Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

正交泛素转移至 E3 底物特异性

• 批准号: 9811405
• 负责人: HIROAKI KIYOKAWA
• 金额: $ 2.63万
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811405
• 关键词: Affect; Affinity; Affinity Chromatography; Bacteriophages; Binding; Biological Assay; Biological Process; C-terminal; Catalysis; Cell Cycle; Cell physiology; Cells; Cellular biology; Complex; Diabetes Mellitus; Disease; Engineering; Ensure; Enzymes; Excision; Gene Activation; Goals; Human; Human Genome; Hybrids; Individual; Label; Length; MDM2 gene; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Modification; Neurodegenerative Disorders; Neurons; Pathologic; Pathway interactions; Peptides; Phage Display; Polyubiquitin; Protein Engineering; Proteins; Proteomics; Reaction; Role; Signal Transduction; Signal Transduction Pathway; Site-Directed Mutagenesis; Structure; Substrate Specificity; TP53 gene; Testing; Two-Dimensional Gel Electrophoresis; Ubiquitin; Ubiquitination; Work; Yeasts; base; carboxylate; combinatorial; cross reactivity; design; insight; mutant; prevent; protein degradation; recruit; thioester; ubiquitin-protein ligase; uptake; virtual

Project Summary/Abstract The poly-ubiquitin (UB) chains attached to the cellular proteins carry diverse signals that regulate virtually all aspect of cell biology including protein stability, enzyme catalysis, and gene activation. The poly UB chain is built by UB transfer through a E1-E2-E3 enzymatic cascade to the cellular proteins. E1 activates UB and loads it on E2 as a thioester conjugate. The E2~UB conjugate is then bound to E3 that recruit cellular proteins for the UB transfer reaction. There are more than 45 E2s and 1,000 E3s in the cell. Each E2 can pair with multiple E3s. Each E3 can pair with different E2s to transfer UB to multiple target proteins in the cell. Due to the complex cross reactivity among the E2 and E3 enzymes, it has been a significant challenge to identify the substrate proteins of a specific E3. It has also not been possible to compare the substrate pool of the same E3 pairing with different E2s in order to reveal the effect of various E2 on the reactivity of E3. The lack of efficient approaches to profile the substrate specificity of an E3 enzyme or an E2-E3 pair prevents the elucidation of the function of E2 and E3 enzymes in the regulatory circuits of the cell. As a result the ubiquitination targets of many E3 enzymes such as Mmd2 and Smurf2 are poorly characterized despite their strong connection with cancer or neurodegenerative diseases. In this application, we plan to develop a method to profile the substrate specificities of a E3 or a E2-E3 pair in the protein ubiquitination reaction. We have proven by preliminary results that we can use protein engineering methods based on phage display, structure-based design and site-directed mutagenesis to create pair wise interactions between UB and E1, and E1 and E2. These engineered pairs shares no cross reactivity with native E1 and E2 enzymes in the cell and we have demonstrated that the engineered E1 (xE1) can transfer engineered UB (xUB) to a specific E2 (xE2) that is engineered to match with xE1. We plan to engineer specific xE2-xE3 interactions so that a UB transfer pathway through the xE1-xE2-xE3 cascade can be installed in the cell to transfer xUB to the substrate proteins of an engineered E3 (xE3). xUB is fused to an affinity tag to allow the identification of the ubiquitination targets of xE3 by affinity purification. The orthogonality of the xE1-xE2-xE3 cascade with their native counterparts ensures xUB can only be utilized by the xE2-xE3 pair to be attached to the substrate proteins of xE3. We thus call such a method to profile E3 substrate specificity “Orthogonal UB Transfer” or “OUT”. By engineering various E2s to create specific xE2-xE3 pairs with the same xE3, we will be able to use OUT to compare the difference of the UB transfer targets of various xE2-xE3 pairs in order to reveal the regulatory role of E2 on E3. We plan to use OUT to profile the ubiquitination targets of Mdm2 and Smurf2 in combination with various E2s. Overall our work will generate a platform to map the UB transfer networks associated with key E3 enzymes for the discovery of new signal transduction pathways in the cell.

项目摘要/摘要 附着在细胞蛋白上的多泛素（UB）链带有调节的不同信号 实际上，细胞生物学的所有方面，包括蛋白质稳定性，酶催化和基因激活。多个 UB链是通过UB通过E1-E2-E3酶级联反应到细胞蛋白的转移而构建的。 E1激活 UB并将其加载在E2上，作为硫酯结合。然后，E2〜UB共轭与募集的E3绑定 UB转移反应的细胞蛋白。细胞中有超过45个E2和1,000个E3。每个 E2可以与多个E3配对。每个E3可以与不同的E2配对以将UB传递到多个目标 细胞中的蛋白质。由于E2和E3酶之间的复杂交叉反应性，它一直是 确定特定E3的底物蛋白的重大挑战。也不可能 比较同一E3配对的基板池与不同的E2，以揭示各种效果 E2关于E3的反应性。缺乏有效的方法来介绍E3的底物特异性 酶或E2-E3对可防止调节中E2和E3酶的功能 电池的电路。结果，许多E3酶的泛素化靶标，例如MMD2和Smurf2 特征很差的使命他们与癌症或神经退行性疾病的牢固联系。 在此应用程序中，我们计划开发一种介绍E3或E2-E3的底物规范的方法 蛋白质泛素化反应中配对。我们已经通过初步结果证明了我们可以使用蛋白质 基于噬菌体显示，基于结构的设计和定向诱变的工程方法 在UB和E1以及E1和E2之间创建对的互动。这些工程配对没有交叉 与细胞中天然E1和E2酶的反应性，我们已经证明了工程的E1（XE1） 可以将工程的UB（XUB）传输到与XE1匹配的特定E2（XE2）。我们计划 工程师特定的XE2-XE3相互作用，以便通过XE1-XE2-XE3 CASCADE的UB传输途径可以 将其安装在单元格中以将XUB传递到工程E3（XE3）的底物蛋白。 xub融合到 一个亲和力标签，允许通过亲和力纯化识别XE3的泛素化靶标。这 XE1-XE2-XE3级联的正交性及其本地对应物可确保只能使用XUB 由XE2-XE3对附着在XE3的底物蛋白上。因此，我们将这种方法称为个人资料 E3底物特异性“正交UB转移”或“ OUT”。通过设计各种E2来创建特定 XE2-XE3与相同的XE3对，我们将能够用来比较UB传输的差异 为了揭示E2在E3上的调节作用，各种XE2-XE3对的靶标。我们计划用来 配置MDM2和SMURF2的泛素化靶标与各种E2结合使用。总体而言，我们的工作 将生成一个平台来映射与键E3酶相关的UB传输网络以进行发现 细胞中新的信号传输途径的。