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来自多个实验室的Cohen证据表明，该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了连接功能相关酶的有组织阵列的支架。该项目的目的是使用尿素周期作为实验模型来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，这些酶在两个细胞室中起作用。前两个反应是用线粒体基质中的酶和接下来的三种反应催化的。尽管所有酶都是可溶的（在没有洗涤剂的情况下会破坏细胞或细胞器时，它们会进入溶液），但该途径的原位高度组织；它作为一个功能单元，其中中间体在酶和隔室之间引导，并且三种细胞质酶在线粒体膜上依次组织。此外，与它们各自的蛋白一样，两种细胞质酶的mRNA，精氨酸酶酸酸合成酶（ASS）和精氨酸酶酶酸酯裂解酶（ASL）（ASL）位于线粒体旁边，表明这些蛋白质的翻译发生在其最终的细胞内肠内。这项研究的目的是：1）研究屁股和ASL mRNA定位的机制，通过：a）确定这些mRNA是否富集在特定的多多层人群中，其中一些人可能与线粒体的原位相关，以及/或与细胞骨架的特定成分相关； b）识别靶向涉及的mRNA序列； c）识别作为这些mRNA靶标的细胞成分。 2）确定如何保持ASS和ASL蛋白的定位。蛋白质蛋白质与彼此之间的蛋白质蛋白相互作用，或与线粒体外膜的成分，或在M Itochrial附近的内质网或细胞骨架的相互作用。 b）基因表达的两个杂交系统。目的是确定参与相互作用的特定蛋白质区域，并检测和表征与锚定酶有关的任何其他蛋白质。 %% %%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被组织成细胞中特定位置的多组分复合物。因为它允许它们更有效地发挥作用，所以可溶性酶系统的细胞内组织是细胞的重要和基本特征。确定组织的基础机制是细胞功能许多方面的一般利益的重要问题。这项研究将增加我们对尿素合成调节的了解和理解，这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，这些酶途径可能取决于特定的细胞内组织和本地化。 ***