Elucidating the Activation Cycle of stress-response kinase GCN2

整合应激反应(ISR)是一种细胞内信号通路，其功能是解决细胞应激，如氨基酸缺乏，并与病毒感染、阿尔茨海默氏症和癌症等多种疾病的发生有关。GCN2在ISR中起着关键作用，它通过减少蛋白质的合成而增加氨基酸的合成，从而恢复蛋白质的动态平衡[2]。GCN2全长380 kDa，由5个结构域组成，包括一个假性激酶域、一个功能激活域和一个类似组氨酰-tRNA合成酶(HisRS)的结构域。GCN2是通过不带电荷的tRNA与HisRS样结构域结合而激活的，允许激活域从惰性反平行构象到功能平行构象的二聚化和切换[3，4]。然后，GCN2将翻译起始因子2的α亚基(eIF2α)磷酸化，从而抑制鸟嘌呤交换因子B(EIF2B)[1，3]将eIF2-GDP循环为eIF2-GTP。这减少了甲硫基启动子tRNA(Met-tRNAiMet)的招募和eIF2-GTP-Met-tRNAiMet三元复合体的形成，减少了蛋白质的合成[3]。EIF2α的磷酸化也触发了一般控制不可降压4(GCN4)的翻译和第二个细胞反应，称为一般氨基酸控制，最终导致氨基酸生物合成[5]。GCN2活性的异常具有广泛的影响，包括肠道炎症、肺动脉高压和肿瘤增殖[6-8]。因此，更详细地了解GCN2的结构和功能可能有助于开发治疗这些疾病的方法。然而，虽然GCN2的单个结构域的结构已经被解析[3]关于它的全长和结构域结构的信息仍然缺乏。该项目的目的是与药物发现中心(CMD)和阿斯利康合作，阐明GCN2的多结构域结构，说明GCN2活性的不同阶段，特别是GCN2假激活域，以阐明其调控。可以研究GCN2与相关结合伙伴形成的复合体中GCN2的结构和功能特征，以实现对GCN2结构域如何识别和响应ISR信号的机械理解，从而实现对ISR的调控。为此，我将在CMD的行业领先的自动化设施中使用分子生物学和蛋白质生物化学技术，在钻石光源(DLS)使用蛋白质结晶学和小角X射线散射，在DLS和/或阿斯利康(AstraZeneca)使用冷冻电子显微镜，以及在小分子药物发现中使用基于片段的命中查找方法1。科斯塔-马蒂奥利，M.和P.沃尔特，《综合应激反应：从机制到疾病》。科学，2020年。368(6489)1.Gcn4的翻译调控和酵母的一般氨基酸调控。《微生物年鉴》，2005。59：第407-50.3页。Lageix，S.等人，酵母GCN2的tRNA结合域和C-末端结构域之间的相互作用调节活体中的激酶活性。PLOS Genet，2015年。11(2)：第E1004991.4页。Padana，A.K.，等，真核细胞起始因子2α蛋白激酶GCN2的自抑制和突变激活的结构基础。生物化学杂志，2005。第280(32)页：29289-99.5.Dever，T.E.和A.G.Hinnebusch，GCN2刺激了人们对氨基酸的胃口。摩尔细胞，2005。18(2)：第141-2.6页。GCN2-ATF4通路对于肿瘤细胞在营养缺乏时的生存和增殖至关重要。EMBO J，2010年。29(12)：第2082-96.7页。EIF2AK4突变导致肺静脉闭塞疾病，这是一种隐性的肺动脉高压。纳特·吉内特，2014。46(1)：第65-9.8页。Ravindran，R.等人，氨基酸传感器GCN2通过抑制炎症体激活来控制肠道炎症。《自然》，2016年。531(7595)：第523-527页。

The integrated stress response (ISR) is an intracellular signaling pathway which functions to resolve cell stress such as amino acid deficiency, and is associated with the development of several diseases including viral infection, Alzheimer's and cancer [1]. The kinase general control non-derepressible 2 (GCN2) plays a key role in the ISR, by reducing protein production while increasing amino acid synthesis, thereby restoring protein homeostasis [2].GCN2 is 380 kDa, consisting of five domains including a pseudo-kinase domain, a functional kinase domain, and a Histidyl-tRNA synthetase (HisRS)-like domain. GCN2 is activated by binding of uncharged tRNA to the HisRS-like domain, allowing dimerization and switching of the kinase domain from the inert anti-parallel conformation to the functional parallel conformation [3, 4]. GCN2 then phosphorylates the alpha subunit of the translation initiation factor 2, (eIF2 alpha) which inhibits the recycling of eIF2-GDP to eIF2- GTP by guanine exchange factor B (eIF2B) [1, 3]. This decreases recruitment of the methionyl initiator tRNA (Met-tRNAiMet) and formation of the eIF2-GTP- Met-tRNAiMet ternary complex, reducing protein synthesis [3]. eIF2 alpha phosphorylation also triggers general control non-derepressible 4 (GCN4) translation and a second cellular response known as general amino acid control, culminating in amino acid bio-synthesis [5]. Aberrations in GCN2 activity have widespread effects including intestinal inflammation, pulmonary hypertension and tumour proliferation [6-8]. As such, a more detailed structural and functional understanding of GCN2 may assist the development of therapies to treat these diseases. However, while structures of GCN2 individual domains have been resolved [3] structural information regarding its full-length and domain architecture is still lacking.In collaboration with the Centre for Medicines Discovery (CMD) and AstraZeneca, the aim of this project is to elucidate the structures of multi-domain constructs of GCN2 illustrating different stages of GCN2 activity, with particular focus on the GCN2 pseudo-kinase domain, to shed light on its regulation. Structural and functional characterisation of GCN2 in complex with relevant binding partners may be investigated to achieve a mechanistic understanding of how GCN2 domains enable recognition of and respond to ISR signals, allowing regulation of the ISR. To this end, I will employ molecular biology and protein biochemistry techniques in an industry-leading automated facility at the CMD, protein crystallography and small-angle X-ray scattering at Diamond Light Source (DLS), cryo-electron microscopy at DLS and/or AstraZeneca, and fragment-based hit-finding approaches in small-molecule drug discovery.1. Costa-Mattioli, M. and P. Walter, The integrated stress response: From mechanism to disease. Science, 2020. 368(6489).2. Hinnebusch, A.G., Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol, 2005. 59: p. 407-50.3. Lageix, S., et al., Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo. PLoS Genet, 2015. 11(2): p. e1004991.4. Padyana, A.K., et al., Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2alpha protein kinase GCN2. J Biol Chem, 2005. 280(32): p. 29289-99.5. Dever, T.E. and A.G. Hinnebusch, GCN2 whets the appetite for amino acids. Mol Cell, 2005. 18(2): p. 141-2.6. Ye, J., et al., The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation. EMBO J, 2010. 29(12): p. 2082-96.7. Eyries, M., et al., EIF2AK4 mutations cause pulmonary veno-occlusive disease, a recessive form of pulmonary hypertension. Nat Genet, 2014. 46(1): p. 65-9.8. Ravindran, R., et al., The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature, 2016. 531(7595): p. 523-527.