Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

正交泛素转移至 E3 底物特异性

• 批准号: 10228557
• 负责人: HIROAKI KIYOKAWA
• 金额: $ 34.24万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228557
• 关键词: Affinity; Antineoplastic Agents; Autoimmune Diseases; Bacteriophages; Binding; Biological Process; Cancer Biology; Cell Cycle; Cell Cycle Regulation; Cell Signaling Process; Cell surface; Cells; Cellular biology; Chimera organism; Complex; Creativeness; Cytokinesis; Data; Development; Disease; Engineering; Enzymes; Funding; Goals; Grant; Knowledge; Lead; Location; Malignant Neoplasms; Maps; Mediating; Methods; Mission; Monitor; Mutation; Natural Killer Cells; Nerve Degeneration; Network-based; Outcome; Parkinson Disease; Pathogenesis; Pathway interactions; Plug-in; Positioning Attribute; Post-Translational Protein Processing; Protein Engineering; Proteins; Public Health; Reaction; Regulation; Research; Research Personnel; Role; Signal Transduction; Substrate Specificity; Testing; Tumor Suppression; UBE3A gene; Ubiquitin; Ubiquitination; United States National Institutes of Health; Work; Yeasts; base; cancer cell; cross reactivity; design; effective therapy; inhibitor/antagonist; innovation; interest; multicatalytic endopeptidase complex; parkin gene/protein; recruit; ubiquitin ligase; ubiquitin-protein ligase; Affinity; Antineoplastic Agents; Autoimmune Diseases; Bacteriophages; Binding; Biological Process; Cancer Biology; Cell Cycle; Cell Cycle Regulation; Cell Signaling Process; Cell surface; Cells; Cellular biology; Chimera organism; Complex; Creativeness; Cytokinesis; Data; Development; Disease; Engineering; Enzymes; Funding; Goals; Grant; Knowledge; Lead; Location; Malignant Neoplasms; Maps; Mediating; Methods; Mission; Monitor; Mutation; Natural Killer Cells; Nerve Degeneration; Network-based; Outcome; Parkinson Disease; Pathogenesis; Pathway interactions; Plug-in; Positioning Attribute; Post-Translational Protein Processing; Protein Engineering; Proteins; Public Health; Reaction; Regulation; Research; Research Personnel; Role; Signal Transduction; Substrate Specificity; Testing; Tumor Suppression; UBE3A gene; Ubiquitin; Ubiquitination; United States National Institutes of Health; Work; Yeasts; base; cancer cell; cross reactivity; design; effective therapy; inhibitor/antagonist; innovation; interest; multicatalytic endopeptidase complex; parkin gene/protein; recruit; ubiquitin ligase; ubiquitin-protein ligase

Project Summary / Abstract Ubiquitin (UB) transfer through the E1-E2-E3 cascade mediates signal transduction in almost all aspects of cell biology. E3 UB ligases catalyze UB transfer from E2s to the substrate proteins and ultimately decide the targets, location, and timing of the ubiquitination reaction. Identifying the substrate proteins is the key to elucidating the biological functions of an E3. However, its has been a significant challenge to profile E3 substrate specificity because of the transient formation of E3-substrate complex and the cross-reactivity among various E3s. Our long-term goal is to elucidate the important roles of E3-catalyzed protein ubiquitination in cell biology and diseases. Working toward this goal, we developed a method that we called “orthogonal UB transfer (OUT)” to identify the direct ubiquitination targets of an E3. In this innovative method, an engineered UB (xUB) is exclusively transferred through an engineered cascade of xE1-xE2-xE3 to the substrates of a specific E3 (“x” designates engineered enzymes free of cross creativities with native partners in the cell). We have constructed OUT cascades of HECT E3 E6AP and U-box E3 E4B and CHIP to profile their substrate specificities and validated OUT as an efficient platform to identify E3 substrates. The objective of this application is to generate OUT cascades with Ring E3s Cbl-b and Parkin to identify their substrate proteins. We will also follow the leads in the substrate profiles of E6AP and CHIP generated by OUT to establish the roles of these E3s in cell cycle and cancer cell invasion. Our central hypothesis is that the OUT cascades of E3s can identify new regulatory relationships between the E3 and substrates and elucidate the roles of E3 in cell signaling. Such a hypothesis is supported by our strong preliminary data demonstrating the feasibility of OUT in profiling the ubiquitination targets of E6AP, E4B, and CHIP. The rationale of our proposed work is that the 600 Ring E3s in the cell used a highly homologous Ring domain to bind to E2 and mediate UB transfer from E2 to their substrate proteins. Once we develop OUT platform with Ring E3s Cbl-b and Parkin, we can use similar protein engineering strategies to build the OUT platform for other Ring E3s to reveal their biological functions. We will pursue three specific aims: 1) Extending the OUT cascades to Ring E3s Cbl-b and Parkin to profile their substrate proteins; 2) Extending the OUT cascade to BRUCE to investigate its role in cytokinesis; 3) Studying the function of E6AP and CHIP based on their substrate profile generated by OUT. The expected outcome of our work is the development of the OUT platform to profile ubiquitination targets of all class of E3s including HECT, U-box and Ring types. Based on the knowledge of their substrate specificity, we will elucidate the role of Cbl-b in regulating NK cell activity against cancer cells, reveal the mechanism of Parkin in tumor suppression, and define the role the BRUCE in cytokinesis. We will establish new cellular circuits mediated by E6AP and CHIP in cell cycle control and cancer cell invasion. The positive impact of our work is that we will develop the OUT platform for other researchers to plug in their E3s of interest and map the E3s on the cell signaling network by profiling their substrate specificity.

