HIV-1 Nef regulates activity of the ER chaperone calnexin

HIV-1 Nef 调节 ER 伴侣钙联蛋白的活性

• 批准号: 8605707
• 负责人: MICHAEL Ilya BUKRINSKY
• 金额: $ 22.19万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2016-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8605707
• 关键词: Activities of Daily Living; Adaptor Signaling Protein; Address; Affect; Anti-HIV Agents; Anti-HIV Therapy; Binding; Bioinformatics; Biological Assay; CD28 gene; CXCR4 gene; Calnexin; Carrier Proteins; Cell Surface Receptors; Cells; Cholesterol; Collaborations; Data; Degradation Pathway; Down-Regulation; Drug Targeting; Endoplasmic Reticulum; Exploratory/Developmental Grant; Glycoproteins; Goals; HIV; HIV Infections; HIV vaccine; HIV-1; Immune; Infection; Integral Membrane Protein; Knowledge; Laboratories; Link; Mass Spectrum Analysis; Mediating; Membrane; Molecular Chaperones; Molecular Models; Mutagenesis; Organism; Phosphorylation; Physiological; Proteins; Public Health; Quality Control; Recording of previous events; Regulation; Research; Research Personnel; Russia; Series; Testing; Transgenic Mice; Vaccine Design; Viral; Viral Proteins; Virus; base; cholesterol transporters; design; glycosylation; gp160; high risk; innovation; molecular modeling; nef Protein; new therapeutic target; novel; palmitoylation; protein folding; public health relevance; receptor; research study; screening; small molecule; translational study; virtual

DESCRIPTION (provided by applicant): HIV-1 protein Nef is a multifunctional protein involved in regulation of viral infectivity and responsible for many pathogenic effects of HIV infection, including downregulation of CD4, MHC I and ABCA1. The effect of Nef on host cell proteins has been ascribed to the ability of Nef to function as a bridge between the host cell transport proteins and the target protein, thus promoting transport and degradation of the latter by the cellular lysosomal or proteasomal machinery. In our preliminary experiments, we identified a novel interaction partner of Nef, the endoplasmic reticulum (ER) chaperone calnexin. Calnexin is an integral ER transmembrane protein the main function of which is to assist protein folding and perform quality control during maturation of N-linked glycoproteins. We found that Nef disrupts interaction between calnexin and cholesterol transporter ABCA1, resulting in release from ER of non-functional ABCA1 and inhibition of cholesterol efflux. However, this effect of Nef was selective, as calnexin interaction with another glycoprotein, HIV-1 gp160, was not disrupted. The mechanism responsible for this selectivity is unknown, but Nef is known to affect localization of proteins to which it binds. Based on these preliminary studies, we hypothesize that Nef interaction with calnexin alters calnexin localization promoting its interaction with gp16 at the expense of a certain repertoire of host cell proteins whose maturation and function are impaired. This hypothesis will be tested in Aim 1 of the proposal. In Aim 2, we will use bioinformatics, molecular modeling and mutagenesis of Nef and calnexin, to identify the motifs and domains responsible for the interaction between these proteins and will test available Nef-targeting drugs and new compounds identified by virtual screening for the ability to interfere with this interaction. The bioinformatics studies will be performed by our Russian collaborator, Dr. Adzhubei. In Aim 3, we will characterize functional consequences of Nef effects on calnexin for the virus, cell and host organism. Using mass-spectrometry, we will assess glycosylation of gp160 and ABCA1 in the presence and absence of Nef. We will also test drugs targeting Nef-calnexin interaction in Nef-transgenic mice available in the laboratory of another Russian collaborator, Dr. Nedospasov. These studies will characterize novel mechanism behind the effects of Nef on host cell and viral proteins, may provide an explanation for the stimulatory effect of Nef on HIV infectivity, will identify new anti-HIV agents, and may inform anti-HIV vaccine design efforts.

描述（由申请人提供）：HIV-1蛋白Nef是一种多功能蛋白，参与病毒感染性的调节，并负责HIV感染的许多致病作用，包括下调CD 4、MHC I和ABCA 1。Nef对宿主细胞蛋白质的作用归因于Nef作为宿主细胞转运蛋白和靶蛋白之间的桥梁的能力，从而促进后者通过细胞溶酶体或蛋白酶体机制的转运和降解。在我们的初步实验中，我们确定了一个新的相互作用伙伴的Nef，内质网（ER）伴侣钙连接蛋白。钙连接蛋白是一种完整的内质网跨膜蛋白，其主要功能是在N-连接糖蛋白成熟过程中帮助蛋白质折叠并进行质量控制。我们发现Nef破坏钙连接蛋白和胆固醇转运蛋白ABCA 1之间的相互作用，导致非功能性ABCA 1从ER释放并抑制胆固醇流出。然而，Nef的这种作用是选择性的，因为钙连接蛋白与另一种糖蛋白HIV-1 gp 160的相互作用没有被破坏。负责这种选择性的机制尚不清楚，但已知Nef会影响其结合的蛋白质的定位。基于这些初步研究，我们假设Nef与钙连接蛋白的相互作用改变了钙连接蛋白的定位，促进其与gp 16的相互作用，以牺牲某些成熟和功能受损的宿主细胞蛋白的库为代价。这一假设将在提案的目标1中得到检验。在目标2中，我们将使用Nef和钙连接蛋白的生物信息学、分子建模和诱变，以鉴定负责这些蛋白质之间相互作用的基序和结构域，并将测试可用的Nef靶向药物和通过虚拟筛选鉴定的新化合物，以确定其干扰 这种互动。生物信息学研究将由我们的俄罗斯合作者Adzhubei博士进行。在目标3中，我们将表征Nef对病毒、细胞和宿主生物体的钙连接蛋白的作用的功能后果。使用质谱法，我们将评估存在和不存在Nef的情况下gp 160和ABCA 1的糖基化。我们还将在另一位俄罗斯合作者Nedospasov博士的实验室中测试针对Nef转基因小鼠中Nef-钙连接蛋白相互作用的药物。这些研究将表征Nef对宿主细胞和病毒蛋白影响背后的新机制，可能为Nef对HIV感染性的刺激作用提供解释，将识别新的抗HIV药物，并可能为抗HIV疫苗设计工作提供信息。