Regulation of Gene Expression During Stress

描述(申请人提供)：在疾病状态下，未折叠的蛋白质由于超出折叠能力而积聚在内质网中，启动细胞应激反应(未折叠蛋白质反应或UPR)。我们的长期兴趣是了解允许细胞承受压力的分子机制，以及在长期压力下导致病理变化的分子机制。转录激活因子和转录抑制因子对基因表达的调控是应激反应的一个重要特征。在之前的资助期间，我们发现对营养饥饿的适应性反应增加了氨基酸转运蛋白基因的表达，这有助于从应激中恢复。我们还发现，在持续的内质网应激过程中，精氨酸/赖氨酸转运蛋白基因Cat-1和其他参与氨基酸代谢的基因的转录在CCAAT/增强子结合蛋白家族转录因子C/EBP2的介导下减弱。C/EBP2 mRNA的调节翻译产生了LAP和LIP，LAP是一种转录激活因子，LIP是一种抑制因子。LAP/LIP比率在细胞命运和新陈代谢中起着关键作用。我们发现，在UPR过程中，LAP/LIP比率通过蛋白质的蛋白酶体降解和C/EBP2 mRNA的翻译控制的机制发生变化；这为这一提议提供了推动力。在内质网应激过程中，通过蛋白酶体途径调节转录因子水平是调控细胞应激反应的新机制。我们假设LAP/LIP比率在控制应激反应基因的转录中发挥作用。我们还假设，在应激反应早期，LIP水平的调节促进了促生存基因的表达，并在长时间的应激中限制了促凋亡基因的表达。在这项提案中，我们将研究在培养细胞和小鼠ER应激过程中调节嘴唇合成和降解的机制。使用压力诱导药物和人类疾病模型的实验将揭示这一调节的生理意义。我们的具体目标是：(I)确定内质网应激早期(生存)阶段LIP水平降低的机制(Ii)研究在内质网应激前存期调节蛋白酶体介导的LIP降解的信号通路。(Iii)确定在内质网应激的后期(促凋亡)阶段增加LIP水平的机制。(4)利用C/EBP2基因缺失的MEF，确定ER应激过程中LAP/LIP比值的生理意义；(5)确定C/EBP2基因突变在ER应激介导疾病动物模型中的作用。我们的长期目标是利用最先进的细菌人工染色体系统，通过C/EBP2基因的敲入突变来产生只表达LAP和LIP的转基因小鼠。敲入小鼠将是一个有价值的工具，以确定LAP和LIP在内质网应激介导的细胞凋亡中的功能，并使我们能够在与人类疾病相关的生理背景下测试AIMS 1-5的发现。细胞应激在许多疾病中都很重要，如糖尿病、神经退行性变、癌症和肥胖并发症。这些疾病的一个共同特征是受损的分泌蛋白在内质网(ER)中积累，内质网是负责正常细胞功能和新陈代谢的重要细胞器。在之前的资助期间，我们发现对营养饥饿的适应性反应增加了氨基酸转运蛋白基因的表达，这有助于从应激中恢复。项目健康相关性：这项提案将研究C/EBP2，应激反应的重要调节因子，以及该调节因子如何控制细胞生存和死亡之间的平衡。我们的研究将为许多应激介导的疾病产生新的治疗靶点，并提供一种新的机制，在这些疾病中调节生存和死亡之间的平衡。

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.