{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/283217"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/283217","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"V-ATPase regulation of Hypoxia Inducible transcription Factors","abstract":"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.","abstract_html":"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-<span class=\"etd-inline-math\">&alpha;</span> 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-<span class=\"etd-inline-math\">&alpha;</span> 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-<span class=\"etd-inline-math\">&alpha;</span>, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-<span class=\"etd-inline-math\">&alpha;</span> isoforms by altering the intracellular iron pool and preventing HIF-<span class=\"etd-inline-math\">&alpha;</span> 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.","abstract_has_math":true,"creators":["Miles, Anna Louise"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Nathan, James"],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-10-20","date_published":"2018-10-20","updated_at":"2026-07-22T22:24:10Z","subjects":["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"],"languages":["en"],"rights":["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)."],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1600eb22-80f1-45ea-9464-d75cac8005fe/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.30585","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Nathan, James"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Medical Research Council Funding MR/K50127X/1"]},{"key":"dc:creator","label":"Author","values":["Miles, Anna Louise"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2018-10-20"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/283217"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["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"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1600eb22-80f1-45ea-9464-d75cac8005fe/download","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)."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.30585"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cc7b6590-d1dc-442e-a813-2071ae1d2719/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["15d024959ae1a850c73b50110d9fa162","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["V-ATPase regulation of Hypoxia Inducible transcription Factors"]}]}],"canonical_facts":{"dc:contributor.advisor":["Nathan, James"],"dc:contributor.sponsor":["Medical Research Council Funding MR/K50127X/1"],"dc:creator":["Miles, Anna Louise"],"dc:date.issued":["2018-10-20"],"dc:description.abstract":["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."],"dc:format.checksum.md5":["15d024959ae1a850c73b50110d9fa162","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["10.17863/CAM.30585"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cc7b6590-d1dc-442e-a813-2071ae1d2719/download"],"dc:language":["en"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/283217"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1600eb22-80f1-45ea-9464-d75cac8005fe/download","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)."],"dc:subject":["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"],"dc:title":["V-ATPase regulation of Hypoxia Inducible transcription Factors"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:10Z"}