The Role of PtdIns-5-P in Cell Function and Signaling

PtdIns-5-P 在细胞功能和信号转导中的作用

基本信息

批准号：
8623255
负责人：
LUCIA RAMEH-PLANT
金额：
$ 19.45万
依托单位：
BOSTON MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8623255
关键词：
Role PtdIns Cell Function Signaling

项目摘要

DESCRIPTION (provided by applicant): Phosphoinositides (PIs) play critical roles in mediating a wide range of cellular responses to multiple hormones and growth factors. PI-modifying enzymes, such as kinases and phosphatases, along with PI-binding proteins, are directly involved in diseases such as cancer, diabetes and myopathies. Phosphatidylinositol-5-phosphate (PI-5-P) is the most recent PI to be identified; hence its function is poorly understood. PI-5-P has properties of a signaling molecule - its levels are low and can be rapidly regulated by extracellular cues. Remarkably, PI-5-P levels are up-regulated upon bacterial invasion of host cells, due to the activity of the bacterial virulence factors IpgD (from Shigella flexneri) and SigD/SopB (from Salmonella enterica), which are phosphatases that use PI-4,5-P2 as substrate. This pathological increase in PI-5-P levels is accompanied by dramatic changes in the actin cytoskeleton and in phosphoinositide 3-kinase (PI3k) signaling. On the other hand, PI-5-P levels can be down-regulated by PIP4k, an enzyme that converts PI-5-P into PI-4,5-P2 . The ultimate goal of this project is to understand the biological role of PI-5-P under physiological or pathological conditions. Our current hypothesis is that PI-5-P facilitates PI3k signaling by promoting local changes in the actin cytoskeleton. This hypothesis is based on evidence that both PI-5-P and PI-4,5-P2, together with the enzymes that catalyze the inter-conversion of these two lipids (PIP4k and IpgD), have been directly associated with actin rearrangements and PI3k signaling. Our specific aims are: (i) To identify the enzymes that regulate basal or inducible levels of PI-5-P. Using HPLC analysis of metabolically labeled lipids, we will measure the temporal changes in PI-5-P levels caused by extracellular stimuli and how expression or knockdown of various lipid kinases or phosphatases can affect this lipid. Using cell fractionation, we will determine the subcellular compartments where PI-5-P is present. (ii) To characterize the effect of IpgD-induced PI-5-P on vesicle trafficking. We will express wild-type IpgD or membrane-targeted-IpgD to induce PI-5-P accumulation in specific membrane compartments. Using cell fractionation, we will determine the subcellular localization of IpgD-induced PI-5-P. Using live-cell microscopy, we will monitor the effect of IpgD expression in membrane structure and vesicle trafficking. (iii) To determine the role of PI-5-P in actin rearrangement and in PI-3,4,5-P3 internalization. The effect of PI-5-P on actin filament dynamics will be determined in a cell-free system, using fluorescence and TIRF microscopy. Using cells stably expressing GFP-actin, we will analyze the changes in actin dynamics in real-time at the specific sub-membrane compartments where PI-5-P is being generated. Using PI-3,4,5-P3- specific probes we will investigate the effect of PI-5-P in the internalization and degradation of PI-3,4,5-P3. Unraveling the molecular mechanisms for PI-5-P function in cells will contribute to the resources available for safely intervening in disease states associated with malfunctions in cell-to-cell signaling. PUBLIC HEALTH RELEVANCE: Phosphoinositides are lipid molecules that play critical roles in cell-to-cell communication by working as intracellular messengers of hormones (e.g., insulin). In 1997, we discovered a new phosphoinositide in cells (PI-5-P) and, since then, we have found evidence that PI-5-P improves cellular response to insulin and promotes bacterial invasion. Our aim is to understand the function of this lipid under physiological and pathological conditions, with the goal of designing new strategies to prevent and treat diseases such as diabetes and bacterial infections.

描述（由申请人提供）：磷酸肌醇（PI）在介导多种细胞对多种激素和生长因子的反应中发挥着关键作用。 PI 修饰酶，例如激酶和磷酸酶，以及 PI 结合蛋白，直接参与癌症、糖尿病和肌病等疾病。 5-磷酸磷脂酰肌醇 (PI-5-P) 是最新确定的 PI；因此对其功能知之甚少。 PI-5-P 具有信号分子的特性 - 其水平较低并且可以通过细胞外信号快速调节。值得注意的是，由于细菌毒力因子 IpgD（来自福氏志贺菌）和 SigD/SopB（来自肠沙门氏菌）的活性，PI-5-P 水平在细菌入侵宿主细胞时上调，这些因子是使用 PI 的磷酸酶-4,5-P2 作为底物。 PI-5-P 水平的病理性增加伴随着肌动蛋白细胞骨架和磷酸肌醇 3-激酶 (PI3k) 信号传导的显着变化。另一方面，PI-5-P 水平可以被 PIP4k 下调，PIP4k 是一种将 PI-5-P 转化为 PI-4,5-P2 的酶。该项目的最终目标是了解 PI-5-P 在生理或病理条件下的生物学作用。我们目前的假设是 PI-5-P 通过促进肌动蛋白细胞骨架的局部变化来促进 PI3k 信号传导。该假设基于以下证据：PI-5-P 和 PI-4,5-P2 以及催化这两种脂质相互转化的酶（PIP4k 和 IpgD）与肌动蛋白重排直接相关，并且PI3k 信号传导。我们的具体目标是： (i) 鉴定调节 PI-5-P 基础或诱导水平的酶。使用代谢标记脂质的 HPLC 分析，我们将测量细胞外刺激引起的 PI-5-P 水平的时间变化，以及各种脂质激酶或磷酸酶的表达或敲低如何影响该脂质。通过细胞分级分离，我们将确定 PI-5-P 所在的亚细胞区室。 (ii) 表征 IpgD 诱导的 PI-5-P 对囊泡运输的影响。我们将表达野生型 IpgD 或膜靶向 IpgD 以诱导 PI-5-P 在特定膜区室中积累。使用细胞分级分离，我们将确定 IpgD 诱导的 PI-5-P 的亚细胞定位。使用活细胞显微镜，我们将监测 IpgD 表达对膜结构和囊泡运输的影响。 (iii) 确定 PI-5-P 在肌动蛋白重排和 PI-3,4,5-P3 内化中的作用。 PI-5-P 对肌动蛋白丝动力学的影响将在无细胞系统中使用荧光和 TIRF 显微镜来确定。使用稳定表达 GFP-肌动蛋白的细胞，我们将实时分析生成 PI-5-P 的特定亚膜区室的肌动蛋白动力学变化。使用 PI-3,4,5-P3 特异性探针，我们将研究 PI-5-P 在 PI-3,4,5-P3 内化和降解中的作用。阐明细胞中 PI-5-P 功能的分子机制将有助于提供可用于安全干预与细胞间信号传导故障相关的疾病状态的资源。公共健康相关性：磷酸肌醇是脂质分子，通过充当激素（例如胰岛素）的细胞内信使，在细胞间通讯中发挥关键作用。 1997年，我们在细胞中发现了一种新的磷酸肌醇（PI-5-P），从那时起，我们发现了PI-5-P改善细胞对胰岛素的反应并促进细菌侵袭的证据。我们的目标是了解这种脂质在生理和病理条件下的功能，目标是设计新的策略来预防和治疗糖尿病和细菌感染等疾病。