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Posttranslational hydroxylation of ankyrin repeats in IkappaB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH).

ABSTRACT: 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.

SUBMITTER: Cockman ME

PROVIDER: S-EPMC1578504 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.

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.

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Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803.

Project description:Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 alpha-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1alpha/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the beta-carbon of Asn-803 and imply production of the threo -isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro -isomer.

| S-EPMC1222951 | biostudies-other

Uncoupled O2-activation in the human HIF-asparaginyl hydroxylase, FIH, does not produce reactive oxygen species.

Project description:The factor inhibiting HIF (FIH) is one of the primary oxygen sensors in human cells, controlling gene expression by hydroxylating the ?-subunit of the hypoxia inducible transcription factor (HIF). As FIH is an alpha-ketoglutarate dependent non-heme iron dioxygenase, oxygen activation is thought to precede substrate hydroxylation. The coupling between oxygen activation and substrate hydroxylation was hypothesized to be very tight, in order for FIH to fulfill its function as a regulatory enzyme. Coupling was investigated by looking for reactive oxygen species production during turnover. We used alkylsulfatase (AtsK), a metabolic bacterial enzyme with a related mechanism and similar turnover frequency, for comparison, and tested both FIH and AtsK for H(2)O(2), O(2)(-) and OH formation under steady and substrate-depleted conditions. Coupling ratios were determined by comparing the ratio of substrate consumed to product formed. We found that AtsK reacted with O(2) on the seconds timescale in the absence of prime substrate, and uncoupled during turnover to produce H(2)O(2); neither O(2)(-) nor OH were detected. In contrast, FIH was unreactive toward O(2) on the minutes timescale in the absence of prime substrate, and tightly coupled during steady-state turnover; we were unable to detect any reactive oxygen species produced by FIH. We also investigated the inactivation mechanisms of these enzymes and found that AtsK likely inactivated due to deoligomerizion, whereas FIH inactivated by slow autohydroxylation. Autohydroxylated FIH could not be reactivated by dithiothreitol (DTT) nor ascorbate, suggesting that autohydroxylation is likely to be irreversible under physiological conditions.

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Factor inhibiting HIF (FIH) recognizes distinct molecular features within hypoxia-inducible factor-? (HIF-?) versus ankyrin repeat substrates.

Project description:Factor Inhibiting HIF (FIH) catalyzes the ?-hydroxylation of asparagine residues in HIF-? transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-? is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterize a conserved RLL motif in HIF and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo and, therefore, have important consequences for HIF regulation.

| S-EPMC3308777 | biostudies-literature

Host FIH-Mediated Asparaginyl Hydroxylation of Translocated <i>Legionella pneumophila</i> Effectors.

Project description:FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function.

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Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH).

Project description:Two primary O(2)-sensors for humans are the HIF-hydroxylases, enzymes that hydroxylate specific residues of the hypoxia inducible factor-? (HIF). These enzymes are factor inhibiting HIF (FIH) and prolyl hydroxylase-2 (PHD2), each an ?-ketoglutarate (?KG) dependent, non-heme Fe(II) dioxygenase. Although the two enzymes have similar active sites, FIH hydroxylates Asn(803) of HIF-1? while PHD2 hydroxylates Pro(402) and/or Pro(564) of HIF-1?. The similar structures but unique functions of FIH and PHD2 make them prime targets for selective inhibition leading to regulatory control of diseases such as cancer and stroke. Three classes of iron chelators were tested as inhibitors for FIH and PHD2: pyridines, hydroxypyrones/hydroxypyridinones and catechols. An initial screen of the ten small molecule inhibitors at varied [?KG] revealed a non-overlapping set of inhibitors for PHD2 and FIH. Dose response curves at moderate [?KG] ([?KG]~K(M)) showed that the hydroxypyrones/hydroxypyridinones were selective inhibitors, with IC(50) in the ?M range, and that the catechols were generally strong inhibitors of both FIH and PHD2, with IC(50) in the low ?M range. As support for binding at the active site of each enzyme as the mode of inhibition, electron paramagnetic resonance (EPR) spectroscopy were used to demonstrate inhibitor binding to the metal center of each enzyme. This work shows some selective inhibition between FIH and PHD2, primarily through the use of simple aromatic or pseudo-aromatic chelators, and suggests that hydroxypyrones and hydroxypyridones may be promising chelates for FIH or PHD2 inhibition.

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Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

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.

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The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism.

Project description:Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis and are resistant to high-fat-diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, and enhanced insulin sensitivity and are also protected against high-fat-diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.

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

Project description: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 FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

| S-EPMC9189129 | biostudies-literature

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

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

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