Structural insights into the eukaryotic General Amino Acid Control pathway

所有活细胞都必须以一种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），这是一个中枢信号网络，使细胞维持细胞稳态或进入凋亡。在后生动物中，ISR由四种不同的激酶组成，每种激酶磷酸化真核起始因子2 (eIF2) α亚基的丝氨酸51 （Ser51）。在酵母和哺乳动物细胞中，祖先的Gcn2（一般控制非抑制-2）激酶调节对营养剥夺的反应。在哺乳动物中，Gcn2对长期记忆的形成、摄食行为和免疫系统调节很重要，并且还与各种疾病有关，包括神经系统疾病（如阿尔茨海默氏症）、癌症以及病毒感染。Gcn2在营养剥夺过程中激活的主流模型是，Gcn2识别并结合在翻译过程中由于未带电（去酰化）trna结合到核糖体a位点的积累而停滞的核糖体。Gcn2的激活严格要求其共激活物Gcn1，这是一种从酵母到人类保存的大蛋白（酵母中有2,672个氨基酸或297 kDa）。尽管Gcn通路对ISR具有高度的保守性和重要性，并且对Gcn蛋白进行了数十年的研究，但其作用于核糖体的机制的结构基础一直缺乏。在这项提议中，我们计划利用携带内源性标记蛋白的菌株，结合亲和层析法，确定天然gcn1 - gcn2核糖体复合物的低温电镜结构。这些结构将为Gcn1如何监测和感知停滞的核糖体，以及招募和激活Gcn2以引发下游ISR提供急需的见解。

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.