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Identification of Novel Protein Important for NAADP-Evoked Calcium Signaling

鉴定对 NAADP 诱发的钙信号传导重要的新型蛋白质

基本信息

批准号：
9344708
负责人：
Jiusheng Yan
金额：
$ 20万
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：
2016
资助国家：
美国
起止时间：
2016-09-15 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9344708
关键词：
Affinity Chromatography Amino Acids Binding Binding Sites Biological Assay Breast Carcinoma Calcium Signaling Cell Culture Techniques Cell Line Cell physiology Cells Complex Cyclic ADP-Ribose Defect Disease Embryo Endoplasmic Reticulum Endosomes Functional Imaging Goals Human Image Immobilization Individual Kidney Knock-out Lipid Bilayers Liquid Chromatography Lysosomes Malignant Epithelial Cell Mammalian Cell Mass Spectrum Analysis Mediating Membrane Molecular NAADP NADP Organelles Pancreas Permeability Phosphate-Binding Proteins Physiological Processes Process Proteins Proteomics Receptor Signaling Research Role Ryanodine Receptors SKBR3 Second Messenger Systems Signal Transduction Source Stable Isotope Labeling System analog base crosslink design genetic regulatory protein imaging modality knock-down link protein nanomolar new therapeutic target novel overexpression patch clamp receptor reconstitution response tandem mass spectrometry targeted treatment

项目摘要

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

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是一个重要的机制。细胞质Ca 2+的重要来源。烟酸腺嘌呤二核苷酸磷酸（NAADP）是最有效的迄今为止确定的Ca 2+动员第二信使;它独特地从酸性内溶酶体动员Ca 2 + 细胞器NAADP已被证明可以有效地在多种不同的细胞中引起Ca 2+释放哺乳动物细胞和NAADP信号传导的缺陷现在与许多疾病有关。尽管 NAADP诱发的Ca 2+信号传导的重要性，NAADP受体和Ca 2 +- 这一进程所涉及的释放渠道尚未明确界定。累积证据提示内溶酶体双孔通道（TPC）可能参与NAADP诱导的Ca ~（2+）释放。然而，最近的直接膜片钳记录的TPC通道电流的内溶酶体表明，TPC 是具有非常有限的Ca 2+渗透性的Na+选择性通道，并且通道活性对 NAADP。目前，关于TPC在NAADP信号传导中的作用的统一假设是TPC不是NAADP信号传导的一部分。直接参与NAADP结合，但也是NAADP受体/Ca 2+释放通道复合物的一部分，其中其他关键蛋白质的分子身份仍然未知。这些新的发现和推测探索可能对NAADP诱发的Ca 2+信号传导重要的其他蛋白质。拟议在本研究中，我们将使用TPC和NAADP作为诱饵，从哺乳动物中分离它们的相互作用伴侣， HEK 293和SKBR 3细胞，然后使用无偏定量蛋白质组学分析和功能测定，筛选和鉴定对NAADP诱导的Ca 2+信号传导重要的新蛋白。我们将继续努力两个具体目标。具体目标1。鉴定对NAADP诱导的Ca 2+重要的新型TPC相互作用蛋白信号我们的目标是使用基于SILAC的定量分析系统地确定TPC的相互作用组。蛋白质组学方法与先进的质谱。然后我们将进行Ca 2+成像功能测定，确定TPC相互作用的蛋白质，以确定新的蛋白质重要的NAADP诱发的Ca 2+信号。具体目标2。确定NAADP相互作用蛋白质的重要NAADP诱发的Ca 2+信号。直接针对NAADP受体，我们将从HEK 293中进行NAADP相互作用蛋白的亲和纯化和SKBR 3细胞，然后进行定量蛋白质组学分析和功能测定，以鉴定对NAADP诱导的Ca 2+释放重要的新蛋白质。拟议研究旨在使用系统的方法来无偏地筛选和鉴定重要的新蛋白质， NAADP诱发的Ca ~（2+）释放。我们的研究结果可能会在分子基础上产生突破， NAADP信号转导机制，通过鉴定新的蛋白质，可以作为NAADP受体，Ca 2 +- 释放通道或NAADP诱发的Ca 2+释放所必需的调节蛋白。 1