Control of Calcium Entry Signals in B Cells

描述（由申请方提供）：B细胞的主要作用（抗体产生和抗原消除）由B细胞受体（BCR）复合物控制。BCR交联激活最近发现的由ER膜蛋白STIM 1和STIM 2介导的强大信号传导系统。直接与PM相互作用，STIM蛋白暴露出一个反应域，该反应域贪婪地结合并捕获一个专门的通道蛋白家族Orai 1、Orai 2和Orai 3。Orai 1通道是非常选择性的Ca 2+通道，其在直接结合到STIM传感器后被激活以将Ca 2+离子传导到连接的胞质空间中。这种高度受控的Ca 2+进入是至关重要的，原因有两个：（i）补充ER内的Ca 2+，防止细胞应激蛋白质错误折叠，并允许维持Ca 2+释放信号;（ii）提供更长期和空间限定的Ca 2+信号，介导对转录、生长或凋亡的控制。STIM-Orai信号通路在B细胞中具有特别的意义-Ca 2+释放和进入信号的精确协调介导振荡Ca 2+信号，其幅度和持续时间决定了B细胞如何响应BCR抗原结合以经历增殖、无反应性或凋亡。这项工作结合了分子、生物物理和细胞方法，使用DT 40 B细胞系和HEK 293人肾衍生细胞研究STIM和奥赖蛋白。DT 40 B细胞保留了功能性的BCR偶联信号机制，并且我们有每个STIM和奥赖蛋白被敲除的细胞系。使用这些细胞，我们的三个具体目标是：1。研究STIM 1和STIM 2蛋白在介导Ca 2+内流信号中的不同功能作用。 2.确定STIM 1和STIM 2蛋白如何与奥赖Ca 2+通道相互作用并控制其功能。 3：检测B细胞中STIM-Orai Ca 2+信号微环境。这些研究剖析了一种新的钙离子信号传导过程，从根本上与BCR偶联机制有关，对B细胞功能发挥关键的调控作用。STIM-Orai信号通路及其在B细胞中的Ca 2+信号产生中的中心作用提供了新的且重要的药理学靶点。Ca 2+信号的大小和持续时间是B细胞命运的主要决定因素，以响应BCR激活-细胞分裂、维持，或在自我识别的情况下，细胞死亡。定义这种长效Ca 2+信号通路的机制运作并检查其通过硼酸盐2-AP B的药理学修饰，提供了一个靶点，通过该靶点可以改变B细胞功能和发育，从而提供控制主要免疫疾病的潜力，包括原发性B细胞缺陷、淋巴细胞增生性疾病（例如慢性淋巴细胞白血病）和自身免疫性疾病。

DESCRIPTION (provided by applicant): The primary role of B cells, antibody production and antigen elimination, is controlled by the B cell receptor (BCR) complex. BCR crosslinking activates a recently discovered and powerful signaling system mediated by the ER membrane proteins, STIM1 and STIM2. Interacting directly with the PM, STIM proteins expose a reactive domain that avidly binds and traps a specialized family of channel proteins, Orai1, Orai2, and Orai3. The Orai1 channels are exceedingly selective Ca2+ channels that, upon direct binding to STIM sensors, become activated to conduct Ca2+ ions into the junctional cytosolic space. This highly controlled entry of Ca2+ is crucial for two reasons: (i) to replenish Ca2+ within the ER preventing cell stress from protein misfolding, and allowing Ca2+ release signals to be maintained; (ii) to provide longer term and spatially defined Ca2+ signals mediating control over transcription, growth, or apoptosis. The STIM-Orai signaling pathway has particular significance in B cells - the precise coordination of Ca2+ release and entry signals mediates oscillatory Ca2+ signals, the amplitude and duration of which determine how B cells respond to BCR antigen-binding to undergo either proliferation, anergy, or apoptosis. The work combines molecular, biophysical, and cellular approaches to study STIM and Orai proteins using the DT40 B cell line and HEK293 human kidney-derived cells. DT40 B cells retain functional BCR-coupled signaling machinery and we have lines in which each STIM and Orai protein is knocked out. Using these cells our three specific aims are: 1. To examine the distinct functional roles of STIM1 and STIM2 proteins in mediating Ca2+ entry signals. 2. To ascertain how STIM1 and STIM2 proteins interact with and control Orai Ca2+ channels. 3: To examine the STIM-Orai Ca2+ signaling microenvironment in B cells. The studies dissect a novel Ca2+ signaling process fundamentally connected to the BCR-coupled machinery, exerting crucial regulatory control over B cell function. The STIM-Orai signaling pathway and its central role in Ca2+ signal generation in B cells provides a novel and important pharmacological target. The size and duration of Ca2+ signals are primary determinants of B cell fate in response to BCR activ- ation - cell division, maintenance, or, in the case of self-recognition, cell death. Defining the mechanistic operation of this long-acting Ca2+ signaling pathway and examining its pharmacological modification by the borate, 2-APB, provides a target through which B cell function and development can be modified providing the potential to control major immunological diseases including primary B cell deficiencies, lymphoproliferative disorders such as chronic lymphocytic leukemia, and autoimmune diseases.