Dataset Information

Factor Inhibiting HIF catalyses two asparaginyl-hydroxylations in VNVN motifs of ankyrin proteins

ABSTRACT: The aspariginyl-hydroxylase activity of Factor Inhibiting HIF (FIH) is a key regulator of the transcriptional activity of the hypoxia inducible factor. FIH also catalyses the hydroxylation of asparaginyl- and other-residues in ankyrin repeat domain (ARD) containing proteins, including Apoptosis stimulating of p53 (ASPP) protein family members. ASPP2 is reported to undergo a single FIH catalysed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing FIH can catalyse the unprecedented post-translational hydroxylation of both asparaginyl-residues in “VNVN” and related motifs of ankyrin repeat domains in apoptosis-stimulating of p53 (ASPP) proteins (i.e. ASPP1, ASPP2 and iASPP) and the related ASB11 and p18-INK4C proteins. The results extend the substrate scope of FIH catalysis and may have implications for its role in the hypoxic response and, ASPP protein function.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Leg Bone, Bone Marrow

DISEASE(S): Pediatric Osteosarcoma

SUBMITTER: Roman Fischer

LAB HEAD: Roman Fischer

PROVIDER: PXD017278 | Pride | 2022-08-11

REPOSITORIES: Pride

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Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins.

Leissing Thomas M TM Hardy Adam P AP Chan Hokfung H Wang Yihua Y Tumber Anthony A Chowdhury Rasheduzzaman R Feng Tianshu T Coleman Mathew L ML Cockman Matthew E ME Kramer Holger B HB Berridge Georgina G Fischer Roman R Kessler Benedikt M BM Ratcliffe Peter J PJ Lu Xin X Schofield Christopher J CJ

The Journal of biological chemistry 20220507 6

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that ...[more]

PMID: 35537551

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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:Hypoxia occurs when tissue or cellular oxygen demand exceeds its supply and is a frequent condition in health and disease. Metazoans have developed a regulatory system that allows them to sense changes in oxygen levels in their microenvironment and adapt to them. The asparagine hydroxylase factor inhibiting HIF (FIH) regulates the transcriptional activity of the hypoxia-inducible factor (HIF), the master regulator of the cellular adaptive response to hypoxia [1, 2]. We recently identified the deubiquitinating enzyme ovarian tumour domain containing, ubiquitin aldehyde binding protein 1 (OTUB1) as novel bona fide FIH target protein with the hydroxylation occurring at the asparagine residue N22 [3, 4]. Surprisingly, in a follow-up investigation we observed a SDS-PAGE resistant interaction between FIH and OTUB1, resulting in a FIH-OTUB1 heterodimer (HD). Further analysis of the FIH-OTUB1 HD demonstrated that it is not linked by a disulfide bond, oxyester or thioester but likely through an amide bond. Interestingly, mutation of the hydroxylation acceptor site N22 in OTUB1 and genetic and pharmacologic inhibition of FIH prevented the formation of the heterodimer, demonstrating that HD formation is a consequence of FIH activity. To investigate if FIH-dependent stable protein complex formation was exclusive for OTUB1, we carried out a mass spectrometry (MS)-based FIH interactome analyses with denatured and non-denatured cell lysates (with or without SDS treatment and boiling). The results indicate the existence of further novel denaturing condition resistant protein complexes. 1. Mahon, P.C., K. Hirota, and G.L. Semenza, FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes & development, 2001. 15(20): p. 2675-86. 2. Lando, D., et al., FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes & development, 2002. 16(12): p. 1466-71. 3. Scholz, C.C., et al., Regulation of IL-1beta-induced NF-kappaB by hydroxylases links key hypoxic and inflammatory signaling pathways. Proc Natl Acad Sci U S A, 2013. 110(46): p. 18490-5. 4. Scholz, C.C., et al., FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1. PLoS biology, 2016. 14(1): p. e1002347.

Dataset Information

Factor Inhibiting HIF catalyses two asparaginyl-hydroxylations in VNVN motifs of ankyrin proteins

Publications

Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins.

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