Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

项目总结/摘要 泛素（UB）通过E1-E2-E3级联传递介导几乎所有方面的信号转导。 细胞生物学E3 UB连接酶催化UB从E2 s转移到底物蛋白，并最终决定UB的功能。 泛素化反应的目标、位置和时间。识别底物蛋白是 阐明E3的生物学功能。然而，对E3基底进行轮廓化一直是一个重大挑战 由于E3-底物复合物的瞬时形成和各种之间的交叉反应性， E3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，我们开发了一种称为“正交UB传输（OUT）”的方法。 来鉴定E3的直接泛素化靶点。在这种创新方法中， 通过xE 1-xE 2-xE 3的工程级联专门转移到特定E3（“x”）的底物上， 表示与细胞中的天然配偶体没有交叉创造性的工程化酶）。我们已经构建 HECT E3 E6 AP和U-box E3 E4 B和CHIP的OUT级联，以分析其底物特异性， 验证OUT作为鉴定E3底物的有效平台。此应用程序的目标是生成 OUT级联与环E3的Cbl-b和帕金，以确定其底物蛋白。我们也会追踪线索 在OUT产生的E6 AP和CHIP的底物谱中，以确定这些E3在细胞周期中的作用 和癌细胞的侵袭。我们的中心假设是，E3的OUT级联可以识别新的调控因子， E3与底物之间的关系，阐明E3在细胞信号转导中的作用。这样的假设是 我们强有力的初步数据证明了OUT在分析泛素化过程中的可行性 E6 AP、E4 B和CHIP的目标。我们所提出的工作的基本原理是，单元中的600个Ring E3使用了 与E2结合并介导UB从E2转移到其底物蛋白的高度同源的环结构域。一旦 我们用环E3 s Cbl-b和Parkin开发OUT平台，我们可以使用类似的蛋白质工程策略， 为其他环E3构建OUT平台，揭示其生物学功能。我们将追求三个具体目标： 1)将OUT级联延伸至环E3 Cbl-b和Parkin以分析其底物蛋白; 2）将OUT级联延伸至环E3 Cbl-b和Parkin以分析其底物蛋白; OUT级联到布鲁斯，探讨其在胞质分裂中的作用; 3）研究E6 AP和CHIP的功能 基于由OUT生成的它们的衬底轮廓。我们工作的预期成果是开发 OUT平台，用于分析所有E3类的泛素化靶标，包括HECT、U-box和Ring类型。基于 基于对它们底物特异性的了解，我们将阐明Cbl-b在调节NK细胞活性中的作用 研究了BRUCE对肿瘤细胞的抑制作用，揭示了Parkin在肿瘤抑制中的作用机制，并确定了BRUCE在肿瘤抑制中的作用。 胞质分裂我们将建立E6 AP和CHIP介导的细胞周期调控和癌症的新的细胞回路 细胞入侵我们工作的积极影响是，我们将为其他研究人员开发OUT平台， 插入它们感兴趣的E3，并通过描绘它们的底物特异性将E3定位在细胞信号网络上。

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.