Regulation of intracellular calcium signaling and phosphoinositide metabolism by IRBIT

项目摘要 癫痫和阿尔茨海默病影响着美国900多万人。尽管 尽管经过了几十年的研究，我们仍然缺乏对这些疾病分子机制的理解。 控制这些疾病和其他相关的神经病理状况。我们建议，最近 发现的蛋白质，伊尔比特（IP 3 R结合蛋白与肌醇1，4，5-三磷酸释放），功能 作为两种主要信号通路的调节剂，基于脂质的磷酸肌醇信号通路和 钙离子从细胞内的细胞器进入细胞质，这两个都被证明是 严重参与这些神经系统疾病和其他病理条件。基于此 假设，我们将表征伊尔比特如何调节这些信号通路，通过（i）表征 野生型和伊尔比特-/-人胚肾（HEK 293）细胞中的钙信号传导，（ii）利用这些 细胞系，通过脂质质谱法分析磷酸肌醇组成，并确定 使用荧光磷酸肌醇结合蛋白在肝中的磷酸肌醇代谢动力学 细胞共聚焦成像实验，和（iii）使用无偏的全基因组表达分析， 在野生型和伊尔比特-/-人胚肾细胞中的RNA-Seq，以评估伊尔比特的活性 影响参与这两个信号网络的基因的表达。来自拟议的数据 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，伊尔比特， 调节两个信号通路，这两个信号通路在几乎所有哺乳动物细胞中都至关重要。 这增强了对这些信号通路及其调节机制的理解， 有可能进一步了解使人衰弱的疾病的分子本质， 癫痫和阿尔茨海默氏症，使我们能够为受影响的患者制定更好的治疗策略， 这些条件。

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.