signal of protein misfolding that in turn activates the ERAD pathway, resulting in misfolded proteins being degraded. Here, we will study the mechanism of how HIV-1 Env is degraded by the ERAD pathway, and we propose the following two specific aims to understand this mechanism: 1) To elucidate the role of ERManI and EDEM proteins in HIV-1 Env degradation. We will study the ERManI activity in ERAD-mediated Env degradation after ectopic expression and identify its critical molecular determinants for its activity. In addition, we will test how EDEM proteins contribute to the ERManI activity using the advantageous CRISPR/Cas9 knockout (KO) technology. 2) To identify the critical ubiquitin E3 ligase for HIV-1 degradation. We will screen the known E3 ligase library in the ERAD pathway using small interfering RNA (siRNA) knockdown to identify the E3 ligase. The identity of the E3 ligase will be further confirmed by CRISPR/Cas9 KO followed by biochemical analyses. These studies will define novel endogenous and potential therapeutically applicable antiretroviral targets, which specifically inhibits Env expression and blocks HIV-1 replication.

描述（由申请人提供）：内质网相关蛋白降解（ERAD）途径通过泛素/蛋白酶体系统处理错误折叠的糖蛋白，作为 ER 中细胞糖蛋白折叠的质量控制。，其本质上加速了其折叠和稳定性，并且是必需的或者其功能涉及许多酶和分子伴侣。值得注意的是，内质网中的糖蛋白折叠容易出错，导致错误折叠的产生。 GH47 酶是 I 类甘露糖苷酶，包括 ERManI、EDEM1、EDEM2、EDEM3、Golgi ManIA、Golgi ManIB 和 Golgi ManIC 1。来自 N 连接高甘露糖聚糖前体的 2 连接甘露糖残基N-糖基化，重要的是，它们在ERAD中也发挥着不可或缺的作用。一般来说，ERAD分为三个步骤：底物识别、细胞质逆转位和蛋白水解，GH47酶参与识别步骤并启动降解。表明 EDEM1 从钙联蛋白/钙网蛋白循环中提取错误折叠的蛋白质，并且错误折叠的蛋白质被靶向 ER 衍生的质量控制区室 (ERQC)，其中ERManI 和可能的 EDEM 蛋白随后催化广泛的去甘露糖基化，这构成了蛋白质错误折叠的信号，进而激活 ERAD 途径，导致错误折叠的蛋白质被降解。在这里，我们将研究 HIV-1 Env 的机制。被 ERAD 途径降解，我们提出以下两个具体目标来理解该机制：1）阐明 ERManI 和 EDEM 的作用我们将研究异位表达后 ERAD 介导的 Env 降解中的 ERManI 活性，并确定其活性的关键分子决定因素。此外，我们将使用有利的优势测试 EDEM 蛋白如何促进 ERManI 活性。 CRISPR/Cas9 敲除（KO）技术 2）鉴定 HIV-1 降解的关键泛素 E3 连接酶我们将筛选 ERAD 中已知的 E3 连接酶库。使用小干扰 RNA (siRNA) 敲低来鉴定 E3 连接酶的身份将通过 CRISPR/Cas9 KO 进一步确认，然后进行生化分析，这些研究将确定新的内源性和潜在的治疗适用的抗逆转录病毒靶标。抑制 Env 表达并阻断 HIV-1 复制。