Regulation of intracellular calcium signaling and phosphoinositide metabolism by IRBIT

项目摘要 癫痫和阿尔茨海默氏症在美国影响着900多万人。尽管 他们的严重性和几十年的研究，我们仍然缺乏对其分子机制的了解 管理这些疾病和其他相关的神经病理情况。我们建议最近的一次 发现的蛋白质Irbit(IP3R结合蛋白与1，4，5-三磷酸肌醇一起释放)具有功能 作为两条主要信号通路的调节者，基于脂质的肌醇磷脂信号转导和释放 钙离子从细胞内细胞器进入细胞质，两者都已被证明是 严重参与这些神经系统疾病和其他病理情况。在此基础上 假设，我们将通过(I)表征IRBIT如何调节这些信号通路 野生型和IRBIT人胚胎肾(HEK293)细胞中的钙信号，(Ii)利用这些信号 脂质联用法分析肌醇磷脂组成及测定细胞系 用荧光肌醇磷脂结合蛋白研究肌醇磷脂在活体内的代谢动力学 细胞共聚焦成像实验，以及(Iii)使用无偏全基因组表达分析，通过 野生型和Irbit人胚胎肾细胞中的RNA-Seq来评估Irbit的活性 影响这两个信号网络中涉及的基因的表达。建议中的数据 实验将使我们深入了解一种新的机制，通过这种机制，一种蛋白质，Irbit， 调节两条在几乎所有哺乳动物细胞中都至关重要的信号通路。 这增强了对这些信号通路及其调节机制的理解 有可能进一步了解致衰性疾病的分子本质，如 癫痫和阿尔茨海默氏症，使我们能够为受影响的患者制定改进的治疗策略 在这些条件下。

Project Summary Epilepsy and Alzheimer’s disease affect more than nine million people in the United States. Despite their severity and decades of research, we still lack an understanding of the molecular mechanisms governing these diseases and other related neuropathological conditions. We propose that a recently discovered protein, IRBIT (IP3R binding protein released with inositol 1,4,5-trisphosphate), functions as a regulator of two major signaling pathways, lipid-based phosphoinositide signaling and release of calcium ions from intracellular organelles into the cytoplasm, that have both been shown to be critically involved in these neurological diseases and other pathological conditions. Based on this hypothesis, we will characterize how IRBIT regulates these signaling pathways by (i) characterizing calcium signaling in wild-type and IRBIT-/- human embryonic kidney (HEK293) cells, (ii) utilizing these cell lines to analyze phosphoinositide composition by lipid mass spectrometry and determine the kinetics of phosphoinositide metabolism using fluorescent phosphoinositide-binding proteins in live- cell confocal imaging experiments, and (iii) using an unbiased whole-genome expression analysis via RNA-Seq in wild-type and IRBIT-/- human embryonic kidney cells to assess how IRBIT’s activity influences expression of genes involved in these two signaling networks. The data from the proposed experiments will allow us to gain insight into a novel mechanism by which one protein, IRBIT, regulates two signaling pathways that are of critical importance in practically all mammalian cells. This enhanced understanding of these signaling pathways and their regulator mechanisms has the potential to further our knowledge about the molecular nature of debilitating diseases such as epilepsy and Alzheimer’s and allow us to develop improved treatment strategies for patients affected by these conditions.