The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α.

Prolyl hydroxylase domain protein 2 (PHD2)-catalyzed modification of hypoxia-inducible factor (HIF)-α is a key event in oxygen sensing. We previously showed that the zinc finger of PHD2 binds to a Pro-Xaa-Leu-Glu (PXLE) motif. Here, we show that the zinc finger binds to this motif in the ribosomal chaperone nascent polypeptide complex-α (NACA). This recruits PHD2 to the translation machinery to cotranslationally modify HIF-α. Importantly, this cotranslational modification is enhanced by a translational pause sequence in HIF-α. Mice with a knock-in Naca gene mutation that abolishes the PXLE motif display erythrocytosis, a reflection of HIF pathway dysregulation. In addition, human erythrocytosis-associated mutations in the zinc finger of PHD2 ablate interaction with NACA. Tibetans, who have adapted to the hypoxia of high altitude, harbor a PHD2 variant that we previously showed displays a defect in zinc finger binding to p23, a PXLE-containing HSP90 cochaperone. We show here that Tibetan PHD2 maintains interaction with NACA, thereby showing differential interactions with PXLE-containing proteins and providing an explanation for why Tibetans are not predisposed to erythrocytosis.

High-Altitude Andean H194R HIF2A Allele Is a Hypomorphic Allele.

For over 10,000 years, Andeans have resided at high altitude where the partial pressure of oxygen challenges human survival. Recent studies have provided evidence for positive selection acting in Andeans on the HIF2A (also known as EPAS1) locus, which encodes for a central transcription factor of the hypoxia-inducible factor pathway. However, the precise mechanism by which this allele might lead to altitude-adaptive phenotypes, if any, is unknown. By analyzing whole genome sequencing data from 46 high-coverage Peruvian Andean genomes, we confirm evidence for positive selection acting on HIF2A and a unique pattern of variation surrounding the Andean-specific single nucleotide variant (SNV), rs570553380, which encodes for an H194R amino acid substitution in HIF-2α. Genotyping the Andean-associated SNV rs570553380 in a group of 299 Peruvian Andeans from Cerro de Pasco, Peru (4,338 m), reveals a positive association with increased fraction of exhaled nitric oxide, a marker of nitric oxide biosynthesis. In vitro assays show that the H194R mutation impairs binding of HIF-2α to its heterodimeric partner, aryl hydrocarbon receptor nuclear translocator. A knockin mouse model bearing the H194R mutation in the Hif2a gene displays decreased levels of hypoxia-induced pulmonary Endothelin-1 transcripts and protection against hypoxia-induced pulmonary hypertension. We conclude the Andean H194R HIF2A allele is a hypomorphic (partial loss of function) allele.

Tibetan PHD2, an allele with loss-of-function properties.

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

High-altitude deer mouse hypoxia-inducible factor-2α shows defective interaction with CREB-binding protein.

Numerous mammalian species have adapted to the chronic hypoxia of high altitude. Recent genomic studies have identified evidence for natural selection of genes and associated genetic changes in these species. A major gap in our knowledge is an understanding of the functional significance, if any, of these changes. Deer mice (Peromyscus maniculatus) live at both low and high altitudes in North America, providing an opportunity to identify functionally important genetic changes. High-altitude deer mice show evidence of natural selection on the Epas1 gene, which encodes for hypoxia-inducible factor-2α (Hif-2α), a central transcription factor of the hypoxia-inducible factor pathway. An SNP encoding for a T755M change in the Hif-2α protein is highly enriched in high-altitude deer mice, but its functional significance is unknown. Here, using coimmunoprecipitation and transcriptional activity assays, we show that the T755M mutation produces a defect in the interaction of Hif-2α with the transcriptional coactivator CREB-binding protein. This results in a loss of function because of decreased transcriptional activity. Intriguingly, the effect of this mutation depends on the amino acid context. Interchanges between methionine and threonine at the corresponding position in house mouse (Mus musculus) Hif-2α are without effects on CREB-binding protein binding. Furthermore, transfer of a set of deer mouse-specific Hif-2α amino acids to house mouse Hif-2α is sufficient to confer sensitivity of house mouse Hif-2α to the T755M substitution. These findings provide insight into high-altitude adaptation in deer mice and evolution at the Epas1 locus.

Identification of Small-Molecule PHD2 Zinc Finger Inhibitors that Activate Hypoxia Inducible Factor.

