Identification of Novel Protein Important for NAADP-Evoked Calcium Signaling

细胞内钙信号通过胞浆钙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CA2动员是一种 胞浆钙的重要来源。烟酸腺嘌呤二核苷酸磷酸(NAADP)是最有效的 CA2-动员第二信使；它独一无二地动员酸性内溶酶体内的钙 细胞器。NAADP已被证明能有效地在多种不同的细胞中引起钙释放 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受体和钙离子的分子同一性 这一过程中涉及的发布渠道尚未得到明确定义。积累的证据 提示内溶酶体双孔通道(TPC)可能参与了NAADP诱导的钙释放。 然而，最近对内溶酶体TPC通道电流的直接膜片钳记录表明，TPC 是钙通透性非常有限的钠选择性通道，通道活性对 NAADP。目前，关于TPC在NAADP信号中的作用的一个统一的假设是TPC不是 直接参与NAADP结合，但属于NAADP受体/钙释放通道复合体的一部分，其中 其他关键蛋白质的分子特性尚不清楚。这些新的发现和推测证明了 探索可能对NAADP诱导的钙信号转导重要的其他蛋白质。在建议的 研究中，我们将使用TPC和NAADP作为诱饵将它们的相互作用伙伴从哺乳动物中分离出来 HEK293和SKBR3细胞，然后使用无偏定量蛋白质组分析和功能分析 筛选和鉴定对NAADP诱导的钙信号转导重要的新蛋白。我们将致力于以下工作 有两个明确的目标。具体目的1.鉴定与NAADP诱导的钙离子有关的新的TPC相互作用蛋白 发信号。我们的目标是使用基于SILAC的定量方法系统地确定TPC的相互作用组 蛋白质组学方法与先进的质谱学。然后我们将进行钙离子成像功能分析 对已鉴定的TPC相互作用蛋白进行鉴定，以确定对NAADP诱导的钙信号转导重要的新蛋白。 特定目的2.鉴定NAADP相互作用蛋白对NAADP诱导的钙信号转导具有重要意义。直接 针对NAADP受体，我们将从HEK293中进行NAADP相互作用蛋白的亲和纯化 和SKBR3细胞使用固定化NAADP及其类似物，然后进行定量蛋白质组学分析 以及功能分析，以确定对NAADP诱导的钙释放至关重要的新蛋白。建议数 研究的目的是使用系统的方法无偏见地筛选和识别新的重要蛋白质 对NAADP诱导的钙释放有抑制作用。我们的结果可能会在分子基础上产生突破， 识别可作为NAADP受体的新蛋白的NAADP信号转导机制 释放通道，或NAADP引起的钙释放所必需的调节蛋白。 1

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 is an important source of cytosolic Ca2+. 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 identities of the NAADP receptors and Ca2+- release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that endolysosomal two-pore channels (TPCs) might participate in NAADP-evoked Ca2+ release. However, recent direct patch-clamp recordings of TPC channel currents in endolysosomes showed that TPCs were Na+-selective channels with very limited Ca2+ permeability, and the channel activity was not sensitive to NAADP. Currently, a unifying hypothesis about the role of TPCs in NAADP signaling is that TPCs are not directly involved in NAADP binding but are part of the NAADP receptor/Ca2+-release channel complex, in which the molecular identities of other key proteins remain unknown. These new findings and speculations warrant an exploration of additional proteins that may be important for NAADP-evoked Ca2+ signaling. In the proposed study, we will use both TPCs and NAADP as baits to isolate their interacting partners from mammalian HEK293 and SKBR3 cells and then use unbiased quantitative proteomic analysis and functional assays to screen and identify novel proteins important for NAADP-evoked Ca2+ signaling. We will pursue the following two specific aims. Specific Aim 1. Identify novel TPC-interacting proteins important for NAADP-evoked Ca2+ signaling. We aim to systematically determine the interactome of TPCs using a SILAC-based quantitative proteomic approach with advanced mass spectrometry. We will then perform Ca2+ imaging functional assays on identified TPC-interacting proteins to identify novel proteins important for NAADP-evoked Ca2+ signaling. Specific Aim 2. Identify NAADP-interacting proteins important for NAADP-evoked Ca2+ signaling. To directly target the NAADP receptor, we will perform affinity purification of NAADP-interacting proteins from HEK293 and SKBR3 cells using immobilized NAADP and its analogue and then employ quantitative proteomic analysis and functional assays to identify novel proteins important for NAADP-induced Ca2+ release. The proposed studies are designed to use systematic approaches to unbiasedly screen and identify novel proteins important for NAADP-evoked Ca2+ release. Our results will likely generate a breakthrough in the molecular basis and mechanisms of NAADP signaling by identifying novel proteins that could serve as NAADP receptors, Ca2+- release channels, or regulatory proteins necessary for NAADP-evoked Ca2+ release. 1