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V-ATPase regulation of Hypoxia Inducible transcription Factors 10.17863/CAM.30585 Miles, Anna Louise Nathan, James Metazoans have evolved conserved mechanisms to promote cell survival under low oxygen tensions by initiating a transcriptional cascade centered on the action of Hypoxia Inducible transcription Factors (HIFs). In aerobic conditions, HIFs are inactivated by ubiquitin-proteasome-mediated degradation of their a subunit, which is dependent on prolyl hydroxylation by 2-oxoglutarate (2-OG) and Fe(II)-dependent prolyl hydroxylases (PHDs). In hypoxia, HIF-$\alpha$ is no longer hydroxylated and is therefore stabilised, activating a global transcriptional response to ensure cell survival. Interestingly, HIFs can also be activated in aerobic conditions, however the mechanisms of this oxygen-independent regulation are poorly understood. Here, I have explored the role of the vacuolar H+-ATPase (V-ATPase), the major proton pump for acidifying intracellular vesicles and facilitating lysosomal degradation, in regulating HIF-$\alpha$ turnover. Unbiased forward genetic screens in near-haploid human cells identified that disruption of the V-ATPase leads to activation of HIFs in aerobic conditions. Rather than preventing the lysosomal degradation of HIF-$\alpha$, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-$\alpha$ isoforms by altering the intracellular iron pool and preventing HIF-$\alpha$ prolyl hydroxylation. In parallel, I characterised two putative mammalian V-ATPase assembly proteins, TMEM199 and CCDC115, identified by the forward genetic screen and subsequent mass spectrometry analysis. I confirmed that both TMEM199 and CCDC115 are required for V-ATPase function, and established assays to determine how TMEM199 and CCDC115 associate with components of the core V-ATPase complex. Lastly, to measure how V-ATPase activity leads to changes in the labile iron pool, I developed an endogenous iron reporter using CRISPR-Cas9 knock-in technology. This approach confirmed that iron homeostasis is impaired during V-ATPase inhibition, and demonstrated that exogenous ferric iron can restore the labile iron pool in a transferrin-independent manner. Together my studies highlight a crucial link between V-ATPase activity, iron homeostasis, and the hypoxic response pathway. University of Cambridge 2018-10-20 Thesis Doctoral Doctor of Philosophy (PhD) en Medical Research Council Funding MR/K50127X/1 https://www.repository.cam.ac.uk/handle/1810/283217 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cc7b6590-d1dc-442e-a813-2071ae1d2719/download 15d024959ae1a850c73b50110d9fa162 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1600eb22-80f1-45ea-9464-d75cac8005fe/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Figure 1.5 (Page 36) has been adapted from Peters et al., 2015, which is licensed under CC BY 3.0: The Company of Biologists Ltd, Biol. Open, Modeling dioxygenase enzyme kinetics in familial paraganglioma. Peters, J.P., Her, Y.F., and Maher, L.J. 4, 1281–1289 © (2015). Figure 1.6 (Page 37) has been adapted from Elkins et al., 2003. This research was originally published in the Journal of Biological Chemistry: J. Biol. Chem. Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha. Elkins, J.M., Hewitson, K.S., McNeill, L.A., Seibel, J.F., Schlemminger, I., Pugh, C.W., Ratcliffe, P.J., and Schofield, C.J. 278, 1802–1806. © (2003) the American Society for Biochemistry and Molecular Biology. Figure 1.8 (Page 41) has been adapted from Arosio et al., 2015. This figure was originally published in the Biochemical Journal: The importance of eukaryotic ferritins in iron handling and cytoprotection. Arosio, P., Carmona, F., Gozzelino, R., Maccarinelli, F., and Poli, M. 472, 1–15. © (2015). Figures 1.11 (Page 50) and 5.12 D (Page 151) have been adapted by permission from Zhao et al. 2015 (License Number 4437060560789): Springer Nature, Nature, Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Zhao, J., Benlekbir, S., and Rubinstein, J.L. 521, 241–245 © (2015). CCDC115 HIF Iron PHD TMEM199 Vacuolar ATPase Vma12p Vma22p Ferritinophagy Prolyl hydroxylation Transferrin Transferrin receptor Lysosomes Acidification Hypoxia Inducible Factors V-ATPase Proteasome Hypoxic response pathway Endo-lysosomal degradation IRP2 IRE