Structural insights into the eukaryotic General Amino Acid Control pathway

真核通用氨基酸控制途径的结构见解

• 批准号: 468673669
• 负责人: Professor Dr. Daniel Nicodemus Wilson
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/468673669?language=en
• 关键词: Structural; insights; into; eukaryotic; General

All living cells must adapt to a variety of different environmental stresses in a rapid and efficient way to survive. Critical to this adaptation is the integrated stress response (ISR), a central signalling network that enables cells to maintain cellular homeostasis or enter into apoptosis. In metazoans, the ISR comprises four different kinases that each phosphorylate serine 51 (Ser51) of the alpha subunit of eukaryotic initiation factor 2 (eIF2). In yeast and mammalian cells, the ancestral Gcn2 (general control nonderepressible-2) kinase modulates the response to nutrient deprivation. In mammals, Gcn2 is important for long-term memory formation, feeding behaviour and immune system regulation, and has also been implicated in various diseases, including neurological disorders (such as Alzheimers), cancer as well as viral infection. The prevailing model for Gcn2 activation during nutrient deprivation is that Gcn2 recognizes and binds ribosomes that have become stalled during translation due to the accumulation of uncharged (deacylated) tRNAs binding to the ribosomal A-site. The activation of Gcn2 strictly requires its co-activator Gcn1, a large protein (2,672 amino acids or 297 kDa in yeast) conserved from yeast to humans. Despite the high conservation and importance of the Gcn pathway for the ISR, as well as decades of research into the Gcn proteins, a structural basis for their mechanism of action on the ribosome has been lacking. In this proposal, we plan to determine cryo-EM structures of native Gcn1-Gcn2-ribosome complexes using strains bearing endogenously tagged proteins, coupled with affinity chromatography. These structures will provide much needed insight into how Gcn1 monitors and sense stalled ribosomes, as well as recruit and activate Gcn2 to elicit the downstream ISR.

所有活着的细胞都必须以快速有效的方式适应各种不同的环境压力才能生存。这种适应的关键是综合应激反应(ISR)，这是一个中央信号网络，使细胞能够维持细胞内稳态或进入凋亡。在后生动物中，ISR由四种不同的激酶组成，每一种都磷酸化真核生物起始因子2(EIF2)的α亚基的丝氨酸51(Ser51)。在酵母和哺乳动物细胞中，祖先的GCN2(一般控制的不可降压-2)激酶调节对营养缺乏的反应。在哺乳动物中，GCN2对长期记忆形成、摄食行为和免疫系统调节非常重要，也与多种疾病有关，包括神经系统疾病(如阿尔茨海默氏症)、癌症以及病毒感染。在营养剥夺过程中，GCN2激活的流行模式是GCN2识别并结合核糖体，这些核糖体在翻译过程中因积累与核糖体A位点结合的无电荷(脱酰)tRNA而停滞不前。GCN2的激活严格需要它的辅助激活因子Gcn1，这是一种从酵母到人类保存的大蛋白(2672个氨基酸或297 kDa)。尽管GCN途径对ISR具有高度的保守性和重要性，以及对GCN蛋白数十年的研究，但它们对核糖体的作用机制缺乏结构基础。在这项建议中，我们计划使用携带内源性标记蛋白的菌株，结合亲和层析来确定天然Gcn1-GCN2-核糖体复合体的冷冻-EM结构。这些结构将为Gcn1如何监测和感测停滞的核糖体提供亟需的洞察力，以及招募和激活GCN2以引发下游的ISR。