Regulation of intracellular calcium signaling and phosphoinositide metabolism by IRBIT

IRBIT 对细胞内钙信号传导和磷酸肌醇代谢的调节

基本信息

批准号：
10702168
负责人：
Martin Kruse
金额：
$ 41.54万
依托单位：
BATES COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-07 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10702168
关键词：
Action Potentials Affect Alzheimer&apos s Disease Binding Proteins Calcium Calcium Signaling Calcium ion Candidate Disease Gene Catalytic Domain Cell Line Cells Confocal Microscopy Critical Pathways Cytoplasm Data Disease Embryo Endoplasmic Reticulum Epilepsy Family Gene Expression Generations Genes Health Human ITPR1 gene Image Inositol Ions Kidney Kinetics Knowledge Lipid Binding Lipids Mammalian Cell Mass Spectrum Analysis Mediating Metabolism Minor Molecular Nature Neurotransmitters Organelles Parkinson Disease Pathologic Patients Persons Phosphatidylinositol Phosphates Phosphatidylinositols Phospholipids Phosphotransferases Process Proteins Publications Purinoceptor RNA Regulation Research Research Personnel Research Technics Role Severities Signal Pathway Signal Transduction Small Interfering RNA Students System United States Western Blotting antagonist confocal imaging experience experimental study gene product improved inorganic phosphate insight kidney cell knock-down meetings nervous system disorder neuropathology novel novel therapeutic intervention overexpression transcriptome sequencing treatment strategy whole genome

项目摘要

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.

项目概要癫痫和阿尔茨海默病影响着美国超过 900 万人。尽管其严重性和数十年的研究，我们仍然缺乏对其分子机制的了解我们最近建议采取一项措施来控制这些疾病和其他相关的神经病理学病症。发现蛋白质 IRBIT（与肌醇 1,4,5-三磷酸一起释放的 IP3R 结合蛋白）的功能作为两个主要信号传导途径的调节剂，基于脂质的磷酸肌醇信号传导和释放钙离子从细胞内细胞器进入细胞质，这两者都被证明是基于此，严重参与了这些神经系统疾病和其他病理状况。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-/- 人胚肾 (HEK293) 细胞中的钙信号传导，(ii) 利用这些细胞系通过脂质质谱分析磷酸肌醇成分并确定使用荧光磷酸肌醇结合蛋白在活体中进行磷酸肌醇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在野生型和 IRBIT-/- 人胚胎肾细胞中进行 RNA 测序，以评估 IRBIT 的活性影响这两个信号网络中涉及的基因的表达。实验将使我们能够深入了解一种新的机制，通过这种机制，一种蛋白质 IRBIT 调节几乎在所有哺乳动物细胞中都至关重要的两条信号传导途径。增强对这些信号通路及其调节机制的理解具有以下意义：进一步了解衰弱性疾病的分子性质的潜力，例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通过这些条件。