CELL CYCLE CONTROL OF ENDOPLASMIC RETICULUM STRUCTURE

内质网结构的细胞周期控制

• 批准号: 3282844
• 负责人: William Edward Balch
• 金额: $ 16.92万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282844
• 关键词: Golgi apparatus; adenosine triphosphate; cell cycle; cell free system; cell growth regulation; clone cells; endoplasmic reticulum; enzyme linked immunosorbent assay; enzyme substrate complex; guanine nucleotide binding protein; hybridomas; immunoglobulin G; laboratory mouse; laboratory rabbit; laboratory rat; membrane fusion; membrane structure; membrane transport proteins; molecular cloning; monoclonal antibody; okadaic acid; phosphorylation; posttranslational modifications; protein folding; protein kinase; protein purification; protein structure function; vesicle /vacuole

A complete understanding of the complexities of cellular proliferation and neoplasia will require a thorough analysis of the biochemical and molecular events governing the structure and function of organelles of the secretory pathway in eukaryotic cells. The long range objective of our research is to understand the structural and functional organization of the endoplasmic reticulum (ER). The research supported by this grant presents a new quantitative assay we have developed to study the molecular mechanisms governing ER structure in a cell-free system. This new assay is based on the concept that one of the major functions of the ER is to catalyze the folding and assembly of homo- and heterooligomeric proteins. Using interphase ER prepared from two different hybridoma cell lines which express either the heavy (H) or light (L) chains of the mature IgG molecule, we have established in vitro conditions which support the fusion and mixing of the lumenal contents of the two ER compartments leading to the oligomerization of the H and L subunits into the mature IgG molecule (H2L2). Our specific aims are to thoroughly characterize the basic biochemical requirements for ER fusion and IgG oligomerization to define the soluble and membrane-associated components which regulate the structure of interphase ER.These studies will include purification of highly enriched ER fractions, optimization of assay conditions which promote ER fusion, biochemical and kinetic analysis of H and L chain oligomerization into mature IgG, characterization of energy requirements, and analysis of specificity of fusion. The soluble cytosolic and membrane-associated proteins promoting ER fusion will be characterized by biochemical fractionation of the key components, their molecular cloning, and preparation of both monoclonal and polyclonal antibody reagents to study the function of these proteins in vitro. NSF, NSF receptor and SNAPs, proteins involved in membrane fusion in vesicular trafficking, will be examined for their potential role in ER assembly. In addition, members of the rab gene family of small GTP-binding proteins will be evaluated for their potential role in regulation of ER structure. To understand the differences in the biochemical mechanisms regulating ER structure during the cell, cycle, we will examine the hypothesis that a kinase/phosphatase cascade initiated by the p34cdc2 kinase is responsible for the phosphorylation and dephosphorylation of key components involved in the disassembly and assembly of the ER during entry and exit from mitosis. Specifically, we will prepare highly enriched metaphase ER and cytosol, and purified p34cdc2 kinase to study cell cycle related modifications leading to the disassembly or assembly of the ER. These studies will be pursued in conjunction with experiments which explore the ability of the phosphatase inhibitor okadaic acid (OKA) to inhibit ER assembly in vitro, potentially revealing a cell cycle specific kinase controlling ER structure.

完全了解细胞增殖的复杂性 和肿瘤将需要对生化和 控制细胞器的结构和功能的分子事件 真核细胞中的分泌途径。 远程目标 我们的研究是了解结构和功能组织 内质网（ER）的。 这项赠款支持的研究 提出了一种新的定量测定，我们已经开发了 在无细胞系统中控制ER结构的分子机制。 这 新测定是基于这样的概念 ER将催化同型和异性物体的折叠和组装 蛋白质。 使用从两个不同杂交瘤细胞制备的相间ER 表达重量（h）或光（l）链的线条 成熟的IgG分子，我们已经建立了体外条件 支持两个ER的腔内含量的融合和混合 将H和L亚基的寡聚化导致隔室 成熟的IgG分子（H2L2）。 我们的具体目的是彻底表征基本的生化 ER融合和IgG低聚以定义可溶性的要求 和与膜相关的组件调节 这些研究将包括纯化高度富集 ER分数，优化促进ER融合的测定条件， H和L链寡聚的生化和动力学分析 成熟的IgG，能量需求的表征和分析 融合的特异性。 可溶性胞质和膜相关 促进ER融合的蛋白质将以生化为特征 关键成分的分馏，它们的分子克隆和 进行研究的单克隆和多克隆抗体试剂的制备 这些蛋白质在体外的功能。 NSF，NSF受体和快照， 在囊泡运输中涉及膜融合的蛋白质将是 检查了它们在ER组装中的潜在作用。 此外，成员 将评估小GTP结合蛋白的Rab基因家族 因为它们在ER结构调节中的潜在作用。 了解调节ER的生化机制的差异 细胞期间的结构，循环，我们将研究以下假设。 由p34CDC2激酶发起的激酶/磷酸酶级联反应是负责 涉及的关键成分的磷酸化和去磷酸化 在进入和退出期间的ER拆卸和组装中 有丝分裂。 具体而言，我们将准备高度富集的中期ER和 细胞质和纯化的p34CDC2激酶研究细胞周期 修改导致ER的拆卸或组装。 这些 研究将与探索探索的实验一起进行。 磷酸酶抑制剂冈田酸（OKA）抑制ER的能力 体外组装，可能揭示细胞周期特异性激酶 控制ER结构。