Regulation of Gene Expression During Stress

应激期间基因表达的调节

• 批准号: 7900752
• 负责人: MARIA HATZOGLOU
• 金额: $ 10万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7900752
• 关键词: 3' Untranslated Regions; Abbreviations; Ablation; Affect; Amino Acid Sequence; Amino Acid Transporter; Amino Acids; Animal Model; Apoptosis; Arts; Attenuated; Bacterial Artificial Chromosomes; Binding Proteins; Boxing; CCAAT-Enhancer-Binding Proteins; Cationic Amino Acid Transporter 1; Cell Death; Cell Line; Cell Nucleus; Cell physiology; Cells; Cellular Stress; Cellular Stress Response; Cessation of life; Cultured Cells; Diabetes Mellitus; Dimerization; Disease; Embryo; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Equilibrium; Eukaryotic Initiation Factors; Felis catus; Fibroblasts; Firefly Luciferases; Future; GADD45; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Glutamic Acid; Goals; Grant; Homologous Gene; Insulin; Internal Ribosome Entry Site; Knock-in Mouse; Liver; Mediating; Membrane; Messenger RNA; Metabolism; Microarray Analysis; Modeling; Molecular; Mus; Mutation; N-terminal; Nerve Degeneration; Nutrient; Obesity; Open Reading Frames; Organelles; Pancreas; Pathology; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Prokaryotic Initiation Factor-2; Proline; Protein Biosynthesis; Protein Family; Proteins; Recovery; Regulation; Response Elements; Ribosomes; Role; Serine; Signal Pathway; Starvation; Stress; System; TNF gene; Testing; Thapsigargin; Threonine; Time; Trans-Activators; Transcription Coactivator; Transgenic Mice; Translating; Translations; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Ubiquitin; Untranslated Regions; activated Protein C; arginyllysine; biological adaptation to stress; cancer complication; driving force; endoplasmic reticulum stress; enhanced green fluorescent protein; factor C; human disease; interest; liver function; mRNA Decay; mouse model; multicatalytic endopeptidase complex; new therapeutic target; novel; overexpression; programs; protein degradation; research study; response; secretory protein; tool; transcription factor

DESCRIPTION (provided by applicant): In disease states, unfolded proteins accumulate in the endoplasmic reticulum because the folding capacity is exceeded, initiating a cellular stress response (the unfolded protein response or UPR). Our long-term interest is to understand the molecular mechanisms that allow cells to withstand stress and that contribute to pathologies during prolonged stress. Regulation of gene expression by transcriptional activators and repressors is a key feature of the stress response. During the previous grant period we found that the adaptive response to nutrient starvation increases expression of amino acid transporter genes, which can facilitate the recovery from stress. We also found that transcription of the arginine/lysine transporter gene, Cat-1, and of other genes involved in amino acid metabolism is attenuated during prolonged ER stress, mediated by the CCAAT/enhancer binding protein family transcription factor, C/EBP2. Regulated translation of the C/EBP2 mRNA produces both LAP, a transcriptional activator, and LIP a repressor. The LAP/LIP ratio plays a critical role in cell fate and metabolism. We found that the LAP/LIP ratios change during the UPR via mechanisms that involve proteasomal degradation of the proteins and translational control of the C/EBP2 mRNA; this provides the driving force behind this proposal. The regulation of transcription factor levels during ER stress via the proteasome pathway is a novel mechanism to modulate the cellular stress response. We hypothesize that the LAP/LIP ratio plays a role in controlling transcription of stress-response genes. We also hypothesize that the regulation of LIP levels promotes expression of prosurvival genes early in the stress response and restricts expression of proapoptotic genes during prolonged stress. In this proposal, we will study the mechanisms that regulate LIP synthesis and degradation during ER stress in cultured cells and in mice. Experiments using stress-inducing drugs and models of human disease will reveal the physiological significance of this regulation. Our Specific Aims are: (i) Determine the mechanism for diminished LIP levels during the early (prosurvival) phase of ER stress (ii) Investigate the signaling pathways that regulate proteasome-mediated degradation of LIP during the prosurvival phase of ER stress. (iii) Determine the mechanisms that increase LIP levels during the late (proapoptotic) phase of ER stress. (iv) Determine the physiological significance of the LAP/LIP ratio during ER stress using MEFs defficient in C/EBP2 (v) Determine the effect of disruption of the C/EBP2 gene in animal models of ER stress-mediated disease. Our long term goal is to generate transgenic mice expressing only LAP and only LIP by knock-in mutations in the C/EBP2 gene, using the state of the art system of Bacterial Artificial Chromosomes. The knock-in mice will be a valuable tool to determining the functions of LAP and LIP in ER- stress mediated apoptosis and enable us to test the findings of Aims 1-5 in a physiological context with relevance to human disease. Cellular stress is important in a large number of diseases, such as diabetes, neurodegeneration, cancer and complications of obesity. A common feature of these diseases is the accumulation of damaged secretory proteins in the endoplasmic reticulum (ER), a vital organelle responsible for proper cellular function and metabolism. During the previous grant period we found that the adaptive response to nutrient starvation increases expression of amino acid transporter genes, which can facilitate the recovery from stress. PROJECT HEALTH RELEVANCE: This proposal will study C/EBP2, an important regulator of the stress response and how this regulator controls the balance between cellular survival and death. Our studies will generate new therapeutic targets for the many stress-mediated diseases and provide a novel mechanism that regulates the balance between survival and death during these diseases.

