Regulation of ER stress-induced cell death

说明（由申请人提供）：糖尿病是一种世界范围流行的疾病，其来源于β细胞不能产生相对充足的胰岛素以维持正常。最近的研究表明，营养波动和胰岛素抵抗驱动β细胞合成胰岛素超过其蛋白质折叠和分泌的能力，并激活未折叠蛋白质反应（UPR）。UPR是一种适应性信号通路，在内质网（ER）中积累未折叠蛋白后促进细胞存活。UPR的子途径通过ER激酶PERK的活化和α亚基上真核翻译起始因子2的磷酸化发出信号，以瞬时减弱蛋白质合成，从而降低β细胞的生物合成负担。最近，我们证明了β细胞选择性地需要eIF 2a的磷酸化和翻译减弱来保护细胞功能。然而，未解决的ER功能障碍和UPR子途径的慢性激活增加了促凋亡转录因子CAAT增强子结合蛋白同源蛋白（CHOP）的表达。胰岛素抵抗小鼠中的Chop缺失显著增加β细胞质量并改善β细胞功能，以维持葡萄糖刺激的胰岛素分泌并预防糖尿病进展。结果表明，抑制CHOP可能在治疗人类糖尿病以增加β细胞的功能和/或质量方面具有治疗价值。为了实现这一潜力，有必要阐明CHOP如何导致β细胞衰竭。为了实现这一目标，我们已经证明，来自Chop-/-小鼠的胰岛受到保护，不受ER中未折叠蛋白积累时发生的氧化应激的影响。此外，我们的初步未发表的研究结果表明，CHOP不显着结合基因的启动子元件编码凋亡功能，而是结合基因的启动子编码蛋白质合成的功能。这些新的发现激发了CHOP通过提高蛋白质合成引起氧化应激而导致β细胞衰竭的假设，并提出了ER中蛋白质错误折叠引起氧化应激的前所未有的联系。这些发现提供了一个前所未有的联系，通过这种联系，ER中的蛋白质合成和蛋白质折叠导致氧化应激。为了验证这一假设，我们提出了五个具体的目标来回答五个基本问题。目的1、CHOP表达如何诱导氧化应激和β细胞衰竭？我们使用遗传学和药理学方法来检验eIF 2a磷酸化通过控制蛋白质合成来防止氧化应激，而CHOP反过来通过促进蛋白质合成来诱导氧化应激的假设。目的2、蛋白质折叠与氧化应激的关系是什么？我们将在这些研究中评估胰岛素原的折叠和加工以及线粒体功能。目的3，eIF 2a磷酸化和CHOP表达如何阻止细胞死亡？我们将评估ATF 4在细胞死亡中的作用，并筛选调控内质网应激诱导细胞死亡的基因。目的4，eIF 2a磷酸化和CHOP表达如何改变基因转录？结合CHOP和ATF 4的基因启动子的mRNA表达谱分析和分析将识别由CHOP通过eIF 2/ATF 4/CHOP信号传导调节的转录网络。目的5，PERK/eIF 2a/CHOP信号通路是否以定量和/或定性的方式改变mRNA翻译？eIF 2a磷酸化和CHOP表达对mRNA翻译效率和AUG起始密码子选择的影响将使用新的核糖体分析技术进行测量。所有这些研究的结果将提供基本的见解和所需的信息，以了解eIF 2a磷酸化和翻译控制如何维持β细胞功能，以及CHOP如何导致β细胞衰竭，并应鼓励开发小分子来调节UPR，以保护人类糖尿病患者的β细胞功能和质量。 公共卫生相关性：糖尿病是一种世界性的流行病，其特征在于由β细胞不能产生胰岛素引起的葡萄糖稳态异常。最近的研究表明，蛋白质合成的调节对于β细胞提高胰岛素产生和β细胞存活是必不可少的。拟议中的蛋白质合成研究将提供重要信息，鼓励开发治疗糖尿病的新疗法。

DESCRIPTION (provided by applicant): Diabetes is a disease of world-wide epidemic proportion derived from a failure of beta cells to produce relatively sufficient insulin to maintain euglycemia. Recent studies indicate that nutrient fluctuations and insulin resistance drive beta cells to synthesize insulin beyond their capacity for protein folding and secretion and activates the unfolded protein response (UPR). The UPR is an adaptive signaling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). A subpathway of the UPR is signaled through activation of the ER kinase PERK and phosphorylation of eukaryotic translation initiation factor 2 on the alpha subunit to transiently attenuate protein synthesis, thereby reducing the biosynthetic burden on the beta cell. Recently, we demonstrated that beta cells selectively require phosphorylation of eIF2a and translation attenuation to preserve cell function. However, unresolved ER dysfunction and chronic activation of UPR subpathways increases expression of the proapoptotic transcription factor CAAT-enhancer binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases beta cell mass and improves beta cell function to maintain glucose-stimulated insulin secretion and prevent progression of diabetes. The results suggest that inhibition of CHOP may have therapeutic value in treatment of human diabetes to increase the function and/or mass of beta cells. To realize this potential, it is necessary to elucidate how CHOP causes beta cell failure. In pursuit of this goal, we have demonstrated that islets from Chop-/- mice are protected from oxidative stress that occurs upon accumulation of unfolded proteins in the ER. In addition, our preliminary unpublished findings show that CHOP does not significantly bind promoter elements of genes that encode apoptotic functions, but rather binds to promoters of genes that encode functions in protein synthesis. These novel findings inspire the hypothesis that CHOP causes beta cell failure by elevating protein synthesis to cause oxidative stress, and suggest an unprecedented link by which protein misfolding in the ER causes oxidative stress. The findings provide an unprecedented link by which protein synthesis and protein folding in the ER causes oxidative stress. In order to test this hypothesis we propose five specific aims to answer five fundamental questions. Aim 1, How does CHOP expression induce oxidative stress and beta cell failure? We use both genetic and pharmacological approaches to test the hypothesis that eIF2a phosphorylation prevents oxidative stress through control of protein synthesis and that CHOP conversely induces oxidative stress by promoting protein synthesis. Aim 2, What is the relationship between protein folding and oxidative stress? We will evaluate proinsulin folding and processing and mitochondrial function in these studies. Aim 3, How do eIF2a phosphorylation and CHOP expression prevent cell death? We will evaluate the role of ATF4 in cell death and screen for genes that regulate ER stress induced cell death. Aim 4, How do eIF2a phosphorylation and CHOP expression alter gene transcription? mRNA expression profiling and analysis of gene promoters that bind CHOP and ATF4 will identify the transcriptional network regulated by CHOP by eIF2/ATF4/CHOP signaling. Aim 5, Does the PERK/eIF2a/CHOP signaling pathway alter mRNA translation in a quantitative and/or qualitative manner? The impact of eIF2a phosphorylation and CHOP expression on mRNA translational efficiency and AUG initiation codon selection will be measured using a new technique of ribosome profiling. The results from all these studies will provide fundamental insight and needed information toward an understanding of how eIF2a phosphorylation and translational control maintain beta cell function and how CHOP causes beta cell failure and should encourage the development of small molecules to modulate the UPR to preserve beta cell function and mass in human diabetes patients. PUBLIC HEALTH RELEVANCE: Diabetes is a world-wide epidemic characterized by an abnormality in glucose homeostasis that results from a failure of beta cells to produce insulin. Recent studies indicate that regulation of protein synthesis is essential for beta cells to elevate insulin production and for beta cell survival. The proposed studies on protein synthesis will provide vital information that should encourage the development of novel therapeutics to treat diabetes.