Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

描述（由申请人提供）：附着于细胞蛋白的聚泛素（UB）链携带多种信号，这些信号调节细胞生物学的几乎所有方面，包括蛋白质 稳定性、酶催化和基因激活。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。然后E2-UB缀合物与E3结合，E3募集细胞蛋白用于UB转移反应。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。由于E2和E3酶之间复杂的交叉反应性，鉴定特定E3的底物蛋白一直是一个重大挑战。我也不可能比较相同E3与不同E2配对的底物库，以揭示不同E2对E3反应性的影响。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酶在细胞调节回路中的功能的阐明。因此，尽管许多E3酶如Mmd 2和Smurf 2与癌症或神经退行性疾病有很强的联系，但其泛素化靶点的特征很差。 在本申请中，我们计划开发一种方法来分析蛋白质泛素化反应中E3或E2-E3对的底物特异性。我们已经通过初步的结果证明，我们可以使用基于噬菌体展示，基于结构的设计和定点突变的蛋白质工程方法来创建UB和E1，E1和E2之间的成对相互作用。这些工程对与细胞中的天然E1和E2酶没有交叉反应性，我们已经证明工程E1（xE 1）可以将工程UB（xUB）转移到与xE 1匹配的特定E2（xE 2）。我们计划设计特定的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，将xUB转移到工程化E3（xE 3）的底物蛋白。xUB与亲和标签融合，以便通过亲和纯化来识别xE 3的遍在蛋白化靶点。xE 1-xE 2-xE 3级联与其天然对应物的正交性确保xUB只能被xE 2-xE 3对利用以连接到xE 3的底物蛋白。因此，我们将这种分析E3底物特异性的方法称为“正交UB转移”或“OUT”。通过工程化不同的E2以产生具有相同xE 3的特定xE 2-xE 3对，我们将能够使用OUT来比较不同xE 2-xE 3对的UB转移靶点的差异，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。总的来说，我们的工作将产生一个平台来映射与关键E3酶相关的UB转移网络，以发现细胞中新的信号转导途径。

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.