Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

项目摘要/摘要 泛素(UB)通过E1-E2-E3级联途径介导了几乎所有方面的信号转导。 细胞生物学。E3UB连接酶催化UB从E2S转移到底物蛋白，并最终决定 泛素化反应的目标、位置和时间。识别底物蛋白质是关键 阐明E3的生物学功能。然而，分析E3底物的ITS一直是一个巨大的挑战 由于E3底物复合体的瞬时形成和不同底物之间的交叉反应而产生的特异性 E3S。我们的长期目标是阐明E3催化的蛋白质泛素化在细胞生物学中的重要作用 和疾病。为了实现这一目标，我们开发了一种方法，我们称之为“正交UB传输(OUT)” 鉴定E3的直接泛素化靶点。在这种创新方法中，工程UB(XUB)是 通过Xe1-Xe2-XE3的工程级联专门转移到特定E3的底物(“x”) 指定不与细胞中的本地伙伴交叉创造的工程酶)。我们已经建造了 级联的Hect E3 E6AP和U-box E3 E4B和芯片，以描述它们的基板特性和 已被证实是鉴定E3底物的有效平台。此应用程序目标是生成 Out与Ring E3s、Cbl-b和Parkin级联以鉴定其底物蛋白。我们也会追随线索 在OUT产生的E6AP和CHIP的底物谱中，建立这些E3在细胞周期中的作用 以及癌细胞的侵袭。我们的中心假设是，E3的外部级联可以识别新的监管 E3与底物的关系，阐明E3在细胞信号转导中的作用。这样的假设是 由我们强有力的初步数据支持，证明了OUT在描述泛素化方面的可行性 E6AP、E4B和CHIP的目标。我们提议的工作的基本原理是，单元中的600个Ring E3使用了 高度同源的Ring结构域与E2结合，并介导UB从E2向其底物蛋白的转移。一次 我们用Ring E3s Cbl-b和Parkin开发了一个平台，我们可以使用类似的蛋白质工程策略来 为其他Ring E3搭建展示其生物学功能的OUT平台。我们将追求三个具体目标： 1)将外级联延伸到Ring E3S Cbl-b和Parkin，以分析其底物蛋白质；2)延伸 3)研究E6AP和CHIP的功能 基于OUT生成的它们的底物分布。我们工作的预期结果是 OUT平台可分析所有类型E3的泛素化靶点，包括Hect、U-box和Ring类型。基座 在了解其底物特异性的基础上，我们将阐明cbl-b在调节nk细胞活性中的作用。 抗癌细胞，揭示Parkin在肿瘤抑制中的机制，并确定Bruce在其中的作用 胞质分裂。我们将在细胞周期控制和癌症中建立由E6AP和芯片介导的新的细胞电路 细胞入侵。我们的工作的积极影响是，我们将开发OUT平台，供其他研究人员 插入他们感兴趣的E3，并通过分析它们的底物专一性将E3映射到细胞信号网络上。

Project Summary / Abstract Ubiquitin (UB) transfer through the E1-E2-E3 cascade mediates signal transduction in almost all aspects of cell biology. E3 UB ligases catalyze UB transfer from E2s to the substrate proteins and ultimately decide the targets, location, and timing of the ubiquitination reaction. Identifying the substrate proteins is the key to elucidating the biological functions of an E3. However, its has been a significant challenge to profile E3 substrate specificity because of the transient formation of E3-substrate complex and the cross-reactivity among various E3s. Our long-term goal is to elucidate the important roles of E3-catalyzed protein ubiquitination in cell biology and diseases. Working toward this goal, we developed a method that we called “orthogonal UB transfer (OUT)” to identify the direct ubiquitination targets of an E3. In this innovative method, an engineered UB (xUB) is exclusively transferred through an engineered cascade of xE1-xE2-xE3 to the substrates of a specific E3 (“x” designates engineered enzymes free of cross creativities with native partners in the cell). We have constructed OUT cascades of HECT E3 E6AP and U-box E3 E4B and CHIP to profile their substrate specificities and validated OUT as an efficient platform to identify E3 substrates. The objective of this application is to generate OUT cascades with Ring E3s Cbl-b and Parkin to identify their substrate proteins. We will also follow the leads in the substrate profiles of E6AP and CHIP generated by OUT to establish the roles of these E3s in cell cycle and cancer cell invasion. Our central hypothesis is that the OUT cascades of E3s can identify new regulatory relationships between the E3 and substrates and elucidate the roles of E3 in cell signaling. Such a hypothesis is supported by our strong preliminary data demonstrating the feasibility of OUT in profiling the ubiquitination targets of E6AP, E4B, and CHIP. The rationale of our proposed work is that the 600 Ring E3s in the cell used a highly homologous Ring domain to bind to E2 and mediate UB transfer from E2 to their substrate proteins. Once we develop OUT platform with Ring E3s Cbl-b and Parkin, we can use similar protein engineering strategies to build the OUT platform for other Ring E3s to reveal their biological functions. We will pursue three specific aims: 1) Extending the OUT cascades to Ring E3s Cbl-b and Parkin to profile their substrate proteins; 2) Extending the OUT cascade to BRUCE to investigate its role in cytokinesis; 3) Studying the function of E6AP and CHIP based on their substrate profile generated by OUT. The expected outcome of our work is the development of the OUT platform to profile ubiquitination targets of all class of E3s including HECT, U-box and Ring types. Based on the knowledge of their substrate specificity, we will elucidate the role of Cbl-b in regulating NK cell activity against cancer cells, reveal the mechanism of Parkin in tumor suppression, and define the role the BRUCE in cytokinesis. We will establish new cellular circuits mediated by E6AP and CHIP in cell cycle control and cancer cell invasion. The positive impact of our work is that we will develop the OUT platform for other researchers to plug in their E3s of interest and map the E3s on the cell signaling network by profiling their substrate specificity.