Organization of the Pathway of Urea Synthesis In Situ

尿素原位合成途径的组织

• 批准号: 9983005
• 负责人: Natalie Cohen
• 金额: $ 33.81万
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9983005&HistoricalAwards=false
• 关键词: Organization; Pathway; Urea; Synthesis; Situ

The cell is a highly structured complex system, in which ultrastructural elements formscaffolds for the attachments of organized arrays of functionally-related enzymes and otherproteins. The purpose of this project is to identify mechanisms underlying the intracellularorganization of soluble enzyme systems, with the urea cycle as a model. Thepathway of urea synthesis in mammalian liver consists of five enzymes in two cellularcompartments; the first two enzymes are in the mitochondrial matrix, and the next three are inthe cytoplasm. Although all the enzymes are soluble (they go into solution when cells ororganelles are disrupted in the absence of detergent), biochemical studies have demonstratedthat the pathway is highly organized in situ, behaving as a functional unit within which intermediates are channeled between enzymes and compartments. Those studies showed thatthe three cytoplasmic enzymes are sequentially organized at the mitochondrial membrane. Itwas also shown that, like their respective proteins, the mRNA's of two of the cytoplasmicenzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), are localizednext to the mitochondria. The 3'untranslated region (3'UTR) of ASS mRNA binds specifically toa protein complex that is located on the outside of liver mitochondria. The specific aims of this project focus on studies of the mechanisms of ASS and ASL mRNA localization and of the macromolecular interactions that maintain the localization of the proteins in situ. There are four aims:1. To determine the sequence(s) in ASS and ASL mRNA's responsible for localizing thesemessages around liver mitochondria in situ, and the functional role of the protein-bindingsequences of the 3'UTR's. This will be done by constructing vectors containing variousmRNA sequences fused to the coding sequence of green fluorescent protein (GFP), transfectingthese into hepatocytes in culture, and determining the location of the GFP in the cells bystandard fluorescence and confocal microscopy.2. To characterize the ASS mRNA 3'UTR binding complex and/or peptides. The complexand any other specifically-binding peptides will be purified on RNA affinity columns, and characterized by SDS-PAGE, isoelectric focusing, and N-terminal sequencing. This will be followed by library screening and cloning and sequencing of the genes.3. To determine if ASS and ASL mRNA localization is required for urea cycle function.The mRNA targeting sequences will be overexpressed in cultured hepatocytes, to compete withendogenous mRNA for localization. After inducing increased endogenous expression of theurea cycle enzymes, the ability of these cells to synthesize urea will be measured.4. To identify and characterize other cellular components that may interact with ASS andASL proteins to maintain the localization of the latter in situ, and to begin to identify theinteracting domains of ASS and ASL. The two-hybrid method will be used to screen a livercDNA library for components interacting with ASS or ASL. These will be cloned, sequenced, andoverexpressed, and the proteins characterized as described above for Aim 2. Site-directedmutagenesis of selected regions of ASS and ASL, and analysis of the effects of the mutationson two-hybrid interactions will be used to identify interacting regions.The intracellular organization of soluble enzyme systems is a significant and basic featureof cells. Identification of the underlying mechanisms is an important matter of general interestin the fields of metabolic biochemistry, cell biology, differentiation, and signal transduction.These studies will increase our knowledge and understanding of the regulation of urea synthesis, a major function of mammalian liver. The studies will also provide basic information directly relevant to other cytoplasmic enzymes known to be associated with the mitochondrial outer membrane, and to other metabolic pathways whose function may be dependent on specific enzyme organization and localization.

细胞是一个高度结构化的复杂系统，其中超微结构元素形成支架，用于附着有组织的功能相关酶和其他蛋白质阵列。该项目的目的是以尿素循环为模型，确定可溶性酶系统的细胞内组织的潜在机制。哺乳动物肝脏中尿素合成的途径由两个细胞室中的五种酶组成；前两种酶位于线粒体基质中，后三种位于细胞质中。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），但生化研究表明，该途径在原位高度组织化，表现为中间体在酶和区室之间引导的功能单元。这些研究表明，三种细胞质酶在线粒体膜上顺序排列。还表明，像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位于线粒体附近。 ASS mRNA 的 3' 非翻译区 (3'UTR) 与位于肝线粒体外部的蛋白质复合物特异性结合。 该项目的具体目标侧重于研究 ASS 和 ASL mRNA 定位的机制以及维持蛋白质原位定位的大分子相互作用。有四个目标： 1．确定 ASS 和 ASL mRNA 中负责将这些信息原位定位在肝线粒体周围的序列，以及 3'UTR 的蛋白质结合序列的功能作用。这将通过构建包含与绿色荧光蛋白（GFP）编码序列融合的各种mRNA序列的载体，将其转染到培养物中的肝细胞中，并通过标准荧光和共聚焦显微镜确定GFP在细胞中的位置来完成。2．表征 ASS mRNA 3'UTR 结合复合物和/或肽。复合物和任何其他特异性结合的肽将在 RNA 亲和柱上纯化，并通过 SDS-PAGE、等电聚焦和 N 末端测序进行表征。随后将进行文库筛选、基因克隆和测序。3．确定尿素循环功能是否需要 ASS 和 ASL mRNA 定位。mRNA 靶向序列将在培养的肝细胞中过表达，与内源 mRNA 竞争定位。诱导尿素循环酶内源表达增加后，将测量这些细胞合成尿素的能力。4．鉴定和表征可能与 ASS 和 ASL 蛋白相互作用的其他细胞成分，以维持后者的原位定位，并开始鉴定 ASS 和 ASL 的相互作用域。两种杂交方法将用于筛选肝cDNA文库中与ASS或ASL相互作用的成分。这些将被克隆、测序和过表达，并且蛋白质的特征如上述目标 2 所示。ASS 和 ASL 选定区域的定点诱变，以及突变对两种杂交体相互作用的影响分析将用于鉴定相互作用区域可溶性酶系统的细胞内组织是细胞的一个重要且基本的特征。识别潜在机制是代谢生物化学、细胞生物学、分化和信号转导领域普遍关注的重要问题。这些研究将增加我们对尿素合成（哺乳动物肝脏的主要功能）调节的认识和理解。这些研究还将提供与已知与线粒体外膜相关的其他细胞质酶以及其功能可能依赖于特定酶组织和定位的其他代谢途径直接相关的基本信息。