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)复合体控制。BCR交联激活了最近发现的一个强大的信号系统，该系统由ER膜蛋白STIM1和STIM2介导。STIM蛋白直接与PM相互作用，暴露出一个反应区域，该区域强烈地结合和捕获一个专门的通道蛋白家族Orai1、Orai2和Orai3。Orai1通道是高度选择性的钙通道，一旦直接与STIM感应器结合，就会被激活，将钙离子引导到连接的胞浆空间。这种高度受控的钙离子进入是至关重要的，原因有两个：(I)补充内质网内的钙离子，防止细胞因蛋白质错误折叠而应激，并允许钙释放信号保持；(Ii)提供更长时间和空间上定义的钙信号，介导对转录、生长或凋亡的控制。STIM-Orai信号通路在B细胞中具有特殊的意义--钙释放和进入信号的精确协调介导了振荡的钙信号，其幅度和持续时间决定了B细胞如何响应BCR抗原结合经历增殖、无能或凋亡。这项工作结合了分子、生物物理和细胞方法，使用DT40B细胞系和HEK293人肾来源细胞来研究STIM和ORAI蛋白。DT40B细胞保留有功能的BCR偶联信号机制，我们有每种STIM和ORAI蛋白被敲除的线路。利用这些细胞，我们的三个特定目的是：1.研究STIM1和STIM2蛋白在介导钙内流信号中的不同功能。2.研究STIM1、STIM2蛋白与Orai钙通道的相互作用及调控机制。3.检测B细胞STIM-Orai钙信号微环境。这些研究剖析了一种新的钙信号传递过程，该过程基本上与BCR偶联机制有关，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细胞钙信号生成中的中心作用提供了一个新的重要的药理靶点。钙信号的大小和持续时间是B细胞在BCR激活时命运的主要决定因素-细胞分裂、维持，或者在自我识别的情况下，细胞死亡。确定这个长效的钙信号通路的机制并研究其被2-APB的药理修饰，提供了一个靶点，通过它可以改变B细胞的功能和发育，从而有可能控制主要的免疫疾病，包括原发的B细胞缺陷，淋巴增殖性疾病，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.