Determination of Protein copy number, slow feedback loops, and protein-protein interaction in the store operated calcium signalling network

确定商店操作的钙信号网络中的蛋白质拷贝数、慢反馈环和蛋白质-蛋白质相互作用

• 批准号: 184485879
• 负责人: Professor Dr. Robert Ahrends, Ph.D.
• 金额: --
• 依托单位: Institut für Analytische Chemie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/184485879?language=en
• 关键词: Determination; Protein; copy; number; slow

Calcium signaling plays a fundamental role in cellular processes that range from muscle contraction and memory formation to triggering activation and differentiation of immune cells. Recent studies in Tobias Meyer’s laboratory at Stanford University have identified STIM as the Ca2+ sensor that monitors endoplasmic reticulum Ca2+ levels and controls calcium influx at ER-plasma membrane junction sites. The discovery of STIM and subsequent identification of the Ca2+ influx channel it activates completed previous molecular models for receptor-triggered Ca2+signaling. However, there are two major shortfalls of the models. 1) The absolute concentrations of the components are not known, and 2) the models are not sufficiently robust, failing to explain how cells can retain signaling capabilities even when significant perturbations are applied. My first specific aim is to use a selective reaction monitoring (SRM) mass spectrometry approach to improve the calcium signaling models. I will quantitate protein copy number of the key components in the calcium signaling network. Then I will carry out system perturbations to understand how feedback loops alter the expression of the signal transduction components in a manner that stabilizes basal Ca2+ signals and allows receptor-triggered Ca2+ signaling to be robustly induced. My second specific aim is to combine SRM mass spectrometry with a cross-linking approach to address another fundamental problem in calcium signaling network – identifying which proteins interact with STIM when it translocates to endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of calcium in the ER. Together, these results will help to discover novel regulatory principles that will significantly improve the modeling and reconstruction of the signaling events controlling calcium signaling in cells.

钙信号在从肌肉收缩和记忆形成到触发免疫细胞的激活和分化的细胞过程中发挥着重要作用。斯坦福大学 Tobias Meyer 实验室最近的研究发现 STIM 是一种 Ca2+ 传感器，可以监测内质网 Ca2+ 水平并控制内质网 - 质膜连接处的钙流入。 STIM 的发现以及随后对其激活的 Ca2+ 流入通道的识别完成了先前受体触发的 Ca2+ 信号转导的分子模型。然而，这些模型有两个主要缺陷。 1）各成分的绝对浓度未知，2）模型不够稳健，无法解释细胞如何在施加显着扰动时保留信号传导能力。我的第一个具体目标是使用选择性反应监测（SRM）质谱方法来改进钙信号传导模型。我将定量钙信号网络中关键成分的蛋白质拷贝数。然后，我将进行系统扰动，以了解反馈环路如何以稳定基础 Ca2+ 信号并允许受体触发的 Ca2+ 信号传导被稳健诱导的方式改变信号转导组件的表达。我的第二个具体目标是将 SRM 质谱与交联方法相结合，解决钙信号网络中的另一个基本问题 - 识别当 STIM 在 ER 中钙耗尽后易位到内质网 (ER)-质膜连接处时哪些蛋白质与 STIM 相互作用。总之，这些结果将有助于发现新的调控原理，从而显着改善控制细胞中钙信号传导的信号事件的建模和重建。