CELL CYCLE CONTROL OF ENDOPLASMIC RETICULUM STRUCTURE

内质网结构的细胞周期控制

• 批准号: 2176967
• 负责人: William Edward Balch
• 金额: $ 19.12万
• 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 1996-07-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176967
• 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.

全面了解细胞增殖的复杂性 肿瘤形成需要对生化和 控制细胞器结构和功能的分子事件 真核细胞的分泌途径。 的长期目标 我们的研究是为了了解结构和功能组织 内质网（ER）。 本次资助资助的研究 提出了我们开发的一种新的定量分析方法来研究 无细胞系统中控制 ER 结构的分子机制。 这 新的检测方法基于以下概念： ER是催化同源和异源寡聚体的折叠和组装 蛋白质。 使用由两种不同杂交瘤细胞制备的间期ER 表达重链（H）或轻链（L）的线 成熟的IgG分子，我们已经建立了体外条件 支持两个 ER 管腔内容物的融合和混合 区室导致 H 和 L 亚基寡聚成 成熟的 IgG 分子 (H2L2)。 我们的具体目标是彻底表征基本生化 ER 融合和 IgG 寡聚化的要求，以确定可溶性 和调节结构的膜相关成分 间期 ER。这些研究将包括高度富集的纯化 ER 组分、促进 ER 融合的测定条件优化、 H 和 L 链寡聚化的生化和动力学分析 成熟 IgG、能量需求表征以及分析 融合的特异性。 可溶性胞质和膜相关 促进 ER 融合的蛋白质将通过生化表征 关键成分的分级分离、分子克隆以及 制备用于研究的单克隆和多克隆抗体试剂 这些蛋白质的体外功能。 NSF、NSF 受体和 SNAP， 参与囊泡运输中膜融合的蛋白质将被 检查它们在内质网组装中的潜在作用。 此外，会员 将评估小 GTP 结合蛋白的 rab 基因家族 因其在 ER 结构调节中的潜在作用。 了解调节 ER 的生化机制的差异 细胞周期中的结构，我们将检验以下假设： 由 p34cdc2 激酶启动的激酶/磷酸酶级联负责 用于相关关键成分的磷酸化和去磷酸化 在进入和退出期间 ER 的拆卸和组装中 有丝分裂。 具体来说，我们将制备高度富集的中期 ER 和 细胞质和纯化的 p34cdc2 激酶用于研究细胞周期相关 导致 ER 拆卸或组装的修改。 这些 研究将与实验相结合，探索 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。