Molecular basis of the NAADP-gated calcium release channel complexes

NAADP 门控钙释放通道复合物的分子基础

• 批准号: 10445514
• 负责人: Jiusheng Yan
• 金额: $ 33.21万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445514
• 关键词: Affect; Affinity; Affinity Chromatography; Agonist; Binding; Binding Sites; Biochemical; Biological; Calcium; Calcium Signaling; Cell Line; Cell physiology; Cells; Complex; Data; Defect; Development; Disease; Electrophysiology (science); Endosomes; Factor V; Human; Immobilization; Ion Channel; Knockout Mice; Knowledge; Label; Light; Lipids; Lysosomes; Mammalian Cell; Mediating; Membrane; Molecular; Mutation; NAADP; NADP; Organelles; Physiological Processes; Play; Process; Property; Protein Dephosphorylation; Proteins; Proteomics; RNA-Binding Proteins; Regulation; Reporting; Research; Role; Second Messenger Systems; Signal Transduction; Source; Testing; analog; base; design; genetic regulatory protein; innovation; new therapeutic target; novel; novel therapeutics; protein function; protein protein interaction; receptor; response; targeted treatment

Project Summary: Intracellular Ca2+ signaling via changes in cytosolic Ca2+ concentration controls a wide range of cellular and physiologic processes. Ca2+ mobilization from intracellular stores mediated by second messengers plays a critical role in regulation of cytosolic Ca2+ levels. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing second messenger identified to date; it uniquely mobilizes Ca2+ from acidic endolysosomal organelles. NAADP has been shown to be effective in evoking Ca2+ release in a multitude of different mammalian cells and defects in NAADP signaling are now being implicated in many diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular basis of NAADP-evoked Ca2+ release remains largely unclear. With immobilized NAADP–based affinity purification and quantitative proteomic analyses of NAADP and TPC interacting proteins, we identified Lsm12 to be a shared interacting partner of NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization. Our findings thus reveal a putative RNA-binding protein functioning as an NAADP receptor and a TPC regulatory protein and provide a new molecular basis for understanding the mechanisms of NAADP signaling. Our further studies showed that Lsm12 has multifaceted function by affecting TPC channel gating properties and functioning in non-TPC dependent NAADP signaling. We hypothesize that: 1) Lsm12 as an NAADP receptor achieves its high selectivity and affinity to NAADP than NADP via its Lsm domain; 2) Lsm12 mediates TPC channel activation by NAADP via protein-protein interactions and/or dephosphorylation; and 3) Lsm12 can regulate multiple ion channels and mediate NAADP-evoked intracellular Ca2+ elevation via shared mechanisms. To test our hypotheses, we will pursue the following 3 specific aims. Aim 1. Determine the molecular mechanism of the Lsm domain in NAADP binding. Aim 2. Determine the molecular mechanisms of Lsm12-mediated TPC activation by NAADP. Aim 3. Determine the multifaceted function of Lsm12 in NAADP-evoked Ca2+ signaling. Findings from the proposed research will elucidate the molecular mechanisms and function of NAADP/Lsm12- mediated Ca2+ signaling and facilitate the development of new drugs for this important Ca2+ signaling process.

项目总结： 细胞内钙信号通过胞内钙离子浓度的变化控制广泛的细胞内 和生理过程。第二信使介导的细胞内钙离子动员起着重要的作用 在细胞内钙离子水平调节中的关键作用。烟酸腺嘌呤二核苷酸磷酸(NAADP)是 迄今为止发现的最有效的钙动员第二信使；它独一无二地从酸性中动员钙 内溶酶体细胞器。NAADP已被证明有效地在许多情况下引起钙离子释放 不同的哺乳动物细胞和NAADP信号缺陷现在与许多疾病有关。尽管 NAADP诱导的钙信号的重要性，NAADP诱导的钙释放的分子基础仍然存在 很大程度上还不清楚。固定化NAADP亲和纯化及定量蛋白质组学分析 NAADP和TPC相互作用蛋白，我们鉴定Lsm12是NAADP、TPC1、 和TPC2。LSM12通过其LSM结构域直接与NAADP结合，与TPC2协同定位，并介导 NAADP与分离的TPC2或含TPC2的膜的明显结合。LSM12是必不可少的， 立即参与NAADP诱导的TPC活化和钙动员。因此，我们的发现揭示了一个 推测的RNA结合蛋白作为NAADP受体和TPC调节蛋白发挥作用，并提供 了解NAADP信号传导机制的新分子基础。我们的进一步研究表明， LSM12通过影响TPC通道选通属性和在非TPC中的功能而具有多方面的功能 依赖NAADP信号。我们假设：1)Lsm12作为一种NAADP受体 对NAADP的选择性和亲和力优于NADP通过其LSM结构域；2)LSM12通过以下方式介导TPC通道激活 NAADP通过蛋白质-蛋白质相互作用和/或去磷酸化；以及3)Lsm12可以调节多种离子 通道，并通过共同的机制介导NAADP引起的细胞内钙升高。测试我们的 假设，我们将追求以下3个具体目标。目的1.确定心绞痛的分子机制 NAADP结合中的LSM结构域。目的2.确定Lsm12介导的TPC的分子机制 NAADP激活。目的3.确定Lsm12在NAADP诱导的钙信号转导中的多方面作用。 拟议的研究结果将阐明NAADP/Lsm12-的分子机制和功能- 介导钙信号转导，促进针对这一重要的钙信号转导过程的新药的开发。