描述（由申请人提供）：在疾病状态下，由于超出了折叠能力，因此在内质网中积累的蛋白质积累，启动了细胞应激反应（未折叠的蛋白质反应或UPR）。我们的长期兴趣是了解允许细胞承受压力的分子机制，并在长时间应激期间导致病理。转录激活因子和阻遏物调节基因表达是应力反应的关键特征。在上一个赠款期间，我们发现对营养饥饿的自适应反应增加了氨基酸转运蛋白基因的表达，这可以促进从应激中恢复。我们还发现，精氨酸/赖氨酸转运蛋白转运蛋白基因CAT-1和其他参与氨基酸代谢的基因的转录在长期的ER应激期间会减弱，这是由CCAAT/增强子结合蛋白结合蛋白家族转录因子C/EBP2介导的。 C/EBP2 mRNA的调节翻译既产生转录激活剂的膝盖，又产生唇部阻遏物。圈/唇比在细胞命运和代谢中起关键作用。我们发现，在UPR期间，LAP/LIP的比率通过涉及蛋白质蛋白质降解的机制和C/EBP2 mRNA的平移控制；这为该提案提供了推动力。通过蛋白酶体途径在ER应力期间转录因子水平的调节是调节细胞应激反应的新机制。我们假设膝上/唇比在控制应激反应基因的转录中起作用。我们还假设，唇部水平的调节在应力反应早期促进了生存基因的表达，并限制了长期应激期间促凋亡基因的表达。在此提案中，我们将研究调节培养细胞和小鼠的ER应激期间唇部合成和降解的机制。使用诱导压力的药物和人类疾病模型的实验将揭示该调节的生理意义。我们的具体目的是：（i）确定ER应力的早期（Provival）阶段（ii）调查调节蛋白酶体介导的唇部降解的信号传导途径在ER胁迫的Prosurvival阶段调节唇部降解的信号传导途径。 （iii）确定在ER应力的晚期（促凋亡）阶段增加唇部水平的机制。 （iv）使用MEF在C/EBP2（V）中脱离MEF在ER应力期间的膝上/唇比确定C/EBP2基因在ER胁迫介导的疾病的动物模型中的破坏作用的生理意义。我们的长期目标是使用细菌性人造染色体的最先进的状态在C/EBP2基因中产生仅表达膝盖的转基因小鼠和仅通过C/EBP2基因中的lip唇。敲门小鼠将是确定膝盖和唇py介导的凋亡中膝盖和唇的功能的宝贵工具，并使我们能够在生理环境中与与人类疾病相关的生理环境中测试目标1-5的发现。细胞应激在大量疾病中很重要，例如糖尿病，神经退行性，癌症和肥胖并发症。这些疾病的一个共同特征是内质网（ER）中受损的分泌蛋白的积累，这是负责适当细胞功能和代谢的重要细胞器。在上一个赠款期间，我们发现对营养饥饿的自适应反应增加了氨基酸转运蛋白基因的表达，这可以促进从应激中恢复。项目健康相关性：该建议将研究C/EBP2，这是压力反应的重要调节因子，以及该调节器如何控制细胞存活与死亡之间的平衡。我们的研究将为许多应力介导的疾病产生新的治疗靶标，并提供一种新的机制，以调节这些疾病期间的生存与死亡之间的平衡。