Molecular basis of the NAADP-gated calcium release channel complexes

项目摘要： 细胞内Ca 2+信号通过改变胞浆Ca 2+浓度控制广泛的细胞内Ca 2+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Ca 2+动员第二信使;它独特地从酸性环境中动员Ca 2 + 内溶酶体细胞器NAADP已被证明是有效的，在引起Ca 2+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诱发的Ca 2+信号的重要性，NAADP诱发的Ca 2+释放的分子基础仍然存在 基本上不清楚。通过固定化NAADP亲和纯化和定量蛋白质组学分析， NAADP和TPC相互作用蛋白，我们确定Lsm 12是NAADP，TPC 1， TPC 2。Lsm 12通过其Lsm结构域直接与NAADP结合，与TPC 2共定位，并介导NAADP的表达。 NAADP与分离的TPC 2或含TPC 2的膜的明显缔合。LSM 12是必要的， 立即参与NAADP诱发的TPC激活和Ca 2+动员。因此，我们的研究结果揭示了 作为NAADP受体和TPC调节蛋白发挥功能的推定RNA结合蛋白，并提供 为理解NAADP信号转导机制提供了新的分子基础。我们进一步的研究表明， Lsm 12通过影响TPC通道门控特性和在非TPC通道中的功能而具有多方面的功能。 依赖NAADP信号。我们假设：1）Lsm 12作为NAADP受体， 与NADP相比，Lsm 12通过其Lsm结构域对NAADP的选择性和亲和力; 2）Lsm 12通过以下途径介导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）Lsm 12可以调节多种离子 通道和介导NAADP诱发的细胞内Ca 2+升高通过共享的机制。来测试我们 假设，我们将追求以下三个具体目标。目标1.确定的分子机制 NAADP结合中的Lsm结构域。目标2.确定Lsm 12介导TPC的分子机制 通过NAADP激活。目标3.确定Lsm 12在NAADP诱发的Ca 2+信号传导中的多方面功能。 该研究的结果将阐明NAADP/Lsm 12 - 1的分子机制和功能。 介导的Ca 2+信号传导，并促进这一重要的Ca 2+信号传导过程的新药的开发。

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.