Regulation of Gene Expression During Stress

描述（由申请人提供）：在疾病状态下，未折叠蛋白质在内质网中积累，因为折叠能力被超过，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分子机制，使细胞能够承受压力，并有助于在长期压力的病理。转录激活因子和抑制因子对基因表达的调控是胁迫反应的一个重要特征。在前期研究中，我们发现，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，这可以促进应激的恢复。我们还发现，转录的精氨酸/赖氨酸转运蛋白基因，猫-1，和其他基因参与氨基酸代谢的衰减在长期的ER应激，介导的CCAAT/增强子结合蛋白家族转录因子，C/EBP 2。C/EBP 2 mRNA的调节翻译产生转录激活因子LIP和抑制因子LIP。LIP/LIP比率在细胞命运和代谢中起关键作用。我们发现，在UPR期间，通过涉及蛋白质的蛋白酶体降解和C/EBP 2 mRNA的翻译控制的机制，EBP/LIP比率发生变化;这提供了该提议背后的驱动力。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节细胞应激反应的一种新机制。我们假设，在控制应激反应基因的转录中，ESTA/LIP比率起着重要作用。我们还推测，LIP水平的调节促进了应激反应早期促生存基因的表达，并在长期应激过程中限制了促凋亡基因的表达。在这个提议中，我们将研究在培养的细胞和小鼠ER应激过程中调节LIP合成和降解的机制。使用应激诱导药物和人类疾病模型的实验将揭示这种调节的生理意义。我们的具体目标是：（i）确定在ER应激的早期（促存活）阶段期间LIP水平降低的机制（ii）研究在ER应激的促存活阶段期间调节蛋白酶体介导的LIP降解的信号传导途径。(iii)确定在ER应激的晚期（促凋亡）阶段增加LIP水平的机制。(iv)使用C/EBP 2缺乏的MEFs确定ER应激期间的EBP/LIP比率的生理意义（v）确定在ER应激介导的疾病的动物模型中破坏C/EBP 2基因的效果。我们的长期目标是使用最先进的细菌人工染色体系统，通过在C/EBP 2基因中敲入突变来产生仅表达LIP和仅表达LIP的转基因小鼠。敲入小鼠将是确定ER-应激介导的细胞凋亡中的LIP和LIP的功能的有价值的工具，并且使我们能够在与人类疾病相关的生理学背景下测试目的1 - 5的发现。细胞应激在许多疾病中是重要的，例如糖尿病、神经变性、癌症和肥胖症的并发症。这些疾病的共同特征是受损的分泌蛋白在内质网（ER）中的积累，内质网是负责适当细胞功能和代谢的重要细胞器。在前期研究中，我们发现，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，这可以促进应激的恢复。项目健康相关性：该提案将研究C/EBP 2，这是应激反应的重要调节因子，以及该调节因子如何控制细胞存活和死亡之间的平衡。我们的研究将为许多压力介导的疾病产生新的治疗靶点，并提供一种新的机制，调节这些疾病中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.