Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

正交泛素转移至 E3 底物特异性

• 批准号: 8867257
• 负责人: HIROAKI KIYOKAWA
• 金额: $ 29.98万
• 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 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8867257
• 关键词: Affect; Affinity; Bacteriophages; Binding; Biological Assay; Biological Process; Boxing; C-terminal; Catalysis; Cell Cycle; Cell physiology; Cells; Cellular biology; Complex; Diabetes Mellitus; Disease; Engineering; Ensure; Enzyme Stability; Enzymes; Excision; Gene Activation; Goals; Health; Human; Human Genome; Hybrids; Individual; Label; Length; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Modification; Neurodegenerative Disorders; Neurons; Pathway interactions; Peptides; Phage Display; Polyubiquitin; Protein Engineering; Proteins; Proteomics; Reaction; Recruitment Activity; Role; Signal Transduction; Signal Transduction Pathway; Site-Directed Mutagenesis; Structure; Substrate Specificity; Testing; Two-Dimensional Gel Electrophoresis; Ubiquitin; Ubiquitination; Work; Yeasts; base; carboxylate; combinatorial; cross reactivity; design; insight; mdm2 protein; mutant; prevent; protein degradation; thioester; ubiquitin-protein ligase; uptake

DESCRIPTION (provided by applicant): 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. I 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 resul 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 purificaton. 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.

描述(申请人提供)：附着在细胞蛋白上的多聚泛素(UB)链携带不同的信号，几乎调控细胞生物学的各个方面，包括蛋白质 稳定性、酶催化和基因激活。多聚UB链是通过UB通过E1-E2-E3酶级联传递到细胞蛋白而构建的。E1激活UB，并将其作为硫酯结合物加载到E2上。然后，E2~UB结合物与E3结合，E3为UB转移反应招募细胞蛋白。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，将UB转移到细胞中的多个靶蛋白上。由于E2和E3酶之间复杂的交叉反应，鉴定特定E3的底物蛋白一直是一个巨大的挑战。我也无法比较同一E3与不同E2配对的底物池，以揭示不同E2对E3反应活性的影响。缺乏有效的方法来分析E3酶或E2-E3对的底物特异性，阻碍了对E2和E3酶在细胞调节电路中的功能的阐明。因此，许多E3酶的泛素化靶点，如Mmd2和SMurf2，尽管它们与癌症或神经退行性疾病有很强的联系，但特征不佳。在这项应用中，我们计划开发一种方法来描述蛋白质泛素化反应中E3或E2-E3对的底物特异性。我们的初步结果证明，我们可以利用基于噬菌体展示、基于结构的设计和定点突变的蛋白质工程方法，在UB和E1之间以及E1和E2之间建立成对的相互作用。这些工程对与细胞中的天然E1和E2酶没有交叉反应，我们已经证明工程E1(XE1)可以将工程UB(XUB)转移到特定的E2(XE2)，该E2(XE2)经过工程设计与XE1匹配。我们计划设计特定的XE2-XE3相互作用，以便通过XE1-XE2-XE3级联的UB转移途径可以安装在细胞内，将XUB转移到工程E3(XE3)的底物蛋白上。将XUB融合到亲和标签上，通过亲和纯化鉴定XE3的泛素化靶标。XE1-XE2-XE3与其天然对应物的正交性确保了xUB只能被XE2-XE3对结合到XE3的底物蛋白上。因此，我们将这种描述E3底物特异性的方法称为“正交UB转移”或“OUT”。通过设计不同的E2来构建具有相同XE3的特定XE2-XE3对，我们将能够用OUT来比较不同XE2-XE3对UB转移靶点的差异，以揭示E2对E3的调控作用。我们计划使用OUT结合各种E2来描述MDM2和S-2的泛素化靶点。总体而言，我们的工作将建立一个平台，以绘制与关键E3酶相关的UB转移网络，以发现细胞中新的信号转导途径。