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（由申请人提供）：连接到细胞蛋白的多泛素（Ub）链带有多种信号，这些信号几乎调节细胞生物学的所有方面 稳定性，酶催化和基因激活。通过UB通过E1-E2-E3酶级联反应到细胞蛋白的UB转移构建。 E1激活UB并将其加载到E2上作为硫酯结合物。然后，E2〜UB偶联物与E3结合，该E3募集了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转移到细胞中的多个靶蛋白。由于E2和E3酶之间的复杂交叉反应性，确定特定E3的底物蛋白是一个重大挑战。我也无法将同一E3配对的底物与不同的E2进行比较，以揭示各种E2对E3反应性的影响。缺乏有效的方法来介绍E3酶或E2-E3对的底物特异性，从而阻止了细胞调节回路中E2和E3酶的功能。作为重塑，尽管它们与癌症或神经退行性疾病的牢固联系，但许多E3酶（例如MMD2和SMURF2）的泛素化靶标具有很差的特征。 在此应用中，我们计划开发一种方法，以介绍蛋白质泛素化反应中E3或E2-E3对的底物特异性。我们已经通过初步结果证明，我们可以基于噬菌体显示，基于结构的设计和定位的诱变来使用蛋白质工程方法，以在UB和E1以及E1和E2之间创建配对的互动。这些工程配对与细胞中的天然E1和E2酶没有交叉反应性，我们已经证明了工程的E1（XE1）可以将工程的UB（XUB）传递到与XE1相匹配的特定E2（XE2）。我们计划设计特定的XE2-XE3相互作用，以便可以在单元格中安装通过XE1-XE2-XE3级联反应的UB传输途径，以将XUB传递到工程E3（XE3）的底物蛋白。 XUB融合到亲和力标签，以允许通过Affinity Purificaton识别XE3的泛素化靶标。 XE1-XE2-XE3与本机对应物的正交性可确保只能通过XE2-XE3对才能使用XUB，以附着在XE3的底物蛋白上。因此，我们将这种方法称为E3底物特异性“正交UB传输”或“ OUT”。通过设计各种E2s以创建具有相同XE3的特定XE2-XE3对，我们将能够使用各种XE2-XE3对的UB传输目标的差异，以揭示E2在E3上的调节作用。我们计划使用与各种E2结合使用MDM2和SMURF2的泛素化靶标。总体而言，我们的工作将生成一个平台，以绘制与关键E3酶相关的UB传输网络，以发现单元格中新的信号传输途径。