Role of heme oxygenase 2 in trafficking and regulation of myristoylated proteins

血红素加氧酶 2 在肉豆蔻酰化蛋白运输和调节中的作用

• 批准号: 10092949
• 负责人: STEPHEN Paine GOFF
• 金额: $ 24.3万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092949
• 关键词: ABCE1 gene; Affect; Antibodies; Antiviral Therapy; Binding; Binding Proteins; Binding Sites; Biological Assay; Cell Line; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Crystallization; Cytoplasm; Endoplasmic Reticulum; Genome; Heme; Hydrophobicity; Hypersensitivity; Immune signaling; Immunofluorescence Immunologic; Immunoprecipitation; Inflammatory Response; Integration Host Factors; Intracellular Transport; Knock-out; Lipopolysaccharides; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Methods; Monitor; Murine leukemia virus; Myristates; Myristic Acids; N-terminal; Pathway interactions; Pattern; Play; Process; Production; Protein Family; Protein Precursors; Proteins; Protocols documentation; RANTES; RNA; RNA Viruses; Receptor Signaling; Regulation; Retroviridae; Role; Sedimentation process; Signal Transduction; Signaling Molecule; Structure; TLR4 gene; Testing; Toll-like receptors; Viral; Virion; Virus; Virus Assembly; Virus Replication; Western Blotting; Work; chemokine; cytokine; experimental study; gag Gene Products; gene therapy; heme oxygenase-2; knock-down; live cell imaging; membrane assembly; mutant; myristoylation; novel; novel strategies; overexpression; particle; receptor; response; small hairpin RNA; trafficking; viral RNA

We will explore the role of a novel host factor in retrovirus replication, heme oxygenase 2 (HO-2), which we discovered binds N-terminally myristoyled proteins and regulates their trafficking to the plasma membrane. We have generated cell lines deficient in HO-2 by shRNA methods, or entirely lacking HO-2 by CRISPR-directed knockout (KO), and documented dramatic increases in retrovirus virion production. We will now characterize the intracellular transport of the myristoylated murine leukemia virus (MLV) Gag protein with and without HO-2 by examination of fixed sections by immunofluorescence, and by live cell imaging. We will study the size of intracellular complexes containing Gag by sedimentation analysis of extracts, followed by Western blots probed with anti-Gag antibodies. We will follow the course of Gag multimerization by co-immunoprecipitation experiments. We will look for alterations in Gag association with its known trafficking partners (including ABCE1, DDX6, and MOV10), and with proteins known to be important in the process of virion assembly and release (notably the ESCRT and CHMP proteins). We will assess the course of Gag interaction with viral RNA by RNA-immunoprecipitation (RIP) assays. These experiments should give us a comprehensive view of the role of HO-2 in controlling Gag localization and function in virion assembly. The work will define a new potential target for antiviral therapies against retroviruses, and other viruses encoding myristoylated proteins. In addition, we will explore the role of HO-2 in trafficking of selected cellular myristoylated proteins. We have developed an extensive hit list of host proteins bound by HO-2 and will focus on two of these proteins: Toll-like receptor adaptor molecule 2 (TRAM), involved in innate immune signaling;; and reticulon 2, a key protein in intracellular trafficking and in promoting membrane curvature. We will characterize the role of HO-2 in controlling the localization of TRAM, the association of TRAM with the TLR4 receptor, and the signaling through TRAM in response to lipopolysaccharide (LPS). We will similarly test for the role of HO-2 in the localization of reticulon 2 to the endoplasmic reticulum. Because of its potential role in membrane bending, we will test for the effect of reticulon 2 KO on ER structure and virion budding. These experiments will reveal new functions of HO-2 in regulating those specific host functions that mediate virus restriction and impact virion assembly. The findings have great potential to reveal entirely new approaches to regulate or inhibit virus replication.

我们将探索一种新型宿主因子血红素加氧酶 2 (HO-2) 在逆转录病毒复制中的作用，我们发现它与 N 末端肉豆蔻酰化蛋白结合并调节其向质膜的运输。我们已经生成了 HO-2 缺陷的细胞系。 2 通过 shRNA 方法，或通过 CRISPR 定向敲除 (KO) 完全缺乏 HO-2，并记录了逆转录病毒病毒体产量的显着增加 我们现在将描述肉豆蔻酰化小鼠的细胞内运输。通过免疫荧光和活细胞成像检查固定切片，我们将通过提取物的沉降分析来研究含有或不含有 HO-2 的白血病病毒 (MLV) Gag 蛋白，然后用抗蛋白进行蛋白质印迹检测。 -Gag 抗体。我们将通过免疫共沉淀实验追踪 Gag 多聚化过程，我们将寻找 Gag 与其已知贩运伙伴（包括 ABCE1、DDX6 和）相关的变化。 MOV10），以及已知在病毒粒子组装和释放过程中重要的蛋白质（特别是 ESCRT 和 CHMP 蛋白质），我们将通过 RNA 免疫沉淀 (RIP) 测定来评估 Gag 与病毒 RNA 相互作用的过程。让我们全面了解 HO-2 在控制 Gag 定位和病毒粒子组装功能中的作用。这项工作将为针对逆转录病毒和其他编码病毒的抗病毒治疗确定一个新的潜在靶点。此外，我们将探讨 HO-2 在选定的细胞豆蔻酰化蛋白运输中的作用。我们已经制定了一份广泛的 HO-2 结合的宿主蛋白列表，并将重点关注其中的两种蛋白：Toll 样蛋白。受体接头分子 2 (TRAM)，参与先天免疫信号传导；以及网状蛋白 2，细胞内运输和促进膜弯曲的关键蛋白。我们将描述 HO-2 在控制定位中的作用。 TRAM 的作用、TRAM 与 TLR4 受体的关联以及通过 TRAM 响应脂多糖 (LPS) 的信号传导 我们将类似地测试 HO-2 在网状蛋白 2 定位到内质网中的作用。为了研究 reticulon 2 KO 在膜弯曲中的潜在作用，我们将测试 reticulon 2 KO 对 ER 结构和病毒颗粒出芽的影响。 HO-2 调节介导病毒限制和影响病毒颗粒组装的特定宿主功能，这些发现具有揭示调节或抑制病毒复制的全新方法的巨大潜力。