Regulation of inositol trisphosphate receptors

肌醇三磷酸受体的调节

• 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
• 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

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.

钙是控制多种细胞反应的通用信使，包括肌肉收缩、 胞吐作用、记忆、受精、新陈代谢、增殖和细胞死亡。多种荷尔蒙， 神经递质和生长因子刺激肌醇 1,4,5-三磷酸 (IP3) 的形成，其作用 位于内质网的受体家族 ( IP3Rs ) 上，其功能是配体门控 Ca2+ 渠道。细胞内 Ca2+ 储存的耗尽也会激活血浆中 Ca2+ 的流入机制 膜。因此，IP3 与其受体的相互作用激活 Ca2+ 信号的所有阶段。 IP3R 是 受 Ca2+ 和磷酸化调节，但这种调节的分子基础知之甚少。钙离子 IP3Rs 释放的物质会局部传输至线粒体并可刺激新陈代谢，并且在较高浓度下 量，也可以引发细胞死亡。癌细胞被认为高度依赖 IP3R- 介导线粒体 Ca2+ 转移以维持其生存。 CRISPR/Cas-9技术的出现使得 来自 HEK293 和 HeLa 宫颈癌细胞的所有三种 IP3R 同工型的基因消融。该提案是 集中于使用这些细胞系来 a) 研究使细胞在环境中生存的适应性机制 完全没有 Ca2+ 信号传导，并且 b) 利用结构功能的零背景 探索IP3打开通道的分子机制和反馈机制的研究 Ca2+的调节。两个具体目标是： 1] 表征 IP3R-3KO 细胞的适应性机制： 将通过测量几种生物能来研究缺乏 Ca2+ 代谢调节的影响 参数和 13C 示踪代谢。增殖、细胞周期状态、细胞死亡和转录重连 将被探索。 2A]IP3如何打开通道？将使用化学交联/质谱法 验证冷冻电镜结构并监测天然 IP3R 中 IP3 介导的构象变化。 关键残基的诱变将用于测试所提出的门控变构机制。 2B] 鉴定 IP3R 中的 Ca2+ 调控位点：基于 IP3R1 和 IP3R3 的最新冷冻电镜结构，我们 已鉴定出 3 簇带负电荷的残基，它们是 Ca2+ 调控位点的候选者。他们的 作为刺激或抑制位点的功能作用将通过诱变进行评估。这些网站的作用将 还可以通过 Capsaspora owczarzaki 的 Ca2+ 不敏感 IP3R 的诱变进行测试。长期目标 该提案的目的是为了更好地了解 Ca2+ 信号在正常细胞、癌细胞和遗传性细胞中的作用 IP3R 功能失活的疾病。该研究还应提供以下方面的基本机制信息： 这些重要渠道如何运作和监管。