Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

项目总结/摘要 多聚泛素（UB）链连接到细胞蛋白携带不同的信号，调节 细胞生物学的几乎所有方面，包括蛋白质稳定性、酶催化和基因活化。聚 UB链是通过E1-E2-E3酶级联将UB转移至细胞蛋白而构建的。E1激活 UB并将其作为硫酯缀合物加载到E2上。E2-UB缀合物然后结合至募集的E3， 用于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传输到多个目标 细胞中的蛋白质。由于E2和E3酶之间复杂的交叉反应性， 这是鉴定特定E3底物蛋白的重大挑战。也不可能 比较具有不同E2的相同E3配对的底物池，以揭示不同E3配对的效应。 E2对E3的反应性。缺乏有效的方法来分析E3的底物特异性 酶或E2-E3对阻止阐明E2和E3酶在调节细胞凋亡中的功能。 细胞的电路。因此，许多E3酶如Mmd 2和Smurf 2的泛素化靶标 尽管它们与癌症或神经退行性疾病有很强的联系，但其特征很差。 在本申请中，我们计划开发一种方法来分析E3或E2-E3的底物特异性， 蛋白质泛素化反应中的一对。我们已经通过初步结果证明，我们可以使用蛋白质 基于噬菌体展示、基于结构的设计和定点诱变的工程方法， 在UB和E1以及E1和E2之间创建成对交互。这些基因对没有交叉 与细胞中天然E1和E2酶的反应性，我们已经证明了工程化的E1（xE 1） 可以将工程化的UB（xUB）转移到被工程化以与xE 1匹配的特定E2（xE 2）。我们计划 设计特异性xE 2-xE 3相互作用，以便通过xE 1-xE 2-xE 3级联的UB转移途径可以 安装在细胞中以将xUB转移到工程化E3（xE 3）的底物蛋白上。xUB融合到 亲和标签，以允许通过亲和纯化鉴定xE 3的泛素化靶标。的 xE 1-xE 2-xE 3级联与它们的本地对应物的正交性确保了xUB只能被利用 通过xE 2-xE 3对连接到xE 3的底物蛋白。因此，我们调用这样的方法来分析 E3底物特异性“正交UB转移”或“OUT”。通过设计各种E2来创建特定的 xE 2-xE 3与相同的xE 3配对，我们将能够使用OUT来比较UB传输的差异 不同xE 2-xE 3对的靶点，以揭示E2对E3的调节作用。我们计划使用OUT来 分析Mdm 2和Smurf 2与各种E2组合的泛素化靶点。我们的工作 将建立一个平台，绘制与关键E3酶相关的UB转移网络， 新的信号传导途径。

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.