Elucidating the Activation Cycle of stress-response kinase GCN2

整合应激反应（ISR）是一种细胞内信号传导途径，其功能是解决细胞应激，如氨基酸缺乏，并与包括病毒感染、阿尔茨海默病和癌症在内的多种疾病的发展相关[1]。激酶一般控制非去阻遏蛋白2（GCN 2）在ISR中起关键作用，通过减少蛋白质产生同时增加氨基酸合成，从而恢复蛋白质稳态[2] XCN 2为380 kDa，由五个结构域组成，包括假激酶结构域、功能性激酶结构域和组氨酰-tRNA合成酶（HisRS）样结构域。GCN 2通过不带电荷的tRNA与HisRS样结构域结合而活化，允许激酶结构域二聚化并从惰性反平行构象转换为功能性平行构象[3，4]。然后，GCN 2磷酸化翻译起始因子2的α亚基（eIF 2 α），其抑制eIF 2-GDP通过鸟嘌呤交换因子B（eIF 2 B）再循环为eIF 2- GTP [1，3]。这减少了甲硫氨酰引发剂tRNA（Met-tRNAiMet）的募集和eIF 2-GTP-Met-tRNAiMet三元复合物的形成，从而减少了蛋白质合成[3]。eIF 2 α磷酸化还触发一般控制非去阻遏4（GCN 4）翻译和称为一般氨基酸控制的第二种细胞应答，最终导致氨基酸生物合成[5]。GCN 2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，对GCN 2更详细的结构和功能理解可能有助于开发治疗这些疾病的疗法。然而，虽然GCN 2的单个结构域的结构已经解析[3]，但关于其全长和结构域结构的结构信息仍然缺乏。与药物发现中心（CMD）和阿斯利康合作，该项目的目的是阐明GCN 2的多结构域构建体的结构，说明GCN 2活性的不同阶段，特别关注GCN 2假激酶结构域，来阐明它的规则。可以研究与相关结合伴侣复合的GCN 2的结构和功能表征，以实现对GCN 2结构域如何能够识别和响应ISR信号的机制理解，从而允许ISR的调节。为此，我将采用分子生物学和蛋白质生物化学技术在CMD，蛋白质晶体学和小角度X射线散射在钻石光源（DLS），低温电子显微镜在DLS和/或阿斯利康，和基于片段的命中发现方法在小分子药物发现的行业领先的自动化设施。Costa-Mattioli，M.沃尔特（P. Walter），《综合应激反应：从机制到疾病》（The Integrated Stress Response：From Mechanism to Disease）。科学，2020年。368（6489）.2. Hinnebusch，A.G.，GCN 4的翻译调控和酵母的一般氨基酸控制。Annu Rev Microbiol，2005. 59：第407-50.3页。Lageix，S.，例如，酵母Gcn 2的tRNA结合和C末端结构域之间的相互作用调节体内激酶活性。PLoS Genet，2015. 11（2）：p. e1004991.4. Padyana，A.K.，例如，真核生物起始因子2 α蛋白激酶GCN 2自身抑制和突变激活的结构基础。J Biol Chem，2005. 280（32）：第29289-99.5页。Dever，T. E.和A.G. Hinnebusch，GCN 2激起了对氨基酸的食欲。Mol Cell，2005. 18（2）：第141-2.6页。是的，J，例如，GCN 2-ATF 4通路对于肿瘤细胞在营养剥夺下的存活和增殖至关重要。EMBO J，2010年。29（12）：第2082-96.7页。Eyries，M.，例如，EIF 2AK 4突变导致肺静脉闭塞性疾病，一种隐性形式的肺动脉高压。Nat Genet，2014. 46（1）：第65-9.8页。拉文德兰河例如，氨基酸传感器GCN 2通过抑制炎性小体激活来控制肠道炎症。Nature，2016. 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.