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：
9243500
负责人：
Jiusheng Yan
金额：
$ 24万
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 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9243500
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 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 Research Design Role Ryanodine Receptors SKBR3 Second Messenger Systems Signal Transduction Source Stable Isotope Labeling System analog base crosslink genetic regulatory protein imaging modality interest knock-down link protein nanomolar new therapeutic target novel overexpression patch clamp receptor reconstitution response second messenger 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Ca2+信号传导通过胞质Ca2+浓度的变化控制多种细胞和生理过程。由第二使者介导的细胞内商店的CA2+动员是胞质Ca2+的重要来源。烟酸腺嘌呤二核苷酸磷酸盐（NAADP）是最有效的 CA2+ - 迄今为止确定的第二使者；它独特地从酸性内溶性中动员Ca2+ 细胞器。 NAADP已被证明可以有效唤起Ca2+释放。 NAADP信号传导中的哺乳动物细胞和缺陷现在与许多疾病有关。尽管有 NAADP引起的Ca2+信号传导的重要性，NAADP受体的分子身份和Ca2+ - 涉及此过程的释放通道尚未明确定义。积累的证据表明内溶性两孔通道（TPC）可能参与NAADP诱发的Ca2+释放。然而，内溶液体中TPC通道电流的最新直接贴片钳记录表明TPCS 是Ca2+渗透性非常有限的Na+选择通道，通道活动对 NAADP。当前，关于TPC在NAADP信号传导中的作用的统一假设是TPC不是直接参与NAADP结合，但是NAADP受体/Ca2+ - 弹性通道复合物的一部分，其中其他关键蛋白的分子身份仍然未知。这些新发现和猜测保证探索可能对NAADP诱发的Ca2+信号传导很重要的其他蛋白质。在提议中研究，我们将同时使用TPC和NAADP作为诱饵，将其相互作用的伴侣与哺乳动物分离 HEK293和SKBR3细胞，然后使用公正的定量蛋白质组学分析和功能测定到筛选并确定对NAADP诱发的Ca2+信号传导重要的新型蛋白质。我们将追求以下两个具体的目标。具体目的1。确定对NAADP诱发的CA2+重要的新型TPC相互作用蛋白信号。我们旨在使用基于SILAC的定量来系统地确定TPC的相互作用具有晚期质谱的蛋白质组学方法。然后，我们将执行CA2+成像功能测定在已识别的TPC相互作用蛋白上，鉴定出对NAADP诱发的Ca2+信号传导重要的新型蛋白质。具体目标2。确定对NAADP诱发的Ca2+信号传导重要的NAADP相互作用蛋白。直接靶向NAADP受体，我们将执行HEK293的NAADP相互作用蛋白的亲和力纯化使用固定的NAADP及其类似物，然后采用定量蛋白质组学分析和功能测定，以鉴定对NAADP诱导的Ca2+释放重要的新型蛋白质。提议研究旨在使用系统的方法来公正筛查并确定新颖的蛋白质很重要用于NAADP诱发的Ca2+释放。我们的结果可能会在分子的基础上产生突破通过鉴定可以用作NAADP受体的新型蛋白质，NAADP信号传导的机制，Ca2+ - 释放通道或NAADP引起的Ca2+释放所需的调节蛋白。 1