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Cell survival upon starvation is dependent on the integrated stress response (ISR), a pro-survival signalling pathway which regulates protein transcription and translation when a number of diverse stresses are detected. A lynchpin of this pathway is the protein kinase General Control Non-derepressible 2 (GCN2), which is subject to investigation in my laboratory. GCN2 coordinates the ISR in response to amino acid deprivation, an event which often occurs in intracellular parasite infection  and diseases such as cancer . Activation of GCN2 leads to a global translational restructuring event via phosphorylation of eIF2α and subsequent activation of the transcription factor ATF4.
This studentship will focus on the role that the cellular cytoskeletal protein actin plays in the regulation of GCN2 and the ISR and will determine whether this regulatory system holds therapeutic potential.
The current model of actin-based regulation is dependent on the sequestration of the protein IMPACT (Yih1 in yeast) [3–5] (see Figure 1). However, there are several aspects of this model which require further investigation.
Figure 1: Model of Actin-mediated GCN2 regulation. Monomeric actin binds to IMPACT, sequestering it and allowing the formation of a GCN1/GCN2 heterodimer and the activation of the ISR. Upon filament formation, IMPACT is free to interact with GCN1, preventing GCN1/GCN2 formation and thus the inhibition of the ISR
Firstly, direct binding of IMPACT and actin has never been shown in vitro, and the interface between the two proteins is ambiguous – making mutagenesis studies of limited use . Currently, this interaction has only been inferred in yeast. The above model should also predict that GCN1 has a higher affinity for IMPACT rather than GCN2 – which is curious given that GCN1 and GCN2 are thought to be tightly associated on the eukaryotic ribosome. The student will recombinantly express and purify actin, IMPACT, GCN1 and GCN2 proteins in bacterial and insect cell expression systems. Using microscale thermophoresis and surface plasmon resonance, the relative affinities between IMPACT and GCN1 and actin will be investigated to determine whether the dynamics of this proposed model are feasible.
Secondly, and central to this exploration will be the use of Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to initiate structural biology investigations. The binding interfaces between IMPACT and actin will be investigated using HDX-MS. If stable heterodimers can be produced, these will be further investigated using x-ray crystallography and cryo-electron microscopy. Critically, the actin binding site can be used to design nullifying mutations which can be checked using the molecular interaction experiments described above. Likewise, the GCN1/IMPACT interface will be investigated in a similar manner.
Once interfaces are elucidated, mutagenesis work can be conducted. Select mutations, such as the D56A mutation on actin , are already implicated in regulating this process and will be investigated using MEK and MCF10a cells. Mutagenized cells will be evaluated for an intact stress response by exposing cells to small molecules capable of stimulating the stress response. Stress response activation is determined through use of an ATF4 transcription luciferase assay, as well as checking for the upregulation of downstream products of the ISR through the use of Western blots. Determining whether these mutations are capable of selectively disrupting the ISR will be key in establishing whether they are viable routes to future therapeutic development.
Students will gain expertise in genetic recombination, protein expression and purification, molecular interaction techniques, HDX-MS, mammalian cell culture, as well as potential experience in x-ray crystallography and cryo-EM.
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