Membrane Targeting and Retargeting of Polarity Proteins

极性蛋白的膜靶向和重靶向

• 批准号: 9897539
• 负责人: Yang Hong
• 金额: $ 30.31万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897539
• 关键词: Acute; Address; Adherens Junction; Allosteric Regulation; Apical; Binding; Biological; Biological Models; Brain Hypoxia-Ischemia; Cell Polarity; Cell membrane; Cells; Charge; Complex; Cultured Cells; Cytosol; Data; Defect; Development; Disease; Drosophila genome; Drosophila genus; Drosophila inturned protein; E-Cadherin; Electrostatics; Ensure; Epithelial; Epithelium; Genetic Models; Human Genome; Hypoxia; Integral Membrane Protein; Invertebrates; Ischemia; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Molecular; Nature; PAR-6 protein; Pathologic; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Phosphorylation; Phosphotransferases; Plasma Cells; Play; Proteins; Recovery; Regulation; Research; Role; Stress; Surface; Tertiary Protein Structure; Time; Tissues; Vertebrates; base; beta catenin; cell cortex; human disease; in vivo; novel; protein complex; protein protein interaction; recruit; tumorigenesis

Membrane Targeting and Retargeting of Polarity Proteins ABSTRACT Establishing and maintaining apical-basal polarity is essential for epithelial tissue integrity and function under both normal and stressed conditions. A group of so-called polarity proteins play essential and conserved functions in regulating cell polarity in both invertebrates and vertebrates. One key feature of all polarity proteins is that association with plasma membrane (PM) or cell cortex is critical for their in vivo functions. With few exceptions, most polarity proteins are assumed to be localized to PM/cell cortex through protein-protein interactions. Regulatable molecular mechanisms underlying the potential direct interaction between PM and majority of polarity proteins have long been elusive. Polybasic domains that are rich in positively charged Arg and Lyn residues have a well-established role in PM-specific targeting, based on its electrostatic interactions with negatively charged polyphosphoinositides PI4P and PIP2 that are uniquely enriched on the PM inner surface. However, for decades functional studies on polybasic domain in PM targeting have been limited to a small number of proteins of diverse functions and no polybasic domains had been characterized in any of the polarity proteins. We recently identified Lgl as the first polarity protein that contains an evolutionarily conserved and phosphorylatable polybasic domain whose electrostatic binding to PI4P and PIP2 on the PM directly mediate the PM localization of Lgl. In this proposal we have identified that multiple additional polarity proteins, as well as hundreds to thousands of proteins in Drosophila and human genomes, also contain potential polybasic domains. We will use both Drosophila and cultured mammalian cells to investigate the hypothesis that electrostatic binding between polybasic domain and plasma membrane serves a key regulatable molecular mechanism for controlling the subcellular localizations of multiple polybasic polarity proteins. We will first confirm the electrostatic binding of PM by multiple polybasic polarity proteins. More importantly, we will investigate the molecular mechanisms such as phosphorylation, allosteric regulation and coincident protein interactions that may control the direct binding between PM and polybasic polarity proteins to achieve their polarized subcellular localization and activations. Finally, our research highlighted for the first time that hypoxia and depletion of ATP acutely and reversibly inhibit polybasic domain proteins PM targeting through depleting PI4P and PIP2 on the PM, revealing a previously unappreciated but biologically significant challenge for cells to retarget polybasic polarity proteins to original PM domains after hypoxia/ischemia. We will focus on identifying mechanisms that actively direct the retargeting of polybasic polarity proteins to their original PM domains. Our research aims to establish a new paradigm regarding how regulatable binding between polybasic domains and PM serves as a fundamental mechanism to integrate the protein-protein interactions in achieving polarized PM targeting of polarity protein under both normal and stress conditions.

极性蛋白的膜靶向和重靶向 摘要 建立和维持顶-底极性对上皮组织的完整性和功能至关重要 在正常和压力条件下。一组所谓的极性蛋白质起着重要作用， 在无脊椎动物和脊椎动物中调节细胞极性的保守功能。所有的一个关键特征 极性蛋白的一个重要特征是与质膜（PM）或细胞皮层的结合对于它们在体内的表达至关重要。 功能协调发展的除了少数例外，大多数极性蛋白质被认为是定位于PM/细胞皮层，通过 蛋白质相互作用潜在直接相互作用的可调控分子机制 PM和大多数极性蛋白之间的联系一直是难以捉摸的。多基域富含 带正电荷的Arg和林恩残基在PM特异性靶向中具有良好的作用，这是基于其 与带负电荷的聚磷酸肌醇PI 4P和PIP 2的静电相互作用， 在PM内表面富集。然而，几十年来，对PM中多碱基结构域的功能研究 靶向仅限于少数不同功能的蛋白质， 在任何一种极性蛋白质中都有特征。 我们最近鉴定出Lgl是第一个含有进化上保守的和 可磷酸化的多元结构域，其与PM上的PI 4P和PIP 2的静电结合直接介导 Lgl的PM定位。在这个建议中，我们已经确定了多种额外的极性蛋白，以及 果蝇和人类基因组中的数百到数千种蛋白质， 域.我们将使用果蝇和培养的哺乳动物细胞来研究这一假设， 多碱基结构域与质膜之间的静电结合是一个关键的可调控分子， 控制多种多元极性蛋白亚细胞定位的机制。我们将首先 证实了PM与多种多元极性蛋白的静电结合。更重要的是，我们将 研究磷酸化、变构调节和重合蛋白等分子机制 可以控制PM和多元极性蛋白质之间的直接结合以实现其功能的相互作用。 极化的亚细胞定位和激活。最后，我们的研究首次强调了缺氧 ATP的耗竭通过耗竭多碱性结构域蛋白PM靶向， PM上的PI 4P和PIP 2，揭示了细胞在细胞增殖中以前未被认识到但具有生物学意义的挑战。 缺氧/缺血后将多元极性蛋白重新靶向原始PM结构域。我们将集中精力识别 主动引导多元极性蛋白重新靶向其原始PM结构域的机制。 我们的研究旨在建立一个新的范式，关于多元之间如何可调控的结合 结构域和PM作为整合蛋白质-蛋白质相互作用的基本机制，实现 在正常和胁迫条件下极性蛋白的极化PM靶向。