项目摘要 /摘要 泛素（UB）通过E1-E2-E3级联反应在几乎所有方面介导信号转移 细胞生物学。 E3 UB连接酶催化UB从E2S转移到底物蛋白，并最终决定 泛素化反应的目标，位置和时机。识别底物蛋白是关键 阐明E3的生物学功能。但是，对于剖面E3基板来说，这是一个重大挑战 特异性是因为E3底物复合物的短暂形成以及各种之间的交叉反应性 E3S。我们的长期目标是阐明E3催化蛋白泛素化在细胞生物学中的重要作用 和疾病。朝着这个目标努力，我们开发了一种称为“正交UB转移（OUT）”的方法 确定E3的直接泛素化靶标。在这种创新方法中，工程UB（XUB）是 通过XE1-XE2-XE3的工程级联级别转移到特定E3的底物（“ X”） 指定与细胞中本土合作伙伴无跨创作的工程酶）。我们已经构建了 耗尽级别的E3 E6AP和U-box E3 E4B，并介绍其底物规范和 被验证为识别E3底物的有效平台。此应用程序的目的是生成 用RING E3S CBL-B和Parkin逐渐识别其底物蛋白。我们还将跟随线索 在由OUT生成的E6AP和芯片的底物曲线中，以确定这些E3在细胞周期中的作用 和癌细胞侵袭。我们的中心假设是，E3的级联级别可以识别新的调节 E3与底物之间的关系，并阐明了E3在细胞信号传导中的作用。这样的假设是 在我们强大的初步数据的支持下，证明了在分析泛素化方面的可行性 E6AP，E4B和CHIP的目标。我们提出的工作的理由是，单元格中的600环E使用了 高度同源的环域与E2结合并介导UB从E2转移到其底物蛋白。一次 我们使用RING E3S CBL-B和Parkin开发平台，我们可以使用类似的蛋白质工程策略来进行 建立其他环E3的平台以揭示其生物学功能。我们将追求三个具体目标： 1）将级联反应到E3S CBL-B和Parkin，以介绍其底物蛋白； 2）扩展 布鲁斯（Bruce）的级联研究其在细胞因子中的作用； 3）研究E6AP和芯片的功能 基于其由OUT生成的底物配置文件。我们工作的预期结果是发展 将平台介绍所有类型E3的泛素化目标，包括HECT，U-BOX类型和环类型。基于 关于其底物特异性的了解，我们将阐明CBL-B在调节NK细胞活性中的作用 针对癌细胞，揭示了帕金在肿瘤抑制中的机制，并定义了布鲁斯的作用 细胞因子。我们将建立由E6AP介导的新细胞电路和细胞周期控制和癌症的芯片 细胞入侵。我们工作的积极影响是，我们将为其他研究人员开发一个OUT平台 通过分析其底物特异性，插入其感兴趣的E3并绘制电池信号网络上的E3。