Elucidating the Activation Cycle of stress-response kinase GCN2

Lead Research Organisation: University of Oxford
Department Name: Clinical Medicine

Abstract

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.

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/W509942/1 30/09/2021 29/09/2025
2596273 Studentship BB/W509942/1 30/09/2021 29/09/2025 Melissa Marx