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（由适用提供）：通过在ER中通过泛素/蛋白酶体系统处理错误折叠的糖蛋白，内质网相关蛋白降解（ERAD）途径可作为ER中细胞糖蛋白折叠的质量控制。 HIV-1 Env是大量的N-糖基化，从本质上加速了其折叠和稳定性，并且是必需的或其功能。 N-糖基化涉及ER中的许多酶和伴侣。值得注意的是，ER中的糖蛋白折叠很容易出错，导致产生错误折叠对细胞有毒的蛋白质，因此细胞已经进化了脱落的途径，以特异性地靶向染色的蛋白质以降解。 GH47酶是I类甘露糖苷酶，包括Ermani，Edem1，Edem2，Edem3，Golgi Mania，Golgi Manib和Golgi Manic。他们在N-糖基化期间清除了N连接的高甘露糖聚糖前体中的1,2- lnk的甘露糖残留物，而且重要的是，它们在Erad中也起着不可或缺的作用。通常，ERAD分为三个步骤：底物识别，细胞质逆转录和蛋白水解GH47酶参与识别步骤并启动降解。已经提出EDEM1提取物中钙蛋白/钙网蛋白周期中脱落的蛋白质，并且错误折叠的蛋白针对ER衍生的质量控制室（ERQC），其中Ermani富含Ermani。 Ermani和可能的EDEM蛋白可能会催化广泛的脱氧化，这构成了蛋白质错误折叠的信号，进而激活了ERAD途径，从而导致降解的错误折叠蛋白。在这里，我们将研究HIV-1 Env如何被ERAD途径降解的机制，并提出以下两个具体目的来理解这种机制：1）阐明Ermani和Edem蛋白在HIV-1 ENV降解中的作用。我们将研究生态表达后Erad介导的ENV降解中的Ermani活性，并确定其关键分子确定素的活性。此外，我们将使用有利的CRISPR/CAS9敲除（KO）技术来测试EDEM蛋白如何对ERMANI活性做出贡献。 2）确定HIV-1降解的临界泛素E3连接酶。我们将使用小的干扰RNA（siRNA）敲低筛选E3途径中已知的E3连接酶文库以识别E3连接酶。 E3连接酶的身份将通过CRISPR/CAS9 KO进一步证实，然后进行生化分析。这些研究将定义新的内源性和潜在的治疗性抗逆转录病毒靶标，该靶标特别抑制ENV表达并阻止HIV-1复制。