Molecular regulation of the CRAC channel signaling pathway

项目摘要/摘要 钙(Ca~(2+))释放激活的Ca~(2+)(CRAC)通道介导钙离子内流 内质网(ER)耗尽，产生对许多细胞过程至关重要的钙信号。 CRAC通道的激活需要Orai和STIM蛋白的相互作用，它们是质膜 钙通道和内质网中的钙感受器；然而，其潜在的分子机制仍然存在 不完全理解。哺乳动物，包括人类，有三个Orai通道(Orai1，Orai2和Orai3)和 两种STIM蛋白(STIM1和STIM2)，而果蝇只有一套Orai和STIM。既有损失也有损失 在人类Orai1和STIM1中发现了导致人类疾病的功能获得突变。这个 Orai2和Orai3的生理作用还不是很清楚，但有新的证据表明 它们可以与Orai1形成异构体通道，其作用可能与Orai1形成的通道不同 和STIM1。我的研究计划的长期目标是研究CRAC通道的分子调控 信号通路以了解CRAC通道不同功能的分子机制 在生理和病理条件下，在不同的组织和细胞中发挥作用。最初，我们正在优先考虑 果蝇Orai-STIM复合体和人CRAC通道启动My的结构与功能研究 研究计划。在这项拟议的研究中，我们的目标是解决(1)生物化学中的关键知识空白 和Orai-STIM相互作用的结构生物学，(2)人类CRAC通道的结构和功能，以及 (3)细胞因子和通道调节剂对CRAC通道的调节。我们将带来创新的方法 并应用首席研究员在结构生物学和离子通道结构-功能研究方面的经验 来解决这些问题。具体地说，我们将确定果蝇Orai-STIM的原子结构 以及人Orai通道和人Orai-STIM复合体的结构。我们还将发展 CRAC通道结构导向功能分析的新功能方法和工具。此外, 我们建议研究小分子调节剂对通道结构和功能的调节，包括 脂类和药理药物。最后，我们将使用功能、生化和结构生物学方法 (包括X射线结晶学和低温电子显微镜)来表征蛋白质-蛋白质 Orai或STIM与多种内源性蛋白调节剂的相互作用。在未来五年，我们的目标是 从机制上对CRAC通道信号通路的调控有更深层次的了解 了解和研究更广泛的信令网络的知识和研究工具的基础 CRAC通道驻留。

Project Summary/Abstract The calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediate Ca2+ influx in response to Ca2+ store depletion in the endoplasmic reticulum (ER), generating Ca2+ signals critical for many cellular processes. Activation of CRAC channels requires the interaction of Orai and STIM proteins, which are the plasma membrane Ca2+ channel and the Ca2+ sensor in the ER membrane; however, the underlying molecular mechanism remains incompletely understood. Mammals, including humans, have three Orai channels (Orai1, Orai2, and Orai3) and two STIM proteins (STIM1 and STIM2), whereas Drosophila has only one set of Orai and STIM. Both loss- and gain-of-function mutations that underlie human diseases have been identified in human Orai1 and STIM1. The physiological roles of Orai2 and Orai3 are less well understood, but there is emerging evidence showing that they could form heteromeric channels with Orai1, which might play roles distinct from channels formed by Orai1 and STIM1. The long-term goal of my research program is to study the molecular regulation of the CRAC channel signaling pathway in order to understand the molecular mechanisms of the diverse functions the CRAC channels play in different tissues and cells under physiological and pathological conditions. Initially, we are prioritizing structure-function studies of the Drosophila Orai-STIM complex and human CRAC channels to launch my research program. In this proposed research, we aim to address key knowledge gaps in (1) the biochemistry and structural biology of the Orai-STIM interaction, (2) the structure and function of human CRAC channels, and (3) regulation of CRAC channels by cellular factors and channel modulators. We will bring innovative approaches and apply the Principal Investigator’s experience in structural biology and ion channel structure-function studies to address these questions. Specifically, we will determine the atomic structure of the Drosophila Orai-STIM complex as well as structures of human Orai channels and human Orai-STIM complexes. We will also develop novel functional approaches and tools for structure-guided functional analysis of CRAC channels. In addition, we propose to study the regulation of the channel structure and function by small molecule modulators including lipids and pharmacological drugs. Finally, we will use functional, biochemical, and structural biology methods (including both X-ray crystallography and cryo-electron microscopy) to characterize the protein-protein interaction of Orai or STIM with a multitude of endogenous protein regulators. In the next five years, we aim to gain a deeper mechanistic understanding of the regulation of the CRAC channel signaling pathway and construct the foundation of knowledge and research tools to understand and study the broader signaling network in which the CRAC channels reside.