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Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates

ABSTRACT: Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain (PHD) enzymes catalyse the posttranslational hydroxylation of conserved prolyl residues in the alpha-subunit of the HIF transcription factor. These modifications, which promote the degradation of HIF-alpha subunits by the pVHL E3 ligase complex and impart oxygen-dependent regulation of the HIF transcriptional response, have been extensively characterised at the molecular, cellular and organismal level. Since the discovery of the PHDs, a range of less well-characterised non-HIF substrates have been reported with the potential to confer oxygen-sensitivity to a diverse range of cellular processes. We sought to systematically compare all of the reported non-HIF substrates for their ability to support PHD-catalysed hydroxylation. We performed a comprehensive series of in vitro hydroxylation assays reacting synthetic peptides and full-length protein substrates generated by in vitro transcription and translation with purified recombinant enzyme preparations. Prolyl hydroxylation was assayed directly by mass spectrometry and radiochemical assay for hydroxyproline. Both methods enabled quantitative appraisal of enzyme-catalysed hydroxylation, with liquid chromatography mass spectrometry methods employing NMR-quantified peptide standards to calibrate retention time signatures and determine ionisation efficiencies for each target peptide. Using these approaches we assayed hydroxylation on 23 different proteins. Surprisingly, we did not detect measurable hydroxylation on any of the reported non-HIF substrates using either method. In contrast, control assays with HIF1-alpha substrate supported high stoichiometry (typically >90%) hydroxylation. Our findings suggest that PHD substrates are more restricted than has been reported.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Matthew Cockman

LAB HEAD: Peter John Ratcliffe

PROVIDER: PXD013112 | Pride | 2019-08-30

REPOSITORIES: Pride

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Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.

Cockman Matthew E ME Lippl Kerstin K Tian Ya-Min YM Pegg Hamish B HB Figg William D WD Abboud Martine I MI Heilig Raphael R Fischer Roman R Myllyharju Johanna J Schofield Christopher J CJ Ratcliffe Peter J PJ

eLife 20190910

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivi ...[more]

PMID: 31500697

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Project description:This a model from the article: Hypoxia-dependent sequestration of an oxygen sensor by a widespread structural motif can shape the hypoxic response - a predictive kinetic model Bernhard Schmierer, Béla Novák1 and Christopher J Schofield BMC Systems Biology2010, 4:139 20955552, Abstract: Background The activity of the heterodimeric transcription factor hypoxia inducible factor (HIF) is regulated by the post-translational, oxygen-dependent hydroxylation of its α-subunit by members of the prolyl hydroxylase domain (PHD or EGLN)-family and by factor inhibiting HIF (FIH). PHD-dependent hydroxylation targets HIFα for rapid proteasomal degradation; FIH-catalysed asparaginyl-hydroxylation of the C-terminal transactivation domain (CAD) of HIFα suppresses the CAD-dependent subset of the extensive transcriptional responses induced by HIF. FIH can also hydroxylate ankyrin-repeat domain (ARD) proteins, a large group of proteins which are functionally unrelated but share common structural features. Competition by ARD proteins for FIH is hypothesised to affect FIH activity towards HIFα; however the extent of this competition and its effect on the HIF-dependent hypoxic response are unknown. Results To analyse if and in which way the FIH/ARD protein interaction affects HIF-activity, we created a rate equation model. Our model predicts that an oxygen-regulated sequestration of FIH by ARD proteins significantly shapes the input/output characteristics of the HIF system. The FIH/ARD protein interaction is predicted to create an oxygen threshold for HIFα CAD-hydroxylation and to significantly sharpen the signal/response curves, which not only focuses HIFα CAD-hydroxylation into a defined range of oxygen tensions, but also makes the response ultrasensitive to varying oxygen tensions. Our model further suggests that the hydroxylation status of the ARD protein pool can encode the strength and the duration of a hypoxic episode, which may allow cells to memorise these features for a certain time period after reoxygenation. Conclusions The FIH/ARD protein interaction has the potential to contribute to oxygen-range finding, can sensitise the response to changes in oxygen levels, and can provide a memory of the strength and the duration of a hypoxic episode. These emergent properties are predicted to significantly shape the characteristics of HIF activity in animal cells. We argue that the FIH/ARD interaction should be taken into account in studies of the effect of pharmacological inhibition of the HIF-hydroxylases and propose that the interaction of a signalling sensor with a large group of proteins might be a general mechanism for the regulation of signalling pathways. There are there models described in the paper. 1) Skeleton Model 1 (SKM1) - HIFα CAD-hydroxylation in the absence of the FIH/AR-interaction. 2) Skeleton Model 2 (SKM2) - FIG sequestration by ARD proteins and oxygen-dependent FIH-release. 3) Full Model (Fusion of SKM1 and SKM2) - the effects of the FIH/ARD proteins interaction on HIFα CAD-hydroxylation. This model corresponds to the "Full Model" described in the paper. The model reproduces figure 5 of the publication. This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team. For more information see the terms of use. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:The response of cells to hypoxia is characterised by co-ordinated regulation of many genes. Studies of the regulation of the expression of many of these genes by oxygen has implicated a role for the heterodimeric transcription factor hypoxia inducible factor (HIF). The mechanism of oxygen sensing which controls this heterodimeric factor is via oxygen dependent prolyl and asparaginyl hydroxylation by specific 2-oxoglutarate dependent dioxygenases (PHD1, PHD2, PHD3 and FIH-1). Whilst HIF appears to have a major role in hypoxic regulation of gene expression, it is unclear to what extent other transcriptional mechanisms are also involved in the response to hypoxia. The extent to which 2-oxoglutarate dependent dioxygenases are responsible for the oxygen sensing mechanism in HIF-independent hypoxic gene regulation is also unclear. Both the prolyl and asparaginyl hydroxylases can be inhibited by dimethyloxalylglycine (DMOG). Such inhibition can produce activation of the HIF system with enhanced transcription of target genes and might have a role in the therapy of ischaemic disease. We have examined the extent to which the HIF system contributes to the regulation of gene expression by hypoxia, to what extent 2-oxoglutarate dependent dioxygenase inhibitor can mimic the hypoxic response and the nature of the global transcriptional response to hypoxia. We have utilised microarray assays of mRNA abundance to examine the gene expression changes in response to hypoxia and to DMOG. We demonstrate a large number of hypoxically regulated genes, both known and novel, and find a surprisingly high level of mimicry of the hypoxic response by use of the 2-oxoglutarate dependent dioxygenase inhibitor, dimethyloxalylglycine. We have also used microarray analysis of cells treated with small interfering RNA (siRNA) targeting HIF-1alpha and HIF-2alpha to demonstrate the differing contributions of each transcription factor to the transcriptional response to hypoxia. Candidate transcripts were confirmed using an independent microarray platform and real-time PCR. The results emphasise the critical role of the HIF system in the hypoxic response, whilst indicating the dominance of HIF-1alpha and defining genes that only respond to HIF-2alpha. Keywords: Hypoxia response, gene knockdown, chemical treatment

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Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates

Publications

Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.

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