V-ATPase regulation of Hypoxia Inducible transcription Factors


Type
Thesis
Change log
Authors
Miles, Anna Louise 
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-α 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-α 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-α, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-α isoforms by altering the intracellular iron pool and preventing HIF-α 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.

Description
Date
2018-06-22
Advisors
Nathan, James
Keywords
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
Qualification
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Sponsorship
Medical Research Council Funding MR/K50127X/1