Regulation of inositol trisphosphate receptors

肌醇三磷酸受体的调节

基本信息

批准号：
10542722
负责人：
SURESH K JOSEPH
金额：
$ 32.12万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10542722
关键词：
Ablation Address Adenine Nucleotides Agonist Amoeba genus Animal Model Binding Binding Sites Bioenergetics Biological C-terminal CRISPR/Cas technology Calcium Calcium Signaling Carbon Cell Cycle Cell Death Cell Line Cell division Cell membrane Cell physiology Cells Cervix carcinoma Charge Chemicals Chickens Citric Acid Cycle Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Cryoelectron Microscopy D Cells Dermal Diabetes Mellitus Disease Drug or chemical Tissue Distribution Endoplasmic Reticulum Energy Supply Enzymes Exocytosis FRAP1 gene Family Feedback Fertilization Fibroblasts Frequencies Gene Expression Genetic Genetic Transcription Glucose Glutamine Glycolysis Goals Growth Growth Factor Heart Diseases Hela Cells Hepatocyte Hereditary Disease Homo Hormones Human Cell Line ITPR1 gene Inositol Interruption Knock-out Knockout Mice Ligand Binding Domain Ligands Location Malate-Aspartate Shuttle Pathway Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of cervix uteri Mass Spectrum Analysis Measures Mediating Memory Metabolic Metabolism Mitochondria Molecular Molecular Conformation Monitor Muscle Contraction Mutagenesis Mutate Mutation N-terminal NADH Neurodegenerative Disorders Neurotransmitters Normal Cell Oxidoreductase Oxygen Consumption Patients Periodicals Phase Phosphorylation Phosphotransferases Process Production Proliferating Property Protein Isoforms Protein Kinase Proteins Regulation Respiratory Chain Role Signal Transduction Site Staging Structure Technology Testing Therapeutic Tracer Transfection Transmembrane Domain Work biological research cancer cell cell motility cell transformation crosslink design experimental study extracellular inorganic phosphate receptor response transcription factor transmission process

项目摘要

Calcium is a universal messenger controlling a multitude of cellular responses including muscle contraction, exocytosis, memory, fertilization, metabolism, proliferation and cell death. Numerous hormones, neurotransmitters and growth factors stimulate the formation of inositol 1,4,5-trisphosphate ( IP3 ) which acts on a family of receptors ( IP3Rs ) located in the endoplasmic reticulum that function as ligand-gated Ca2+ channels. The depletion of intracellular Ca2+ stores also activates Ca2+ influx mechanisms in the plasma membrane. Thus, the interaction of IP3 with its receptor activates all phases of a Ca2+ signal. IP3Rs are regulated by Ca2+ and phosphorylation but 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. Cancer cells have been proposed to be highly dependent on IP3R- mediated mitochondrial Ca2+ transfer for their survival. The advent of CRISPR/Cas-9 technology has allowed the genetic ablation of all three IP3R isoforms from HEK293 and HeLa cervix carcinoma cells. The proposal is centered on the use of these cell lines to a) study the adaptive mechanisms allowing the cells to survive in the complete absence of Ca2+ signaling and b) to take advantage of a null background for structure-function studies exploring the molecular mechanism by which IP3 opens the channel and the mechanism of feed-back regulation by Ca2+. The two specific aims are: 1] Characterize adaptive mechanisms in IP3R-3KO cells: The impact of a lack of Ca2+ regulation of metabolism will be investigated by measuring several bioenergetic parameters and 13C-tracer metabolism. Proliferation, cell-cycle status, cell death and transcriptional rewiring will be explored. 2A] How does IP3 open the channel? Chemical crosslinking/mass spectroscopy will be used to validate cryo-EM structures and monitor conformational changes mediated by IP3 in native IP3Rs. Mutagenesis of key residues will be used to test proposed allosteric mechanisms of gating. 2B] Identification of the Ca2+ regulatory sites in IP3Rs: Based on the most recent cryo-EM structures of IP3R1 and IP3R3 we have identified 3 clusters of negatively charged residues that are candidates for Ca2+ regulatory sites. Their functional role as stimulatory or inhibitory sites will be assessed by mutagenesis. The role of these sites will also be tested by mutagenesis of the Ca2+-insensitive IP3R from Capsaspora owczarzaki . The long-term goal of the proposal is to better understand the role of Ca2+signaling in normal cells, cancer cells and in inherited disorders inactivating IP3R function. The study should also provide fundamental mechanistic information on how these important channels work and are regulated.

钙是一个通用的使者，控制了许多细胞反应，包括肌肉收缩，胞吞作用，记忆，受精，代谢，增殖和细胞死亡。许多激素，神经递质和生长因子刺激肌醇1,4,5-三磷酸（IP3）的形成在位于内质网中的受体家族（IP3RS）上，起着配体门控Ca2+的功能频道。细胞内Ca2+存储的耗竭还激活了等离子体中的Ca2+涌入机制膜。因此，IP3与其受体的相互作用激活了Ca2+信号的所有阶段。 IP3RS是受CA2+和磷酸化的调节，但该调节的分子基础知之甚少。 Ca2+ 从IP3RS释放的局部传播到线粒体，可以刺激新陈代谢，并且在较高的情况下数量，也可以引发细胞死亡。癌细胞被认为高度依赖于IP3R- 介导的线粒体Ca2+传递以生存。 CRISPR/CAS-9技术的出现允许 HEK293和HELA子宫颈癌细胞的所有三种IP3R同工型的遗传消融。该提议是集中于使用这些细胞系用于a）研究适应机制，使细胞在完全没有CA2+信号和b）利用无效的结构功能研究探索IP3打开通道和进料机理的分子机制的研究通过Ca2+调节。这两个特定目的是：1]表征IP3R-3KO细胞中的自适应机制：缺乏CA2+代谢调节的影响将通过测量几种生物能量来研究参数和13C追踪代谢。增殖，细胞周期状态，细胞死亡和转录重新布线将被探索。 2A] IP3如何打开频道？化学交联/质谱将使用验证IP3在天然IP3R中介导的构象变化的验证和监测构象变化。关键残基的诱变将用于测试所提出的门控的变构机制。 2b]识别 IP3RS中的CA2+调节站点：基于IP3R1和IP3R3的最新冷冻EM结构已经确定了3个负电荷残基簇，这些残基是CA2+调节部位的候选物。他们的作为刺激或抑制部位的功能作用将通过诱变评估。这些站点的作用将也可以通过Capsaspora Owczarzaki的Ca2+不敏感IP3R的诱变进行测试。长期目标该建议的方法是更好地了解Ca2+信号传导在正常细胞，癌细胞和遗传中的作用疾病灭活IP3R功能。该研究还应提供有关的基本机械信息这些重要渠道如何工作并受到监管。