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

基本信息

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
项目类别：
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

项目摘要

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+ 信号通过胞质 Ca2+ 浓度的变化控制多种细胞和生理过程。由第二信使介导的细胞内储存的 Ca2+ 动员起着重要作用在细胞质 Ca2+ 水平的调节中发挥关键作用。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；它独特地从酸性中调动 Ca2+ 内溶酶体细胞器。 NAADP 已被证明可以有效地在多种细胞中引起 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。尽管 NAADP 诱发的 Ca2+ 信号传导的重要性，NAADP 受体的分子特性仍然存在难以捉摸，并且参与该过程的 Ca2+ 释放通道尚未明确定义。积累的证据表明，内溶酶体双孔通道（TPC）在 NAADP 诱发 Ca2+ 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 Ca2+ 释放的关键通道但他们的 NAADP 敏感性源于一些未知的 NAADP 结合辅助蛋白。为了识别由于难以捉摸的 NAADP 受体，我们使用 TPC 和 NAADP 作为诱饵，将它们的相互作用伙伴与哺乳动物 HEK293 和 SKBR3 细胞，然后使用无偏定量蛋白质组分析和 Ca2+- 成像功能测定法筛选和鉴定对 NAADP 诱发 Ca2+ 重要的新型蛋白质发布。我们已经鉴定出 Lsm12 蛋白是 NAADP 和 TPC 的相互作用蛋白。和 Lsm12敲除细胞，我们发现Lsm12介导NAADP和TPCs之间的相互作用，并且是 HEK293 细胞中 NAADP 诱发 Ca2+ 释放所必需的。我们假设 Lsm12 是必需的并通过作为 NAADP 受体和/或 TPC 直接参与 NAADP 诱发的 Ca2+ 释放调节或辅助蛋白质。我们将追求以下 3 个具体目标：1）检验 Lsm12 的假设重要且直接参与 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、蛋白质生物化学等多学科方法来实现这些目标和电生理学。拟议研究的结果将提供突破，推动我们的研究了解NAADP诱发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。