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Genetic and chemical perturbation of amino acid sensing by the GCN1-GCN2 pathway
Genetic and chemical perturbation of amino acid sensing by the GCN1-GCN2 pathway
Amino acid stress activates the GCN2-regulated branch of the integrated stress response (ISR). This ancient pro-survival signaling network is conserved across eukaryotes to react to cellular stress by controlling proteostasis. Mechanistically, GCN2 blocks translation by phosphorylating the initiation factor eIF2α and simultaneously activating an ATF4-dependent transcriptional program for stress adaptation. The amino acid response was initially discovered in Saccharomyces cerevisiae, where the HEAT-repeat protein GCN1 was suggested to regulate the GCN2 activation at the ribosomal machinery. The master regulator of cell growth, mTORC1, is another amino acid sensing hub, which also modulates translation. However, the molecular and mechanistic events that lead to the mammalian GCN2-ISR activation, its connection to the mTORC1 pathway and the outcomes in terms of cell state adaptation remain elusive. In this PhD thesis, I investigated the GCN2-ISR by genetic and chemical perturbation in diverse murine cell systems. My major focus was to dissect the interplay of GCN1, GCN2 and mTORC1 upon amino acid stress. Using CRISPR/Cas9-genetically modified cell lines, I found that GCN1 acts upstream of GCN2 to regulate its autophosphorylation and ultimately the ATF4-induced transcriptional response in an eIF2α independent way. Using a multi-omics approach, I show that GCN1 and GCN2 are isogenic in regulating the ISR in response to leucine stress by controlling transcriptome and proteome changes over time. Processes involved in mitochondrial 1C- metabolism, amino acid uptake, tRNA synthetases and glutamine metabolism are modulated at the gene and protein level in a GCN1 and/or GCN2 dependent way. Furthermore, I provide evidence that both proteins have a distinct bioenergetic profile – already at steady-state. I also show that GCN1 can be involved in the DNA damage response by physically interacting with the MRN complex in a transient way. I also highlight that the ISR can play a role in ferroptosis regulation in an ATF4-SLC7A11-dependent manner. Moreover, this thesis suggests that a direct interaction of GCN1 with GCN2 and the ribosome is unlikely. In a 3,876 compound GCN2 inhibitor screen, I discovered that dual mTOR inhibitors concurrently block the mTORC1 and the GCN2 branches of amino acid sensing upon amino acid stress. This effect was not mediated by direct GCN2-binding and independent of PERK and eIF2α. Instead, these results suggest a role of mTOR in modulating the activation of GCN2 upon prolonged leucine stress. Finally, I provide new insights on the involvement of GCN1 in the mammalian ISR and potential GCN2- and amino acid stress-independent functions. Moreover, I discovered an unexpected interplay of the GCN1-GCN2 and the mTORC1 amino acid sensing pathways, which is of high importance for understanding how these complex multiprotein kinases integrate nutrient sensing. This finding paves a new frontier for mTOR and GCN2 anti-neoplastic drug development for the selective targeting of amino acid-dependent cell protection pathways in cancer.
GCN2, amino acid response, GCN1, mTORC1, amino acid sensing and signaling, ATF4, GCN2 inhibitors, translational control
Brüggenthies, Johanna Barbara
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Brüggenthies, Johanna Barbara (2021): Genetic and chemical perturbation of amino acid sensing by the GCN1-GCN2 pathway. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Amino acid stress activates the GCN2-regulated branch of the integrated stress response (ISR). This ancient pro-survival signaling network is conserved across eukaryotes to react to cellular stress by controlling proteostasis. Mechanistically, GCN2 blocks translation by phosphorylating the initiation factor eIF2α and simultaneously activating an ATF4-dependent transcriptional program for stress adaptation. The amino acid response was initially discovered in Saccharomyces cerevisiae, where the HEAT-repeat protein GCN1 was suggested to regulate the GCN2 activation at the ribosomal machinery. The master regulator of cell growth, mTORC1, is another amino acid sensing hub, which also modulates translation. However, the molecular and mechanistic events that lead to the mammalian GCN2-ISR activation, its connection to the mTORC1 pathway and the outcomes in terms of cell state adaptation remain elusive. In this PhD thesis, I investigated the GCN2-ISR by genetic and chemical perturbation in diverse murine cell systems. My major focus was to dissect the interplay of GCN1, GCN2 and mTORC1 upon amino acid stress. Using CRISPR/Cas9-genetically modified cell lines, I found that GCN1 acts upstream of GCN2 to regulate its autophosphorylation and ultimately the ATF4-induced transcriptional response in an eIF2α independent way. Using a multi-omics approach, I show that GCN1 and GCN2 are isogenic in regulating the ISR in response to leucine stress by controlling transcriptome and proteome changes over time. Processes involved in mitochondrial 1C- metabolism, amino acid uptake, tRNA synthetases and glutamine metabolism are modulated at the gene and protein level in a GCN1 and/or GCN2 dependent way. Furthermore, I provide evidence that both proteins have a distinct bioenergetic profile – already at steady-state. I also show that GCN1 can be involved in the DNA damage response by physically interacting with the MRN complex in a transient way. I also highlight that the ISR can play a role in ferroptosis regulation in an ATF4-SLC7A11-dependent manner. Moreover, this thesis suggests that a direct interaction of GCN1 with GCN2 and the ribosome is unlikely. In a 3,876 compound GCN2 inhibitor screen, I discovered that dual mTOR inhibitors concurrently block the mTORC1 and the GCN2 branches of amino acid sensing upon amino acid stress. This effect was not mediated by direct GCN2-binding and independent of PERK and eIF2α. Instead, these results suggest a role of mTOR in modulating the activation of GCN2 upon prolonged leucine stress. Finally, I provide new insights on the involvement of GCN1 in the mammalian ISR and potential GCN2- and amino acid stress-independent functions. Moreover, I discovered an unexpected interplay of the GCN1-GCN2 and the mTORC1 amino acid sensing pathways, which is of high importance for understanding how these complex multiprotein kinases integrate nutrient sensing. This finding paves a new frontier for mTOR and GCN2 anti-neoplastic drug development for the selective targeting of amino acid-dependent cell protection pathways in cancer.