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: 10218212
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218212
• 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.

项目摘要： 细胞内Ca2+信号传导通过胞质Ca2+浓度的变化控制多种细胞 和生理过程。由第二使者介导的细胞内商店的CA2+动员 在调节胞质Ca2+水平的调节中的关键作用。烟酸腺嘌呤二核苷酸磷酸盐（NAADP）是 迄今为止，大多数有效的CA2+Mobilizing第二使者都确定了；它独特地从酸性中动员了Ca2+ 内溶血细胞器。 NAADP已被证明可以有效唤起Ca2+释放 NAADP信号传导中的不同哺乳动物细胞和缺陷现在与许多疾病有关。尽管 NAADP诱发的Ca2+信号传导的重要性，NAADP受体的分子身份仍然存在 在此过程中涉及的难以捉摸的和Ca2+释放通道尚未明确定义。 积累的证据表明，内溶性两孔通道（TPC）在 NAADP诱发的Ca2+释放。但是，有力的证据也表明TPC不是NAADP受体。 当前，一个统一的假设是TPC是负责NAADP诱发CA2+释放的关键渠道 但是它们的NAADP敏感性来自一些未知的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，将其相互作用伙伴与 哺乳动物HEK293和SKBR3细胞，然后使用公正的定量蛋白质组学分析和Ca2+ - 成像功能分析以筛选和鉴定对NAADP诱发的Ca2+很重要的新型蛋白质 发布。我们已经确定LSM12蛋白是NAADP和TPC的相互作用蛋白。和 LSM12-KNOCKOUT细胞，我们发现LSM12介导了NAADP和TPC之间的相互作用，并且IS 在HEK293细胞中，基本上需要NAADP诱发的Ca2+释放。我们假设LSM12是必不可少的 并直接通过充当NAADP受体和/或TPC来参与NAADP诱发的Ca2+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LSM12是的假设 必不可少的，直接参与NAADP诱发的Ca2+释放； 2）检验LSM12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 通过使用分子生物学，细胞生物学，蛋白质生物化学的多学科方法，这些目标， 和电生理学。拟议的研究的发现将提供一个突破，这将推动我们的 了解NAADP诱发的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。