Molecular basis of the NAADP-gated calcium release channel complexes

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受体的分子同一性仍然存在 这一过程中涉及的难以捉摸的钙释放通道还没有明确的定义。 越来越多的证据表明，内溶酶体双孔通道(TPC)在多种疾病中起重要作用 NAADP可引起细胞内钙离子释放。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NAADP引起的钙释放的关键通道 但它们对NAADP的敏感性来自于一些未知的NAADP结合辅助蛋白。要确定 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伙伴 哺乳动物HEK293和SKBR3细胞，然后使用无偏定量蛋白质组分析和钙离子- 成像功能分析筛选和鉴定与NAADP诱导的钙离子有关的新蛋白 放手。我们已经鉴定出一个Lsm12蛋白是NAADP和TPC的相互作用蛋白。使用 Lsm12-基因敲除细胞，我们发现Lsm12介导了NAADP和TPC之间的相互作用 NAADP诱导HEK293细胞钙释放所必需的。我们假设LSM12是必不可少的 并通过作为NAADP受体和/或TPC直接参与NAADP诱导的钙释放 调节蛋白或辅助蛋白。我们将追求以下三个具体目标：1)检验Lsm12是 2)检验Lsm12是NAADP的假说 3)验证Lsm12是TPC的调节蛋白或辅助蛋白的假设。我们将实现 这些目标通过使用分子生物学、细胞生物学、蛋白质生物化学、 和电生理学。拟议的研究结果将提供一个突破，将推动我们的 了解NAADP诱导的钙释放的分子基础和机制，并促进 为这一重要的钙信号转导过程开发新药。

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.