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 Ca2+ 信号通过胞质 Ca2+ 浓度的变化控制多种细胞和生理过程。由第二信使介导的细胞内储存的 Ca2+ 动员是一种胞质Ca2+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已确定的 Ca2+ 动员第二信使；它独特地从酸性溶酶体中调动 Ca2+ 细胞器。 NAADP 已被证明可以有效地在多种不同的细胞中引起 Ca2+ 释放。哺乳动物细胞和 NAADP 信号传导缺陷现在与许多疾病有关。尽管 NAADP 诱发的 Ca2+ 信号传导的重要性、NAADP 受体和 Ca2+- 的分子特性此过程中涉及的发布渠道尚未明确定义。积累的证据表明内溶酶体双孔通道 (TPC) 可能参与 NAADP 诱发的 Ca2+ 释放。然而，最近内溶酶体中 TPC 通道电流的直接膜片钳记录表明，TPC 是 Na+ 选择性通道，Ca2+ 通透性非常有限，并且通道活性对全国农业发展计划。目前，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，但属于 NAADP 受体/Ca2+ 释放通道复合体的一部分，其中其他关键蛋白质的分子身份仍然未知。这些新发现和猜测有理由对 NAADP 诱发的 Ca2+ 信号传导可能重要的其他蛋白质的探索。在提议的研究中，我们将使用 TPC 和 NAADP 作为诱饵，从哺乳动物中分离出它们的相互作用伙伴 HEK293 和 SKBR3 细胞，然后使用无偏定量蛋白质组分析和功能测定来筛选并鉴定对 NAADP 诱发的 Ca2+ 信号传导重要的新型蛋白质。我们将追求以下目标两个具体目标。具体目标 1. 鉴定对 NAADP 诱发 Ca2+ 重要的新型 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。具有先进质谱分析的蛋白质组学方法。然后我们将进行 Ca2+ 成像功能测定鉴定出 TPC 相互作用蛋白，以鉴定对 NAADP 诱发的 Ca2+ 信号传导重要的新蛋白。具体目标 2. 鉴定对于 NAADP 诱发的 Ca2+ 信号转导很重要的 NAADP 相互作用蛋白。直接到针对 NAADP 受体，我们将对 HEK293 中的 NAADP 相互作用蛋白进行亲和纯化和 SKBR3 细胞，使用固定化的 NAADP 及其类似物，然后进行定量蛋白质组分析和功能测定来鉴定对 NAADP 诱导的 Ca2+ 释放重要的新蛋白质。拟议的研究旨在使用系统方法公正地筛选和识别重要的新型蛋白质用于 NAADP 诱发的 Ca2+ 释放。我们的结果可能会在分子基础和通过鉴定可作为 NAADP 受体 Ca2+- 的新蛋白来研究 NAADP 信号传导机制释放通道或 NAADP 诱发 Ca2+ 释放所需的调节蛋白。 1