Organization of the Pathway of Urea Synthesis In Situ

尿素原位合成途径的组织

• 批准号: 9601421
• 负责人: Natalie Cohen
• 金额: $ 31.6万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9601421&HistoricalAwards=false
• 关键词: Organization; Pathway; Urea; Synthesis; Situ

9601421 Cohen Evidence from several laboratories has shown that the cell is a highly structured system, in which the cytoskeleton and intracellular membranes form a scaffold for the attachment of organized arrays of functionally related enzymes. The purpose of this project is to identify some of the mechanisms underlying the intracellular organization of soluble enzyme systems, using the urea cycle as an experimental model. The pathway of urea synthesis in mammalian liver consists of five enzymes, which operate in two cellular compartments; the first two reactions are catalyzed by enzymes in the mitochondrial matrix, and the next three by cytoplasmic enzymes. Although all the enzymes are soluble (they go into solution when cells or organelles are disrupted in the absence of detergent), the pathway is highly organized in situ; it behaves as a functional unit within which intermediates are channeled between enzymes and compartments, and the three cytoplasmic enzymes are sequentially organized at the mitochondrial membrane. In addition, like their respective proteins, the mRNAs of two of the cytoplasmic enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are localized next to mitochondria, indicating that the translation of these proteins occurs at their final intracellular site. The aims of this research are: 1) To investigate the mechanisms underlying the localization of ASS and ASL mRNAs, by: a) determining if these mRNAs are enriched in a particular polysome population, some of which may be associated in situ with mitochondria, and/or with a specific component of the cytoskeleton; b) identifying the mRNA sequences involved in targeting; and c) identifying the cellular component(s) that serve as the target for these mRNAs. 2) To determine how the localization of ASS and ASL protein is maintained. Protein-protein interactions of ASS and ASL with each other, or with a component of the mitochondrial outer membrane, or of the endoplasmic reticulum or cytoskeleton in the m itochondrial vicinity, will be studied in situ using: a) reversible cross-linking reagents and immunological methods; and b) the two-hybrid system of gene expression. The aim is to identify the specific protein regions participating in the interactions, and to detect and characterize any other proteins involved in anchoring the enzymes. %%% Living cells are highly structured systems, in which certain enzyme proteins that are related in their overall function are organized into multicomponent complexes in specific locations in the cell. Because it allows them to function more efficiently, the intracellular organization of soluble enzyme systems is a significant and basic feature of cells. Identification of the mechanisms underlying that organization is an important matter of general interest for many aspects of cell function. This research will increase our knowledge and understanding of the regulation of urea synthesis, a major function of mammalian liver. It will also provide basic information directly relevant to other enzyme pathways whose function may be dependent on specific intracellular organization and localization. ***

9601421 Cohen 来自多个实验室的证据表明，细胞是一个高度结构化的系统，其中细胞骨架和细胞内膜形成支架，用于附着有组织的功能相关酶阵列。该项目的目的是使用尿素循环作为实验模型，确定可溶性酶系统细胞内组织的一些机制。哺乳动物肝脏中尿素合成的途径由五种酶组成，它们在两个细胞区室中发挥作用；前两个反应由线粒体基质中的酶催化，接下来的三个反应由细胞质酶催化。尽管所有酶都是可溶的（当细胞或细胞器在没有去污剂的情况下被破坏时，它们会进入溶液），但该途径是在原位高度组织的；它充当一个功能单元，其中中间体在酶和区室之间引导，并且三种细胞质酶在线粒体膜上顺序组织。此外，与它们各自的蛋白质一样，两种细胞质酶精氨基琥珀酸合成酶 (ASS) 和精氨基琥珀酸裂解酶 (ASL) 的 mRNA 位于线粒体旁边，表明这些蛋白质的翻译发生在它们的最终细胞内位点。本研究的目的是： 1) 通过以下方式研究 ASS 和 ASL mRNA 定位的机制：a) 确定这些 mRNA 是否在特定的多核糖体群体中富集，其中一些可能与线粒体原位相关，以及/或具有细胞骨架的特定成分； b) 鉴定参与靶向的 mRNA 序列； c) 鉴定作为这些 mRNA 靶点的细胞成分。 2) 确定ASS和ASL蛋白的定位是如何维持的。 ASS 和 ASL 之间的蛋白质-蛋白质相互作用，或与线粒体外膜成分、或线粒体附近的内质网或细胞骨架的蛋白质-蛋白质相互作用，将使用以下方法进行原位研究：a) 可逆交联试剂和免疫学方法； b) 基因表达的双杂交系统。目的是识别参与相互作用的特定蛋白质区域，并检测和表征参与锚定酶的任何其他蛋白质。 %%% 活细胞是高度结构化的系统，其中某些与其整体功能相关的酶蛋白在细胞的特定位置组织成多组分复合物。由于可溶性酶系统的细胞内组织使它们能够更有效地发挥作用，因此它是细胞的一个重要且基本的特征。识别该组织的潜在机制是细胞功能许多方面普遍关注的重要问题。这项研究将增加我们对尿素合成调节的认识和理解，尿素合成是哺乳动物肝脏的主要功能。它还将提供与其他酶途径直接相关的基本信息，这些酶途径的功能可能依赖于特定的细胞内组织和定位。 ***