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: 10000113
• 负责人: Jiusheng Yan
• 金额: $ 30.8万
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000113
• 关键词: Affect; Affinity; Affinity Chromatography; Amino Acids; Binding; Biological Assay; CRISPR/Cas technology; Calcium; Calcium Signaling; Cell Line; Cell membrane; Cell physiology; Cells; Cellular biology; Complex; Cytoplasmic Protein; Defect; Development; Disease; Electrophysiology (science); Endoplasmic Reticulum; Endosomes; Goals; Image; Immobilization; Immunofluorescence Immunologic; In Situ; Knock-out; Knockout Mice; Label; Ligation; Light; Lysosomes; Mammalian Cell; Mediating; Membrane; Methods; Molecular; Molecular Biology; Mutation Analysis; N-terminal; NAADP; Nicotinic Receptors; Organelles; Physiological Processes; Play; Preparation; Process; Property; Protein Biochemistry; Proteins; Proteomics; Regulation; Reporting; Research; Role; SKBR3; Second Messenger Systems; Signal Transduction; Small Interfering RNA; Structure of beta Cell of islet; Structure-Activity Relationship; System; Testing; analog; design; genome editing; innovation; interdisciplinary approach; knock-down; live cell imaging; mutant; new therapeutic target; novel; novel therapeutics; patch clamp; protein distribution; 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 identity of the NAADP receptor remains elusive and the Ca2+-release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that endolysosomal two-pore channels (TPCs) play an important role in NAADP-evoked Ca2+ release. However, strong evidence also suggests that TPCs are not NAADP receptors. Currently, a unifying hypothesis is that TPCs are the key channels responsible for NAADP-evoked Ca2+ release but that their NAADP sensitivity arises from some unknown NAADP-binding accessory protein. To identify the elusive NAADP receptor, we used both TPCs and NAADP as bait to isolate their interacting partners from mammalian HEK293 and SKBR3 cells and then used an unbiased quantitative proteomic analysis and a Ca2+- imaging functional assay to screen and identify novel proteins that are important for NAADP-evoked Ca2+ release. We have identified an Lsm12 protein to be an interacting protein of both NAADP and TPCs. With Lsm12-knockout cells, we found that Lsm12 mediates the interaction between NAADP and TPCs and is essentially required for NAADP-evoked Ca2+ release in HEK293 cells. We hypothesize that Lsm12 is essential and directly involved in NAADP-evoked Ca2+ release by functioning as a NAADP receptor and/or a TPC regulatory or auxiliary protein. We will pursue the following 3 specific aims: 1) test the hypothesis that Lsm12 is essential and directly involved in NAADP-evoked ca2+ release; 2) test the hypothesis that Lsm12 is a NAADP receptor; and 3) test the hypothesis that Lsm12 is a regulatory or auxiliary protein of TPCs. We will achieve these goals by using multidisciplinary approaches from molecular biology, cell biology, protein biochemistry, and electrophysiology. Findings from the proposed research will provide a breakthrough that will advance our understanding of the molecular basis and mechanisms of NAADP-evoked Ca2+ release, and facilitate the development of new drugs for this important Ca2+ signaling process.

项目概要： 细胞内Ca2+信号通过改变胞浆Ca2+浓度控制广泛的细胞内Ca2+信号传导。 和生理过程。由第二信使介导的细胞内钙库的钙动员， 在调节胞质Ca 2+水平中起关键作用。烟酸腺嘌呤二核苷酸磷酸（NAADP）是 迄今为止发现的最有效的Ca2+动员第二信使;它独特地从酸性环境中动员Ca2 + 内溶酶体细胞器NAADP已被证明是有效的，在引起Ca2+释放在许多 不同的哺乳动物细胞和NAADP信号传导中的缺陷现在与许多疾病有关。尽管 NAADP诱导的Ca2+信号的重要性，NAADP受体的分子身份仍然存在 这一过程中涉及的Ca2+释放通道尚未明确定义。 越来越多的证据表明，内溶酶体双孔通道（TPC）在细胞凋亡中起着重要作用。 NAADP诱发的Ca~（2+）释放。然而，强有力的证据也表明TPC不是NAADP受体。 目前，一个统一的假设是TPC是负责NAADP诱发的Ca 2+释放的关键通道 但它们对NAADP的敏感性来自于一些未知的NAADP结合辅助蛋白。识别 难以捉摸的NAADP受体，我们使用TPC和NAADP作为诱饵来分离它们的相互作用伴侣， 哺乳动物HEK293和SKBR3细胞，然后使用无偏定量蛋白质组学分析和Ca2 +- 成像功能测定以筛选和鉴定对NAADP诱发的Ca 2+重要的新蛋白质 release.我们已经确定了Lsm12蛋白是NAADP和TPC的相互作用蛋白。与 在Lsm12敲除细胞中，我们发现Lsm12介导NAADP和TPC之间的相互作用， HEK293细胞中NAADP诱导的Ca2+释放所必需的。我们假设Lsm12是必不可少的， 并通过充当NAADP受体和/或TPC直接参与NAADP诱导的Ca 2+释放 调节或辅助蛋白。我们将追求以下3个具体目标：1）测试Lsm 12是 Lsm 12是NAADP诱导的Ca~（2+）释放的关键和直接参与; 2）检验Lsm 12是NAADP的假设 受体; 3）检验Lsm12是TPC的调节或辅助蛋白的假设。我们将实现 这些目标通过使用来自分子生物学，细胞生物学，蛋白质生物化学， 和电生理学。拟议研究的结果将提供一个突破， 了解NAADP诱导的Ca2+释放的分子基础和机制，并促进 为这一重要的Ca2+信号传导过程开发新药。