Redox Regulation of Intracellular Calcium Signaling

细胞内钙信号传导的氧化还原调节

• 批准号: 9022475
• 负责人: Gyorgy Hajnoczky
• 金额: $ 35.1万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9022475
• 关键词: Address; Affect; Agonist; Apoptosis; Binding; Biochemical; Biological Assay; C-terminal; Calcium; Calcium Signaling; Cardiovascular Diseases; Cell Death; Cell Fractionation; Cell physiology; Cells; Cellular Stress; Collaborations; Cysteine; Data; Diabetes Mellitus; Disease; Drug or chemical Tissue Distribution; Endoplasmic Reticulum; Experimental Models; Fluorescence; Frequencies; Fructose; Gene Expression; Goals; Health; Heart Diseases; Homo; ITPR1 gene; Image; Inositol; Ion Channel; Life; Ligand Binding Domain; Ligands; Liver; Location; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Metabolism; Methods; Mitochondria; Modeling; Modification; Molecular; Mutate; N-terminal; NADPH Oxidase; Neurodegenerative Disorders; Neurotransmitters; Oxidants; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Play; Production; Property; Protein Isoforms; Proteins; Proteome; Regulation; Role; Signal Transduction; Sirolimus; Somatotropin; Source; Stress; Sulfhydryl Compounds; System; Tacrolimus Binding Proteins; Testing; Tissues; Transmembrane Domain; Work; base; catalase; cell motility; design; endoplasmic reticulum stress; imaging modality; in vivo; mutant; novel; novel therapeutic intervention; palmitoylation; receptor; response

 DESCRIPTION (provided by applicant): An elevation of cytosolic free calcium concentration is an integral component of the mechanism by which cells respond to hormones, growth factors and neurotransmitters. D-myo-inositol 1,4,5-trisphosphate ( IP3 ) is an intracellular messenger mediating the mobilization of Ca2+ from intracellular stores by interaction with an ubiquitous receptor ( IP3R ) that acts as a ligand-gated Ca2+ channel. IP3Rs are redox sensitive channels and are sensitized by oxidative stress. However, the molecular basis of this regulation is poorly understood. Ca2+ released from IP3Rs is locally transmitted to the mitochondria and can stimulate metabolism, and in higher amounts, can also initiate cell death. The overarching hypothesis of this study is that redox modulation of IP3Rs is an important component of the regulation of Ca2+ signals in cell death pathways. The proposal encompasses the following three specific aims: 1] To measure and map redox changes in IP3Rs. We have developed methods to determine the redox state of IP3Rs in vivo which will be used to quantitate the effects of exogenous and endogenous agents causing oxidative stress. Preliminary studies using mass-spectroscopy identify a subset of 11 cysteines that become oxidized in IP3R-1. The type of oxidative modifications occurring will be identified. Redox-sensitive thiols will be mutate and the functional sensitivity to oxidative stress will be assessed. 2] To measure IP3R redox state at the ER/mitochondrial junction. We will test the hypothesis that the pool of IP3Rs located at the ER/mito junction is particularly prone to ROS modifications. We will employ subcellular fractionation and imaging methods utilizing targeted IP3Rs, ROS-sensitive fluorescent proteins, ROS-producing photosensitive probes and targeted catalases. 3] To investigate the role of IP3R redox changes in models of ER stress/apoptosis. We will test the hypothesis that ER-resident NADPH oxidases play an important role in IP3R redox regulation. Liver will be used as an experimental model to induce ER stress. The role of IP3R redox regulation in ER stress pathways activated by fructose will be examined. The long-term goal of the proposal is to obtain a detailed understanding of how oxidative stress impacts intracellular Ca2+ signaling under normal and disease conditions.

 描述（通过应用程序提供）：胞质游离钙浓度的升高是细胞对激素，生长因子和神经递质响应的机制的组成部分。 D-myo-Insitol 1,4,5-三磷酸（IP3）是一种细胞内的使者，通过与无处不在的受体（IP3R）相互作用，可介导Ca2+从细胞内存储中的动员，它充当配体配方的Ca2+通道。 IP3R是氧化还原敏感的通道，并且对氧化应激敏感。但是，该调节的分子基础知之甚少。从IP3RS释放的Ca2+局部传播到线粒体，可以刺激新陈代谢，并且以较高的量也可以启动细胞死亡。这项研究的总体假设是IP3RS的氧化还原调节是细胞死亡途径中Ca2+信号调节的重要组成部分。该提案包括以下三个特定目的：1]测量和映射IP3RS中的氧化还原变化。我们开发了确定体内IP3RS氧化还原状态的方法，该方法将用于定量产生氧化应激的外源性和内源性剂的作用。使用质谱镜检查的初步研究确定了11个半胱氨酸的子集，这些子集被IP3R-1氧化。将确定发生氧化修饰的类型。氧化还原敏感的硫醇将是突变的，并且将评估对氧化应激的功能敏感性。 2]在ER/线粒体连接处测量IP3R氧化还原状态。我们将测试以下假设：位于ER/MITO结的IP3R池特别容易进行ROS修饰。我们将采用靶向IP3R，ROS敏感荧光蛋白，产生ROS的光敏感问题和靶向过氧酶的亚细胞分级和成像方法。 3]研究IP3R氧化还原变化在ER应力/凋亡模型中的作用。我们将检验以下假设：ER居民NADPH氧化物在IP3R氧化还原调节中起重要作用。肝脏将用作诱导ER应力的实验模型。将检查IP3R氧化还原调节在果糖激活的ER应力途径中的作用。该提案的长期目标是详细了解氧化应激如何在正常和疾病条件下影响细胞内Ca2+信号。