Defining the role of bilayer lipid composition in nuclear envelope dynamics and genome protection

定义双层脂质成分在核膜动力学和基因组保护中的作用

• 批准号: 10361185
• 负责人: Shirin Bahmanyar
• 金额: $ 33.79万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10361185
• 关键词: Aging; Animal Model; Binding; Biochemistry; Biological Assay; Cells; Chromosome Segregation; Complex; Cytoplasm; DNA Damage; Data; Defect; Disease; Embryonic Development; Endoplasmic Reticulum; Ensure; Environment; Genetic Diseases; Genome; Genome Stability; Goals; Homeostasis; Human; Human Genetics; Human Pathology; Image; Immunoelectron Microscopy; In Vitro; Integral Membrane Protein; Interphase; Interphase Cell; Lead; Link; Lipid Bilayers; Lipid Binding; Lipids; Lipodystrophy; Maintenance; Malignant Neoplasms; Mediating; Membrane; Membrane Lipids; Metabolic; Microscopy; Mitosis; Mitotic; Molecular; Muscle; Muscular Dystrophies; Mutation; Nuclear; Nuclear Envelope; Nuclear Proteins; Nucleoplasm; Organelles; Peripheral; Phosphatidate Phosphatase; Phospholipids; Progeria; Property; Protein Engineering; Protein phosphatase; Proteins; Regulation; Resolution; Role; Rupture; Saccharomycetales; Structure; Testing; Textbooks; Viral; Work; cancer cell; cell motility; confocal imaging; design; env Gene Products; genome integrity; in vivo; lipid metabolism; lipine; live cell imaging; micronucleus; mutant; preservation; prevent; recruit; repaired

PROJECT DESCRIPTION The nuclear envelope is a subdomain of the endoplasmic reticulum (ER) that surrounds and protects the genome. Contrary to prior dogma, the nuclear envelope is not a stable structure, but is highly dynamic in both interphase and mitosis. Defects in nuclear envelope dynamics are common to cancer cells and to genetic disorders caused by mutations in nuclear envelope proteins that include muscular dystrophy and progeria. Thus, elucidating mechanisms that underlie nuclear envelope dynamics will inform on our understanding of disease mechanisms ranging from cancer to accelerated aging. An underlying principle that drives membrane dynamics at nearly all membrane-bound organelles is the composition of bilayer phospholipids, yet little is known about the role of phospholipids in nuclear envelope dynamics. This proposal focuses on a distinct regulator of phospholipid synthesis, CTDNEP1 (formerly known as Dullard), that is enriched at the nuclear envelope and is required for nuclear envelope dynamics. CTDNEP1 and its obligate binding partner NEP1R1 are a ubiquitously conserved protein phosphatase complex that activate the phosphatidic acid phosphatase lipin; lipin's activity is central to the de novo phospholipid synthesis. The work proposed here has two main aims: 1) to establish the functional significance of CTDNEP1-NEP1R1 localization at the nuclear envelope in local regulation of phospholipid synthesis and nuclear envelope dynamics and 2) to elucidate a role for nuclear envelope lipids in protecting genome integrity by regulating mitotic nuclear envelope reformation. Completion of these goals will elucidate a potentially significant, yet understudied role for lipid-mediated regulation of membrane dynamics at the nuclear envelope to ensure genome protection. Aim 1 will focus on the role of a conserved region in NEP1R1 that we discovered targets/retains CTDNEP1 in the nuclear envelope/ER in human cells. We will identify mutants in NEP1R1 that specifically disrupt enrichment of CTDNEP1-NEP1R1 at the nuclear envelope so that it is a more generic ER protein and assess the effects on lipid composition and nuclear envelope dynamics. Aim 2 builds on our preliminary data that CTDNEP1 regulates nuclear envelope reformation and prevents micronucleus formation in dividing cancer cells. These data suggest that core nuclear envelope proteins may be regulated by specific phospholipids to form the nuclear envelope from ER-derived membranes. We will use endogenous tags combined with conventional and super-resolution live-cell imaging to systematically define the localization and dynamics of core nuclear envelope proteins relative to nascent nuclear membranes during nuclear envelope reformation in wild type and ctdnep1-deficient cancer cells. In vitro lipid binding assays will identify domains in nuclear envelope proteins that directly bind to major phospholipids. Such studies will allow the design of mutant proteins with altered lipid binding properties to test for functional significance in ER membrane recruitment and remodeling to generate the nuclear envelope.

项目描述 核被膜是内质网（ER）的一个亚结构域，它包围并保护着细胞核。 基因组与先前的教条相反，核被膜不是一个稳定的结构，但在两者中都是高度动态的。 间期和有丝分裂。核膜动力学缺陷在癌细胞和遗传性肿瘤中是常见的。 由核膜蛋白突变引起的疾病，包括肌肉萎缩症和早衰症。 因此，阐明核膜动力学的基础机制将有助于我们理解 从癌症到加速衰老的疾病机制。驱动膜的基本原理 几乎所有的膜结合细胞器的动力学都是双层磷脂的组成，但很少有 已知磷脂在核膜动力学中的作用。该提案侧重于一个独特的 一种磷脂合成的调节因子，CTDNEP 1（以前称为Dullard），在细胞核中富集 这是核动力学所必需的。CTDNEP 1及其专性结合伴侣NEP 1 R1 是一种普遍保守的蛋白磷酸酶复合物，它激活磷脂酸磷酸酶 脂蛋白;脂蛋白的活性是从头磷脂合成的核心。这里提出的工作有两个主要方面 目的：1）确定CTDNEP 1-NEP 1 R1在核被膜定位的功能意义， 磷脂合成和核膜动力学的局部调节; 2）阐明核内磷脂合成和核膜动力学的作用。 包膜脂质通过调节有丝分裂核膜重组保护基因组完整性。完成 这些目标将阐明一个潜在的重要，但研究不足的作用，脂质介导的调节， 膜动力学在核膜，以确保基因组的保护。目标1将侧重于 在NEP 1 R1中的保守区域，我们发现靶向/保留CTDNEP 1在核膜/ER中， 人体细胞我们将鉴定NEP 1 R1中特异性破坏CTDNEP 1-NEP 1 R1富集的突变体， 核被膜，使其成为一种更通用的ER蛋白，并评估对脂质组成的影响， 核膜动力学目的2建立在我们的初步数据，CTDNEP 1调节核膜 重组和防止分裂癌细胞中微核的形成。这些数据表明， 包膜蛋白可由特定的磷脂调节，以从ER衍生的核被膜形成核膜。 膜。我们将使用内源性标记结合常规和超分辨率活细胞成像 系统地确定核心核膜蛋白相对于新生核的定位和动态， 在野生型和CTDNEP 1缺陷型癌细胞中核膜重组期间的核膜。在 体外脂质结合分析将鉴定核膜蛋白中直接结合主要脂质的结构域。 磷脂这些研究将允许设计具有改变的脂质结合特性的突变蛋白质来测试 在ER膜募集和重塑以产生核膜中的功能意义。