The prolyl hydroxylase domain (PHD) protein:hypoxia inducible factor (HIF) pathway is the main pathway by which changes in oxygen concentration are transduced to changes in gene expression. In mammals, there are three PHD paralogues, and PHD2 has emerged as a particularly critical one for regulating HIF target genes such as erythropoietin (EPO), which controls red cell mass and hematocrit. PHD2 is distinctive among the three PHDs in that it contains an N-terminal MYND-type zinc finger. We have proposed that this zinc finger binds a Pro-Xaa-Leu-Glu (PXLE) motif found in proteins of the HSP90 pathway to facilitate HIF-α hydroxylation. Targeting this motif could provide a means of specifically inhibiting this PHD isoform. Here, we screened a library of chemical compounds for their capacity to inhibit the zinc finger of PHD2. We identified compounds that, in vitro, can inhibit PHD2 binding to a PXLE-containing peptide and induce activation of HIF. Injection of one of these compounds into mice induces an increase in hematocrit. This study offers proof of principle that inhibition of the zinc finger of PHD2 can provide a means of selectively targeting PHD2 to activate the HIF pathway.

Identification of prolyl hydroxylation modifications in mammalian cell proteins.

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen-dependent regulation of hypoxia inducible factor-α (HIF-α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF-α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506-binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C-1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.

Defective Tibetan PHD2 binding to p23 links high altitude adaption to altered oxygen sensing.

The Tibetan population has adapted to the chronic hypoxia of high altitude. Tibetans bear a genetic signature in the prolyl hydroxylase domain protein 2 (PHD2/EGLN1) gene, which encodes for the central oxygen sensor of the hypoxia-inducible factor (HIF) pathway. Recent studies have focused attention on two nonsynonymous coding region substitutions, D4E and C127S, both of which are markedly enriched in the Tibetan population. These amino acids reside in a region of PHD2 that harbors a zinc finger, which we have previously discovered binds to a Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 cochaperone p23, thereby recruiting PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. We herein report that the Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes the interaction of PHD2 with p23, resulting in impaired PHD2 down-regulation of the HIF pathway. The defective binding to p23 depends on both the D4E and C127S substitutions. We also identify a PXLE motif in HSP90 itself that can mediate binding to PHD2 but find that this interaction is maintained with the D4E/C127S PHD2 haplotype. We propose that the Tibetan PHD2 variant is a loss of function (hypomorphic) allele, leading to augmented HIF activation to facilitate adaptation to high altitude.

Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.

Prolyl hydroxylase domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is a key oxygen-sensing protein in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of the hypoxic response, hypoxia-inducible factor-α (HIF-α), thereby targeting HIF-α for degradation. In this report we show that the heat shock protein 90 (HSP90) co-chaperones p23 and FKBP38 interact via a conserved Pro-Xaa-Leu-Glu motif (where Xaa = any amino acid) in these proteins with the N-terminal Myeloid Nervy and DEAF-1 (MYND)-type zinc finger of PHD2. Knockdown of p23 augments hypoxia-induced HIF-1α protein levels and HIF target genes. We propose that p23 recruits PHD2 to the HSP90 machinery to facilitate HIF-1α hydroxylation. These findings identify a link between two ancient pathways, the PHD:HIF and the HSP90 pathways, and suggest that this link was established concurrent with the emergence of the PHD:HIF pathway in evolution.

Mouse knock-out of IOP1 protein reveals its essential role in mammalian cytosolic iron-sulfur protein biogenesis.

Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.

Human ISCA1 interacts with IOP1/NARFL and functions in both cytosolic and mitochondrial iron-sulfur protein biogenesis.

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.

An Erythrocytosis-Associated Mutation in the Zinc Finger of PHD2 Provides Insights into Its Binding of p23.

BACKGROUND: Loss of function mutations in the EGLN1 gene are a cause of erythrocytosis. EGLN1 encodes for prolyl hydroxylase domain protein 2 (PHD2). PHD2 hydroxylates and downregulates hypoxia-inducible factor-2α (HIF-2α), a transcription factor that regulates erythropoiesis. While the large majority of erythrocytosis-associated EGLN1 mutations occur within its catalytic domain, rare mutations reside in its zinc finger. This zinc finger binds a Pro-Xaa-Leu-Glu motif in p23, an HSP90 cochaperone that facilitates hydroxylation of HIF-α, an HSP90 client. Essentially nothing is known about the specific interactions between the PHD2 zinc finger and p23. RESULTS: Here, we characterize an erythrocytosis-associated mutation in the zinc finger, K55N, that abolishes interaction with p23. We provide evidence that the affected residue, Lys-55, interacts with Asp-152 of p23. We also present results that indicate that PHD2 Arg-32 interacts with p23 Glu-160. CONCLUSION: These studies not only reinforce the importance of the PHD2 zinc finger in the control of erythropoiesis, but also lead to a model in which a peptide motif in p23 binds in a specific orientation to a predicted groove in the zinc finger of PHD2.

Author Correction: Loss of Phd2 cooperates with BRAFV600E to drive melanomagenesis.

