Eukaryotic Cell Signaling: Modulation of Molecular Microenvironments through the Scaffold Protein Asc1/RACK1

真核细胞信号转导：通过支架蛋白 Asc1/RACK1 调节分子微环境

• 批准号: 236865030
• 负责人: Dr. Oliver Valerius
• 金额: --
• 依托单位: Abteilung für Molekulare Mikrobiologie und Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/236865030?language=en
• 关键词: Eukaryotic; Cell; Signaling; Modulation; Molecular

In eukaryotes, the scaffold protein Asc1/RACK1 connects ribosomes with the network of signal transduction. Cell proliferation and differentiation are significantly affected by Asc1/RACK1. Both the ribosomal mRNA translation rate and the homeostasis of ribosomes are influenced by the WD40 repeat propeller protein. Asc1/RACK1 locates to the regulatory head region of the ribosomal 40S subunit and is substrate for phosphorylation. This project aims at the identification of kinases phosphorylating Asc1 in Saccharomyces cerevisiae, and the impact of stress/signal-induced Asc1 phosphorylation on its molecular proximity. The composition of protein modules at the head region of the ribosomal 40S subunit will be analyzed with the Biotin-dependent IDentification (BioID) approach, e.g. from the perspective of ribosomal neighbors (i.e. Rps2). Using the BioID approach with hTERT-RPE1 cells the modular composition of the RACK1 microenvironment in human cells will be compared to that of Asc1 in S. cerevisiae. The impact of Epidermal Growth Factor (EGF) and EGF receptor-inhibitors on the molecular proximity of RACK1 will be also studied in hTERT-RPE1 cells. In yeast, also context-specific sub-complexes of Asc1 with already identified proximal proteins (e.g. Bre5-Ubp3, Hel2, Stm1, Def1) will be characterized using Split-BioID and Biomolecular Fluorescence Complementation (BiFC). Moreover, signals for Asc1-homodimer formation and its molecular consequences will be studied. The impact of Asc1 on the ubiquitylation of proximal but also non-proximal proteins will be analyzed using a global ubiquitylome approach. The results of this project will lead to a substantial improvement of the general understanding of the molecular action of beta-propeller proteins during signal transduction and of Asc1/RACK1-specific ribosomal regulation of gene and protein expression.

在真核生物中，支架蛋白Asc1/RACK1连接核糖体和信号转导网络。Asc1/RACK1显著影响细胞的增殖和分化。WD40重复螺旋桨蛋白影响核糖体的mRNA转译率和核糖体的动态平衡。Asc1/RACK1位于核糖体40S亚单位的调控头部区域，是磷酸化的底物。本项目旨在鉴定酿酒酵母中Asc1磷酸化的激酶，以及胁迫/信号诱导的Asc1磷酸化对其分子亲和力的影响。将使用生物素依赖鉴定(BioID)方法，例如从核糖体邻居(即Rps2)的角度，分析核糖体40S亚单位头部区域的蛋白质模块的组成。对hTERT-RPE1细胞使用BioID方法，人类细胞中RACK1微环境的模块组成将与酿酒酵母中Asc1的模块组成进行比较。还将研究表皮生长因子(EGF)和EGF受体抑制剂对hTERT-RPE1细胞中RACK1分子接近程度的影响。在酵母中，也将使用Split-BioID和生物分子荧光互补(BIFC)来表征Asc1与已识别的近端蛋白(例如Bre5-Ubp3、Hel2、Stm1、Def1)的上下文特定的亚复合体。此外，还将研究Asc1-Homodimer形成的信号及其分子后果。Asc1对近端和非近端蛋白质泛素化的影响将使用全球泛素组方法进行分析。该项目的结果将大大提高对β-螺旋桨蛋白在信号转导过程中的分子作用以及Asc1/RACK1特异性核糖体对基因和蛋白质表达的调控的总体理解。