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

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起着重要的作用。托拜厄斯·迈耶在斯坦福大学的实验室最近的研究发现，STIM是一种钙传感器，可以监测内质网钙离子水平，并控制内质网-质膜连接部位的钙内流。STIM的发现及其激活的钙离子内流通道的发现完善了先前受体触发的钙信号传导的分子模型。然而，这些模型有两个主要缺陷。1)组分的绝对浓度是未知的，2)模型不够健壮，无法解释细胞如何在受到显著干扰时仍能保持信号传递能力。我的第一个具体目标是使用选择性反应监测(SRM)质谱学方法来改进钙信号模型。我将对钙信号网络中关键成分的蛋白质拷贝数进行定量。然后，我将进行系统扰动，以了解反馈环路如何改变信号转导成分的表达，从而稳定基本的钙信号，并允许受体触发的钙信号被稳健地诱导。我的第二个具体目标是将SRM质谱学与交联法相结合，以解决钙信号网络中的另一个基本问题-识别当STIM移位到内质网(ER)时哪些蛋白质与STIM相互作用-内质网中钙耗尽时的质膜连接。总之，这些结果将有助于发现新的调控原理，这些原理将显著改进控制细胞内钙信号的信号事件的建模和重建。

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.