Elongation factor 1A1 and the regulation of protein synthesis and cytoskeleton dynamics during lipid-induced stress

延伸因子(EEF)1A1在肝脏、胰腺和脑中表达，并通过促进AA-tRNA-核糖体结合参与蛋白质合成。EEF1A1还在肌动蛋白细胞骨架调节和应激反应中的细胞凋亡/失巢凋亡中发挥兼职作用。EEF1A1的典型和非典型功能是否都参与了细胞对代谢应激的反应尚不清楚。这个项目的目的是阐明蛋白质合成、肌动蛋白细胞骨架的调节和脂质超载诱导的细胞应激反应之间的联系。脂肪毒性是指非脂肪细胞中的异位脂肪沉积导致细胞功能障碍、细胞死亡，最终导致组织功能障碍的过程。由于内质网发生了许多脂质代谢过程，分泌细胞类型的脂毒状态伴随着内质网应激和未折叠蛋白反应(UPR)的快速诱导。UPR通过阻止蛋白质合成来恢复动态平衡，同时促进蛋白质折叠和降解。然而，严重或持续的应激会引发内质网应激反应(ERSR)，导致细胞死亡。ERSR包括重新启动蛋白质合成，以产生促炎症和促凋亡因子，一种有害的合成代谢反应。EEF1A1在内质网应激中被诱导，并被认为是脂毒性细胞死亡的关键介质。我们最近发现，用一种特殊的抑制剂白粉菌素B阻断EEF1A1的蛋白质合成活性，可以减少肝细胞中的脂毒性细胞死亡。此外，白鲜素B的急性干预改善了严重脂肪肝小鼠的肝脏内质网应激和相关炎症。但EEF1A1参与细胞应激反应和促进肝细胞脂毒性的确切机制尚不清楚。目前也不知道EEF1A1是否参与了其他富含内质网的细胞类型的脂毒性，特别是胰岛β细胞。因此，该计划的短期目标是确定EEF1A1在细胞对过度脂肪积累的反应中的比较功能(1)在培养的肝细胞和胰岛β细胞中，以及(2)在饮食诱导的组织脂肪积累的小鼠的肝脏和胰腺中。该计划的长期目标是了解EEF1A1在全身代谢组织(包括肝脏、胰腺、心肌和骨骼肌)对过量脂肪的细胞应激反应中的全球机制(S)，部分方法是使用独特的转基因Eef1a1-EGFP小鼠模型来测试全身Eef1a1的表达模式，以响应高脂饮食诱导的代谢应激。总之，这个项目将确定EEF1A1的规范和非规范功能如何参与内质网应激反应和细胞过度积累所导致的细胞死亡过程，从而加深我们对脂毒性和EEF1A1的不同功能的理解。

Elongation factor (EEF) 1A1 is expressed in liver, pancreas, and brain, and participates in protein synthesis by promoting aa-tRNA-ribosome binding. EEF1A1 also has moonlighting' functions in actin cytoskeleton regulation, and in apoptosis/anoikis in response to stress. Whether both the canonical and non-canonical functions of EEF1A1 are involved in cellular responses to metabolic stress is unclear. The goal of this program is to elucidate the links between protein synthesis, regulation of the actin cytoskeleton, and cellular stress responses induced by lipid overload. Lipotoxicity is the process whereby ectopic lipid deposition in non-adipose cells leads to cellular dysfunction, cell death, and eventual tissue dysfunction. Since many lipid metabolic processes occur at the ER, lipotoxic conditions in secretory cell types are accompanied by rapid induction of ER stress and the unfolded protein response (UPR). The UPR restores homeostasis by halting protein synthesis, while promoting protein folding and degradation. However, severe or prolonged stress initiates an ER stress response (ERSR) which can lead to cell death. The ERSR involves re-initiation of protein synthesis to generate pro-inflammatory and pro-apoptotic factors a deleterious anabolic response. EEF1A1 is induced in upon ER stress, and is known to be a key mediator of lipotoxic cell death. We recently found that blocking the protein synthetic activity of EEF1A1 with a specific inhibitor, didemnin B, decreased lipotoxic cell death in hepatocytes. Moreover, acute intervention with didemnin B improved hepatic ER stress and associated inflammation in mice with severe fatty liver. But the precise mechanisms through which EEF1A1 participates in cellular stress responses and promotes hepatocyte lipotoxicity are unclear. It is also not known whether EEF1A1 participates in lipotoxicity in other ER-rich cell types, particularly pancreatic beta-cells. Thus the short term objectives of this program are to determine the comparative functions of EEF1A1 in cellular responses to excess lipid accumulation (1) in cultured hepatocytes and pancreatic beta-cells, and (2) in liver and pancreas of mice with diet-induced tissue lipid accumulation. The long term program goal is to understand the global mechanism(s) of action of EEF1A1 in cellular stress responses to excess lipid in metabolic tissues throughout the body, including liver, pancreas, myocardium, and skeletal muscle, in part by using a unique transgenic Eef1a1-EGFP mouse model to test whole body Eef1a1 expression patterns in response to metabolic stress induced by high fat diet. Overall, this program will determine how the canonical and non-canonical functions of EEF1A1 are involved in ER stress response and cell death processes induced by excessive cellular lipid accumulation, thereby furthering our understanding of lipotoxicity and the diverse functions of EEF1A1.