Control of Calcium Entry Signals in B Cells

描述（由申请人提供）：B细胞的主要作用是产生抗体和消除抗原，由B细胞受体（BCR）复合物控制。BCR交联激活了最近发现的由内质网膜蛋白STIM1和STIM2介导的一个强大的信号系统。STIM蛋白直接与PM相互作用，暴露出一个活性结构域，该结构域贪婪地结合和捕获一个特殊的通道蛋白家族，Orai1， Orai2和Orai3。Orai1通道是极具选择性的Ca2+通道，在直接与STIM传感器结合后，被激活以将Ca2+离子引导到连接的细胞质空间。这种高度控制的Ca2+进入至关重要，原因有两个：(i)在内质网内补充Ca2+，防止蛋白质错误折叠引起的细胞应激，并允许Ca2+释放信号得到维持；（ii）提供更长期和空间定义的Ca2+信号，介导转录、生长或凋亡的控制。STIM-Orai信号通路在B细胞中具有特别重要的意义- Ca2+释放和进入信号的精确协调介导了振荡Ca2+信号，其振幅和持续时间决定了B细胞如何响应BCR抗原结合以进行增殖，能量或凋亡。这项工作结合了分子、生物物理和细胞方法，利用DT40 B细胞系和HEK293人肾源性细胞研究STIM和Orai蛋白。DT40 B细胞保留功能性bcr偶联信号机制，我们有敲除每种STIM和Orai蛋白的细胞系。利用这些细胞，我们有三个具体目的：研究STIM1和STIM2蛋白在介导Ca2+进入信号中的不同功能作用。2. 确定STIM1和STIM2蛋白如何与Orai Ca2+通道相互作用和控制。3：检测B细胞中STIM-Orai Ca2+信号微环境。这些研究剖析了一种与bcr偶联机制基本相关的新型Ca2+信号传导过程，对B细胞功能施加关键的调节控制。STIM-Orai信号通路及其在B细胞Ca2+信号产生中的核心作用提供了一个新的重要的药理靶点。Ca2+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，或者，在自我识别的情况下，细胞死亡。定义这种长效Ca2+信号通路的机制运作，并通过硼酸盐2-APB检查其药理学修饰，提供了一个靶标，通过该靶标可以修饰B细胞的功能和发育，提供了控制主要免疫疾病的潜力，包括原发性B细胞缺陷、淋巴增生性疾病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.