Elucidation of oxygen sensing mechanisms in human and animal cells - PowerPoint PPT Presentation

Elucidation of oxygen sensing mechanisms in human and animal cells Peter J. Ratcliffe Nobel Lecture - December 2019 Oxford-Yale expedition to Pike’s Peak, Colorado J. S. Haldane and colleagues - July 1911

O 2 Sensor Epo Extra-ordinary sensitivity of the erythropoietin response to changes in blood oxygen availability – responses to donation of blood Lorentz et al. Transfusion 31 650-654 (1991)

Development of a cellular model for study of Epo regulation by Oxygen Transgenic marker gene strategy indicates that erythropoietin producing cells are Interstitial fibroblasts

Widespread operation of hypoxia signalling pathways Mouse Epo gene Mapping of oxygen regulated oxygen sequences by transient transfection Oxygen Oxygen // insensitive sensitive cell cell reporter gene

EPO HIF

Regulation of HIF by oxygen Cellular Oxygen iron/cobalt availability HIF- α Gal4 DNA binding domain HSVP16 transactivator Signal HIF- α Regulated activity Regulated protein stability

Sequencing the human Multiple hypoxia inducible genome suggests existence transcripts identified of HIF paralogues Transcripts constitutively Making anti-EPAS antibodies upregulated in VHL (PM9) proves regulation by oxygen defective cells VHL- VHL+ Role in Cancer Distortion from HIF-1 to Oxygen sensing Treatment by HIF-2 HIF-2 during RCC pathway antagonists development

Signalling modification is prolyl hydroxylation Biochemical analysis Heat labile extract Non-enzymatic oxidation NADH/NADHoxidase Oxygen Iron ATP Treatment HIF with cell peptide extract VHL

HIF prolyl hydroxylation implies a mechanism of oxygen sensing O 2 Iron HIF prolyl 2- oxoglutarate hydroxylase Cobalt Hyp Pro HIF - α VHL ubiquitin Proteolysis ligase

HIF prolyl hydroxylases - a set of Fe(II) and 2-oxoglutarate dioxygenases that are conserved throughout the animal kingdom 6 8 2 4 Fe(II) 3 7 5 1

O 2

HIF-2 mRNA HIF-2 IHC

Protein Oxidation in Signalling hypoxia Evolutionary Origins? All eukaryotic kingdoms use protein oxidation and proteolysis to signal oxygen levels Protists Plants Animals Funghi Dictyostelium Arabidopsis Homo sapiens Schizosaccharomyces discoideum thaliana pombe Cysteine Prolyl 4 Prolyl 4 Ofd1 - Prolyl 3 oxidases hydroxylase hydroxylase hydroxylase Hypoxia Skp1 ubiquitin MCxxx N-end SRE inducible factors ligase rule proteolysis VHL ubiquitin Culmination Ethylene Sterol ligase factors response factors response

A conserved oxygen sensing mechanism in plants and animals? Oxygen dependent destabilizing sequences from Arabidopsis thaliana ERF transcription factor RAP-2.12 operate in human cells A.t. RAP2.12 GFP-V5

Oxygen sensing by enzymatic protein oxidation A conserved N-terminal cysteine dioxygenase regulates G-protein signalling in human cells

Modulation and integration of oxygen sensing systems employing enzymatic protein oxidation linked to degradation ERF tfs Cys N-terminal HIF Prolyl HI Cysteine Dioxygenases Hydroxylases Cys Pro G-protein (G α ) R4 R4 - RGS Signalling HIF tfs Acute oxygen sensing mechanisms?

Clinical trials show efficacy of prolyl hydroxylase inhibitors in raising haemoglobin levels in pre-dialysis and dialysis patients Roxadustat, Fibrogen Daprodustat, GSK Vadadustat, Akebia HIF prolyl hydroxylase inhibitors (so far) show powerful effects on ‘You can't always get what you want But if you try sometime you find renal anaemia with little evidence of general activation of HIF You get what you need’

With grateful thanks!