REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM

脯氨酸代谢的调节和划分

• 批准号: 2624601
• 负责人: MARJORIE C BRANDRISS
• 金额: $ 28.69万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2624601
• 关键词: Saccharomyces cerevisiae; aminoacid metabolism; biological signal transduction; conformation; intermolecular interaction; mass spectrometry; molecular cloning; nitrogen fixation; phosphorylation; proline; protein structure function; transcription factor; ubiquitin; western blottings; yeast two hybrid system

DESCRIPTION:The long term objective of this project is to understand molecular mechanisms that regulate gene expression in nitrogen assimilation, including the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. In the wild, proline is the predominant nitrogen source for this organism. Its utilization requires the exhaustion of preferred, but less abundant, nitrogen sources and induction of the specific genes that encode the enzymes of this pathway. Induction is mediated by the Put3 protein, a transcriptional regulator that sits poised at the promoters of its target genes, remaining inactive unless proline is present. The focus of this study is to determine how Put3p is converted from an "off" to an "on" state and how it senses that proline is present and preferred nitrogen sources are absent from the growth medium. Two general models will be tested: a protein-protein or intermolecular interaction model and a conformational change or intramolecular interaction model. Put3p is modified by phosphorylation and ubiquitination; the role of each in the activity of the protein will be tested. Wild-type, constitutive and non-inducible put3 mutants will be examined for differences in modification. Pure Put3p will be analyzed by mass spectrometry to identify sites of modification. Limited proteolysis will be employed to determine if Put3p undergoes conformational changes in the presence of proline. The effect of proline on activation of Put3p is an in vitro transcription system will be tested. Put3p-interacting proteins will be sought using two-hybrid selections employing fragments of Put3p as the bait, and by the isolation of suppressors of Put3p constitutive mutants. Intramolecular interactions will be examined by a directed two-hybrid approach using fragments of Put3p as both bait and prey. The roles of the five characterized nitrogen repression regulators in the proline utilization system will be studied. This system provides a useful model to understand how changes in the cellular environment are signaled to the elements of a metabolism in humans. The work in Saccharomyces cerevisiae on proline metabolism contributed significantly to the recent isolation of human genes encoding homologous enzymes. Deficiencies in these enzymes cause hyperprolinemias; studies on the human genes will lead to a greater understanding of the molecular basis of these diseases.

描述：该项目的长期目标是了解 在氮同化中调节基因表达的分子机制， 包括调节蛋白的作用，酶的区室化， 底物和代谢物通量的总体功能的代谢 通路 脯氨酸在酿酒酵母中的利用 作为本研究的模型系统。 在野外，脯氨酸是主要的 氮源为这一生物体。 它的使用需要用尽 优选的，但不太丰富的氮源和诱导， 编码这种途径的酶的特定基因。 归纳是 由Put 3蛋白介导，Put 3蛋白是一种转录调节因子， 在其靶基因的启动子上，除非脯氨酸被激活，否则保持不活动。 礼物 本研究的重点是确定Put 3 p是如何转换的 从“关”到“开”状态，以及它如何感觉到脯氨酸存在， 优选的氮源不存在于生长培养基中。 两个一般 将测试模型：蛋白质-蛋白质或分子间相互作用 模型和构象变化或分子内相互作用模型。 Put 3 p被磷酸化和泛素化修饰;每一个在 将测试蛋白质的活性。 野生型、组成型和 检查非诱导型Put 3突变体的修饰差异。 将通过质谱法分析纯Put 3 p以鉴定Put 3 p的位点。 改性 将采用有限的蛋白水解来确定Put 3 p是否 在脯氨酸的存在下经历构象变化。 的影响 脯氨酸对Put 3 p的激活是一种体外转录系统， 测试. Put 3 p相互作用的蛋白质将寻求使用双杂交 采用Put 3 p片段作为诱饵的选择，并通过分离Put 3 p片段， Put 3 p组成型突变体的抑制子。 分子内相互作用将 通过定向双杂交方法使用Put 3 p的片段作为 诱饵和猎物 五种特征性氮阻遏的作用 将研究脯氨酸利用系统中的调节剂。 该系统 提供了一个有用的模型来了解细胞内的变化， 环境的信号传递给人类新陈代谢的元素。 的 酿酒酵母中脯氨酸代谢的工作 重要的是最近分离的人类基因编码同源 内切酶 这些酶的缺陷导致高脯氨酸血症; 人类基因将使我们更好地理解 这些疾病。