title: The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.
abstract: Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.
authors.affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Miles
authors.affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Burr
authors.affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Guinevere L
authors.initials: GL
authors.lastname: Grice
authors.affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: James A
authors.identifier: 0000-0002-0248-1632
authors.initials: JA
authors.lastname: Nathan
mesh_terms: D000332:Aerobiosis; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D064828:Prolyl Hydroxylases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology* / Q000378:metabolism*
chemical_list: C000618034:Ccdc115 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C000618033:TMEM199 protein, human; D007501:Iron; D064828:Prolyl Hydroxylases; D025262:Vacuolar Proton-Translocating ATPases
keywords: CCDC115; HIF; Hypoxia Inducible factors; Iron; PHD; TMEM199; Vacuolar ATPase; Vma12; Vma22; biochemistry; cell biology; ferritinophagy; human; prolyl hydroxylation; transferrin; vATPase