Mechanism of HIV-1 Env Degradation by the ERAD pathway

ERAD 途径降解 HIV-1 Env 的机制

• 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
• 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

 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（由申请方提供）：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。HIV-1 Env是高度N-糖基化的，这本质上加速了其折叠和稳定性，并且是其功能所必需的。N-糖基化涉及ER中的许多酶和分子伴侣。值得注意的是，糖蛋白在内质网中的折叠是容易出错的，导致错误折叠的糖蛋白的产生。 这些蛋白质对细胞有毒，因此细胞已经进化出ERAD途径，专门针对错误折叠的蛋白质进行降解。GH47酶是I类甘露聚糖酶，包括ERManI、EDEM 1、EDEM 2、EDEM 3、Golgi ManIA、Golgi ManIB和Golgi ManIC。它们在N-糖基化过程中从N-连接的高甘露糖聚糖前体切割1，2-连接的甘露糖残基，重要的是，它们在ERAD中也发挥不可或缺的作用。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。已经表明，EDEM 1从钙连接蛋白/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靶向ER衍生的质量控制室（ERQC），其中ERManI富集。ERManI和可能的EDEM蛋白然后催化广泛的甘露糖基化，其构成蛋白质错误折叠的信号，其反过来激活ERAD途径，导致错误折叠的蛋白质被降解。本论文主要研究ERAD途径降解HIV-1 Env的机制，并提出以下两个具体目标：1）阐明ERManI和EDEM蛋白在HIV-1 Env降解中的作用。我们将研究ERAD介导的Env降解后异位表达的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CRISPR/Cas9敲除（KO）技术测试EDEM蛋白如何有助于ERManI活性。2)确定降解HIV-1的关键泛素E3连接酶。我们将使用小干扰RNA（siRNA）敲低筛选ERAD途径中已知的E3连接酶文库以鉴定E3连接酶。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点，特异性抑制Env表达和阻断HIV-1复制。