Regulation of ER stress-induced cell death

描述（由申请人提供）：糖尿病是一种世界性的流行病，由β细胞不能产生相对足够的胰岛素来维持正常血糖引起。最近的研究表明，营养波动和胰岛素抵抗驱动β细胞合成胰岛素超出其蛋白质折叠和分泌能力，并激活未折叠蛋白反应（UPR）。UPR是一种适应性信号通路，通过内质网（ER）中未折叠蛋白的积累来促进细胞存活。UPR的一个亚通路通过ER激酶PERK的激活和α亚基上真核翻译起始因子2的磷酸化来短暂地减弱蛋白质合成，从而减轻β细胞的生物合成负担。最近，我们证明β细胞选择性地需要eIF2a磷酸化和翻译衰减来保持细胞功能。然而，未解决的内质网功能障碍和UPR亚通路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。Chop缺失在胰岛素抵抗小鼠中显著增加β细胞质量和改善β细胞功能，以维持葡萄糖刺激的胰岛素分泌和预防糖尿病的进展。结果表明，抑制CHOP可能在治疗人类糖尿病中具有提高β细胞功能和/或质量的治疗价值。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Chop-/-小鼠的胰岛免受内质网中未折叠蛋白积累时发生的氧化应激的保护。此外，我们的初步未发表的研究结果表明，CHOP不显著结合编码凋亡功能的基因的启动子元件，而是结合编码蛋白质合成功能的基因的启动子。这些新发现激发了CHOP通过提高蛋白质合成引起氧化应激导致β细胞衰竭的假设，并提出了内质网中蛋白质错误折叠导致氧化应激的前所未有的联系。这些发现提供了一种前所未有的联系，即内质网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验证eIF2a磷酸化通过控制蛋白质合成来防止氧化应激的假设，而CHOP反过来通过促进蛋白质合成来诱导氧化应激。目标2：蛋白质折叠和氧化应激之间的关系是什么？我们将在这些研究中评估胰岛素原折叠和加工以及线粒体功能。目的3:eIF2a磷酸化和CHOP表达如何预防细胞死亡？我们将评估ATF4在细胞死亡中的作用，并筛选调节内质网应激诱导细胞死亡的基因。eIF2a磷酸化和CHOP表达如何改变基因转录？结合CHOP和ATF4的基因启动子的mRNA表达谱和分析将通过eIF2/ATF4/CHOP信号通路识别CHOP调控的转录网络。目标5，PERK/eIF2a/CHOP信号通路是否以定量和/或定性的方式改变mRNA的翻译？eIF2a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通过一种新的核糖体分析技术来测量。所有这些研究的结果将为了解eIF2a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细胞衰竭提供基本的见解和所需的信息，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。

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.