Nutritional Control of Asparagine Synthetase

天冬酰胺合成酶的营养控制

• 批准号: 7367121
• 负责人: MICHAEL S. KILBERG
• 金额: $ 31.27万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367121
• 关键词: Affinity Chromatography; Amino Acids; Antibodies; Aspartate-Ammonia Ligase; Binding; Binding Proteins; Cell physiology; Cells; Chromatin Remodeling Factor; Co-Immunoprecipitations; Collection; Complex; Cultured Cells; DNA Binding; DNA Polymerase II; Elements; Embryo; Enhancers; Fibroblasts; Gene Expression; General Transcription Factors; Genes; Genetic Transcription; Glutathione S-Transferase; Histones; Holoenzymes; Human; Hybrids; Immunoblotting; Immunoprecipitation; In Vitro; Individual; Investigation; Length; Leucine Zippers; Mammals; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Metabolism; Modification; Mus; Nuclear; Nuclear Protein; Nuclear Proteins; Nutrient; Nutritional; Pathway interactions; Protein Binding; Protein Biosynthesis; Proteins; RNA; Rate; Recruitment Activity; Research Personnel; Response Elements; Role; Signal Transduction; Small Interfering RNA; Stress; Study models; Testing; Tissue-Specific Gene Expression; Transcriptional Regulation; Translations; Work; Yeasts; acid stress; biological adaptation to stress; cDNA Library; chromatin immunoprecipitation; chromatin remodeling; citrate carrier; deprivation; detection of nutrient; hepatoma cell; in vivo; member; nutrition; programs; promoter; protein S precursor; response; transcription factor

Protein/amino acid availability is a critical factor in general nutrition in mammals, yet there is relatively little known about the impact of amino acid fluctuation on cell functions, such as transcription. We use amino acid deprivation of cultured cells to investigate transcriptional control by nutrients. Limiting the amount of any single amino acid suppresses general protein synthesis, but actually promotes increased translation of a subset of mRNAs, including the transcription factor ATF4. Our hypothesis is that ATF4 is a critical mediator of the amino acid stress response pathway and that the human asparagine synthetase (ASNS) gene is a model for the study of this pathway. Our previous work has documented ATF4-mediated activation of the ASNS gene through the promoter cis-element Nutrient Sensing Response Element-1 (NSRE-1). In association with enhancer binding, a "pre-initiation complex" is assembled at eukaryotic promoters, which is comprised of at least four multi-protein complexes: a chromatin remodeling complex, a Mediator complex, a general transcription factor complex, and the RNA Pol II holoenzyme. The role of ATF4 and its sub-domains in factor recruitment to the ASNS promoter will be studied by chromatin immunoprecipitation (ChIP) in ATF4-/- fibroblasts and ATF4-siRNA treated HepG2 hepatoma cells (Specific Aim I). ATF4 binding proteins will be identified by yeast 2-hybrid and studied by co-IP, GST pull-down, and ChIP (Specific Aim II).To identify other proteins at the ASNS promoter, ATF4-containing complexes will be isolated, individual proteins identified by mass spectrometry, and the ATF4-interacting proteins studied by co-IP and ChIP (Specific Aim III). These studies will yield valuable information about the mechanisms by which nutritional signals mobilize the transcriptional machinery and thereby, alter cellular metabolism through gene expression. Given the central role of ATF4 in the response to amino acid limitation, and other forms of nutritional stress, we believe that it is critical to understand its function in the assembly of the general transcriptional machinery.

蛋白质/氨基酸的可用性是哺乳动物一般营养的关键因素，但相对较少 知道氨基酸波动对细胞功能的影响，例如转录。我们使用氨基酸 剥夺培养细胞以研究营养的转录控制。限制任何数量 单氨基酸抑制一般蛋白质的合成，但实际上促进了A的翻译增加 mRNA的子集，包括转录因子ATF4。我们的假设是ATF4是一个关键的中介者 氨基酸应激反应途径和人天冬酰胺合成酶（ASNS）基因是一种 研究该途径的模型。我们以前的工作已经记录了ATF4介导的激活 ASNS基因通过启动子CIS元素营养感应响应元件1（NSRE-1）。在 与增强子结合的关联，在真核启动子上组装了“预启动络合物”，即 由至少四种多蛋白络合物组成：染色质重塑络合物，介体配合物，A 一般转录因子复合物和RNA Pol II Holoenzyme。 ATF4及其子域的作用 在因子募集到ASNS启动子中，将通过染色质免疫沉淀（CHIP）研究 ATF4 - / - 成纤维细胞和ATF4-SIRNA处理的HEPG2肝癌细胞（特定的AIM I）。 ATF4结合蛋白 将通过酵母2杂交识别，并通过co-IP，GST下拉和芯片（特定的AIM II）进行研究。 在ASNS启动子处识别其他蛋白质，将分离含ATF4的复合物，单个蛋白质 通过质谱法鉴定，以及由Co-IP和CHIP研究的ATF4相互作用蛋白（特定的目标 iii）。这些研究将产生有关营养信号动员的机制的宝贵信息 转录机制，从而通过基因表达改变细胞代谢。鉴于 我们认为，ATF4在对氨基酸限制的反应和其他形式的营养应激中的核心作用，我们认为 了解其在一般转录机械组装中的功能至关重要。