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作用。斯坦福大学的Tobias Meyer实验室最近的研究已经将STIM确定为Ca 2+传感器，其监测内质网Ca 2+水平并控制ER-质膜连接位点处的钙内流。STIM的发现及其激活的Ca 2+内流通道的随后鉴定完成了受体触发的Ca 2+信号传导的先前分子模型。然而，这些模型有两个主要不足。1)这些成分的绝对浓度是未知的，2）这些模型不够稳健，无法解释细胞如何在施加显著扰动时仍能保持信号传导能力。我的第一个具体目标是使用选择性反应监测（SRM）质谱方法来改善钙信号模型。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，以了解反馈回路如何以稳定基础Ca 2+信号并允许受体触发的Ca 2+信号被稳健诱导的方式改变信号转导组分的表达。我的第二个具体目标是将联合收割机SRM质谱与交联方法相结合，以解决钙信号网络中的另一个基本问题-确定当STIM易位到内质网（ER）-质膜连接处时，在ER中钙耗尽时，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则，这将显着改善控制细胞中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.