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.

项目概要： 细胞内 Ca2+ 信号通过胞质 Ca2+ 浓度的变化控制多种细胞 由第二信使介导的细胞内储存的 Ca2+ 动员发挥着重要作用。 烟酸腺嘌呤二核苷酸磷酸 (NAADP) 在调节细胞质 Ca2+ 水平中起关键作用。 迄今为止发现的最有效的 Ca2+ 动员第二信使，它独特地从酸性中动员 Ca2+； 内溶酶体细胞器已被证明可有效引起多种 Ca2+ 释放。 不同的哺乳动物细胞和 NAADP 信号传导缺陷现在与许多疾病有关。 NAADP 诱发的 Ca2+ 信号传导的重要性，NAADP 诱发的 Ca2+ 释放的分子基础仍然存在 基于固定化 NAADP 的亲和纯化和定量蛋白质组分析。 NAADP 和 TPC 蛋白相互作用，我们确定 Lsm12 是 NAADP、TPC1 的共享相互作用伙伴， TPC2 通过其 Lsm 结构域直接与 NAADP 结合，与 TPC2 共定位，并介导 NAADP 与分离的 TPC2 或含有 TPC2 的 Lsm12 膜的明显关联是必不可少的。 立即参与 NAADP 诱发的 TPC 激活和 Ca2+ 动员，因此我们的研究结果揭示了 假定的 RNA 结合蛋白充当 NAADP 受体和 TPC 调节蛋白，并提供 我们的进一步研究表明，了解 NAADP 信号传导机制的新分子基础。 Lsm12 通过影响 TPC 通道门控属性和在非 TPC 中的功能而具有多方面的功能 我们努力解决以下问题：1) Lsm12 作为 NAADP 受体达到其高水平。 通过其 Lsm 结构域对 NAADP 的选择性和亲和力高于 NADP 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；3) Lsm12 可以调节多种离子 通道并通过共享机制介导 NAADP 诱发的细胞内 Ca2+ 升高。 假设，我们将追求以下 3 个具体目标 1. 确定分子机制。 NAADP 结合中的 Lsm 结构域 目的 2. 确定 Lsm12 介导的 TPC 的分子机制。 目标 3. 确定 Lsm12 在 NAADP 诱发的 Ca2+ 信号传导中的多方面功能。 拟议研究的结果将阐明 NAADP/Lsm12- 的分子机制和功能 介导 Ca2+ 信号传导，并促进针对这一重要 Ca2+ 信号传导过程的新药物的开发。