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

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Jul 25, 2023 300 likes •506 views

Elucidation of oxygen sensing mechanisms in human and animal cells Peter J. Ratcliffe Nobel Lecture - December 2019 Oxford-Yale expedition to Pikes Peak, Colorado J. S. Haldane and colleagues - July 1911 O 2 Sensor Epo Extra-ordinary

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Oxford-Yale expedition to Pike’s Peak, Colorado

J. S. Haldane and colleagues - July 1911

Elucidation of oxygen sensing mechanisms in human and animal cells Peter J. Ratcliffe Nobel Lecture - December 2019

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Epo Sensor

O2

Extra-ordinary sensitivity of the erythropoietin response to changes in blood

xygen

availability – responses to donation of blood Lorentz et al. Transfusion 31 650-654 (1991)

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Transgenic marker gene strategy indicates that erythropoietin producing cells are Interstitial fibroblasts

Development of a cellular model for study of Epo regulation by Oxygen

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Mouse Epo gene Mapping of oxygen regulated oxygen sequences by transient transfection

reporter gene

Widespread operation of hypoxia signalling pathways

Oxygen sensitive cell

//

Oxygen insensitive cell

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HIF

EPO

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HIF-α

Gal4 DNA binding domain HSVP16 transactivator

Regulation of HIF by oxygen

Regulated protein stability Oxygen availability Cellular iron/cobalt HIF-α Regulated activity Signal

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Sequencing the human genome suggests existence

f HIF paralogues

Making anti-EPAS antibodies (PM9) proves regulation by oxygen Multiple hypoxia inducible transcripts identified Transcripts constitutively upregulated in VHL defective cells

VHL+ VHL-

Distortion from HIF-1 to HIF-2 during RCC development Role in Cancer Treatment by HIF-2 antagonists

Oxygen sensing pathway

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HIF peptide

VHL

Treatment with cell extract

Signalling modification is prolyl hydroxylation

Biochemical analysis Heat labile extract Non-enzymatic oxidation NADH/NADHoxidase Oxygen Iron ATP

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Iron

Proteolysis

O2

HIF-α Pro Hyp

VHL ubiquitin ligase Cobalt 2- oxoglutarate HIF prolyl hydroxylase HIF prolyl hydroxylation implies a mechanism of oxygen sensing

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HIF prolyl hydroxylases - a set of Fe(II) and 2-oxoglutarate dioxygenases that are conserved throughout the animal kingdom

2 1 8 7 4 3 5 6

Fe(II)

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O2

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HIF-2 IHC HIF-2 mRNA

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Funghi

Schizosaccharomyces pombe

Ofd1 - Prolyl 3 hydroxylase

SRE proteolysis Sterol response Protists

Dictyostelium discoideum

Prolyl 4 hydroxylase

Skp1 ubiquitin ligase Culmination factors Plants

Arabidopsis thaliana

Cysteine

xidases

MCxxx N-end rule Ethylene response factors

All eukaryotic kingdoms use protein oxidation and proteolysis to signal oxygen levels

Animals

Homo sapiens

Prolyl 4 hydroxylase

VHL ubiquitin ligase Hypoxia inducible factors

Protein Oxidation in Signalling hypoxia Evolutionary Origins?

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A conserved oxygen sensing mechanism in plants and animals?

A.t. RAP2.12 GFP-V5

Oxygen dependent destabilizing sequences from Arabidopsis thaliana ERF transcription factor RAP-2.12 operate in human cells

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Oxygen sensing by enzymatic protein oxidation

A conserved N-terminal cysteine dioxygenase regulates G-protein signalling in human cells

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Pro

HI

HIF Prolyl Hydroxylases HIF tfs

ERF tfs

Cys N-terminal Cysteine Dioxygenases

R4

R4 - RGS

Cys G-protein (Gα) Signalling

Modulation and integration of oxygen sensing systems employing enzymatic protein oxidation linked to degradation

Acute oxygen sensing mechanisms?

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Roxadustat, Fibrogen Vadadustat, Akebia Daprodustat, GSK

Clinical trials show efficacy of prolyl hydroxylase inhibitors in raising haemoglobin levels in pre-dialysis and dialysis patients HIF prolyl hydroxylase inhibitors (so far) show powerful effects on renal anaemia with little evidence of general activation of HIF

‘You can't always get what you want But if you try sometime you find You get what you need’

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