Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803.
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ABSTRACT:
SUBMITTER: McNeill LA
PROVIDER: S-EPMC1222951 | biostudies-other | 2002 Nov
REPOSITORIES: biostudies-other
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Project description:Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IkappaB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IkappaBalpha were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.
| S-EPMC1578504 | biostudies-literature
Project description:BackgroundThe roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH).MethodsWe used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD.ResultsIn our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis.ConclusionsThe causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.
| S-EPMC10539236 | biostudies-literature
Project description:Factor-inhibiting hypoxia-inducible factor (FIH) is an Fe(II)/2-oxoglutarate-dependent dioxygenase that acts as a negative regulator of the hypoxia-inducible factor (HIF) by catalysing ?-hydroxylation of an asparaginyl residue in its C-terminal transcriptional activation domain (CAD). In addition to the hypoxia-inducible factor C-terminal transcriptional activation domain (HIF-CAD), FIH also catalyses asparaginyl hydroxylation of many ankyrin repeat domain-containing proteins, revealing a broad sequence selectivity. However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues. Here, we report that histidinyl residues within the ankyrin repeat domain of tankyrase-2 can be hydroxylated by FIH. NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the ?-position. The results further expand the scope of FIH-catalysed hydroxylations.
| S-EPMC3569879 | biostudies-literature
Project description:Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the β-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13-24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification.
| S-EPMC3045019 | biostudies-literature
Project description:Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.
| S-EPMC2649815 | biostudies-literature
Project description:Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor ? (HIF-?) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-? hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-?. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.
| S-EPMC5007793 | biostudies-literature
Project description:The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
| S-EPMC5034057 | biostudies-literature
Project description:Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.
| S-EPMC5887987 | biostudies-literature
Project description:The asparaginyl hydroxylase, factor-inhibiting hypoxia-inducible factor (HIF), is central to the oxygen-sensing pathway that controls the activity of HIF. Factor-inhibiting HIF (FIH) also catalyzes the hydroxylation of a large set of proteins that share a structural motif termed the ankyrin repeat domain (ARD). In vitro studies have defined kinetic properties of FIH with respect to different substrates and have suggested FIH binds more tightly to certain ARD proteins than HIF and that ARD hydroxylation may have a lower K(m) value for oxygen than HIF hydroxylation. However, regulation of asparaginyl hydroxylation on ARD substrates has not been systematically studied in cells. To address these questions, we employed isotopic labeling and mass spectrometry to monitor the accrual, inhibition, and decay of hydroxylation under defined conditions. Under the conditions examined, hydroxylation was not reversed but increased as the protein aged. The extent of hydroxylation on ARD proteins was increased by addition of ascorbate, whereas iron and 2-oxoglutarate supplementation had no significant effect. Despite preferential binding of FIH to ARD substrates in vitro, when expressed as fusion proteins in cells, hydroxylation was found to be more complete on HIF polypeptides compared with sites within the ARD. Furthermore, comparative studies of hydroxylation in graded hypoxia revealed ARD hydroxylation was suppressed in a site-specific manner and was as sensitive as HIF to hypoxic inhibition. These findings suggest that asparaginyl hydroxylation of HIF-1 and ARD proteins is regulated by oxygen over a similar range, potentially tuning the HIF transcriptional response through competition between the two types of substrate.
| S-EPMC3190818 | biostudies-literature
Project description:Objective: This study aimed to explore the global research status, hot topics, and future prospects in the field of the hypoxia inducible factor prolyl hydroxylase inhibitor (HIF-PHI) by bibliometric analysis. Methods: The literatures about HIF-PHI were downloaded from the Web of Science Core Collection and Pubmed database from inceptions to January.10th. 2022. The VOSviewer 1.6.18 was used to explore the bibliometric networks and research priorities of HIF-PHI. Results: A total of 409 papers about HIF-PHI were included, involving 1,674 authors from 548 institutions in 43 countries. The number of HIF-PHI literatures showed an upward trend, with steady growth from 2016 to 2020 and rapid growth in 2021. Tadao Akizawa, Masaomi Nangaku and Alexander R Cobitz published the most literatures. The United States, Japan and China contributed the most publications. The three most contributed institutions are Astellas Pharma Inc., the Showa University and Glaxosmithkline. Therapeutic Apheresis and Dialysis, American Journal of Nephrology and Clinical Pharmacology in Drug Development are the most productive journals. The main hot topics of HIF-PHI field are anemia, chronic kidney disease, hif-phi, epoetin and roxadustat. Conclusion: The United States and Japan are dominant in the field of HIF-PHI research. The discovery and clinical application of HIF-PHI is a great boon for patients with renal anemia. However, due to the short clinical application time of HIF-PHI, and its long-term efficacy and safety still need time to prove. In addition, more cooperation should be carried out between European and American countries and Asian countries to better prove the clinical value of HIF-PHI.
| S-EPMC9549679 | biostudies-literature