The original version of this Article contained an error in the spelling of the author Brett L. Ecker, which was incorrectly given as Brett Ecker. This has now been corrected in both the PDF and HTML versions of the Article.

IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity.

A central means by which mammalian cells respond to low oxygen tension is through the activation of the transcription factor HIF-1 (hypoxia-inducible factor-1). Under normoxic conditions, HIF-1alpha (the alpha subunit of HIF-1) is targeted for rapid degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this degradation is inhibited, thereby leading to the stabilization and activation of HIF-1alpha. Here, we report the identification of IOP1 (iron-only hydrogenase-like protein 1), a protein homologous with enzymes present in anaerobic organisms that contain a distinctive iron-sulfur cluster. IOP1 is present in a broad range of cell types. Knockdown of IOP1 using siRNA (small interfering RNA) in mammalian cells increases protein levels of HIF-1alpha under both normoxic and hypoxic conditions, and augments hypoxia-induced HRE (hypoxia response element) reporter gene and endogenous HIF-1alpha target gene expressions. We find that IOP1 knockdown up-regulates HIF-1alpha mRNA levels, thereby providing a mechanism by which knockdown induces the observed effects. The results collectively provide evidence that IOP1 is a component of the protein network that regulates HIF-1alpha in mammalian cells.

Loss of Phd2 cooperates with BRAFV600E to drive melanomagenesis.

Prolyl hydroxylase domain protein 2 (PHD2) is a well-known master oxygen sensor. However, the role of PHD2 in tumor initiation remains controversial. We find that during the transition of human nevi to melanoma, the expression of PHD2 protein is significantly decreased and lower expression PHD2 in melanoma is associated with worse clinical outcome. Knockdown of PHD2 leads to elevated Akt phosphorylation in human melanocytes. Mice with conditional melanocyte-specific expression of Phd2lox/lox (Tyr::CreER;Phd2lox/lox) fail to develop pigmented lesions. However, deletion of Phd2 in combination with expression of BRafV600E in melanocytes (Tyr::CreER;Phd2lox/lox;BRafCA) leads to the development of melanoma with 100% penetrance and frequent lymph node metastasis. Analysis of tumor tissues using reverse phase protein arrays demonstrates that Phd2 deletion activates the AKT-mTOR-S6 signaling axis in the recovered tumors. These data indicate that PHD2 is capable of suppressing tumor initiation largely mediated through inhibiting of the Akt-mTOR signaling pathway in the melanocyte lineage.

The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) 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-α 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-α. 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.

A role for IOP1 in mammalian cytosolic iron-sulfur protein biogenesis.

The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation.

Hyperdynamic circulation in portal-hypertensive rats is dependent on central c-fos gene expression.

Portal hypertension is associated with hyperdynamic circulation, but the pathogenesis remains unclear. To clarify the role of central cardiovascular regulatory mechanisms, several protocols were conducted in rats with portal hypertension due to portal vein stenosis (PVS). Neuronal activation was quantified by immunohistochemical staining for Fos, the protein product of the c-fos gene. Fos expression in several brain nuclei with cardiovascular-regulatory roles was examined at 1, 3, 5, and 10 days following PVS surgery. This was correlated with development of cardiovascular changes measured at the same time points. Finally, Fos expression in the nucleus tractus solitarius (NTS) was blocked by local microinjection of c-fos antisense oligonucleotides twice daily for 5 days following PVS. The results showed that Fos-positive neurons were significantly increased in the paraventricular nucleus of hypothalamus, supraoptic nucleus, ventrolateral medulla, and NTS, detectable at day 1 and persistently increased at every day examined in the PVS rats. However, the hyperdynamic circulation developed between days 3 to 5. Administration of c-fos antisense oligonucleotides eliminated the hyperdynamic circulation in PVS rats, but had no effect on sham-operated controls. We conclude that the activation of central cardiovascular-regulatory nuclei, through a c-fos-dependent pathway, is necessary for development of hyperdynamic circulation in portal-hypertensive rats.

The role of endothelin-1 and the endothelin B receptor in the pathogenesis of hepatopulmonary syndrome in the rat.

Endothelin-1 (ET-1) stimulation of endothelial nitric oxide synthase (eNOS) via pulmonary endothelial endothelin B (ET(B)) receptors and pulmonary intravascular macrophage accumulation with expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) are implicated in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). Our aim was to evaluate the role of ET-1 in the development of experimental HPS. The time course of molecular and physiological changes of HPS and the effects of selective endothelin receptor antagonists in vivo were assessed after CBDL. Effects of ET-1 on intralobar pulmonary vascular segment reactivity and on eNOS expression and activity in rat pulmonary microvascular endothelial cells (RPMVECs) were also evaluated. Hepatic and plasma ET-1 levels increased 1 week after CBDL in association with a subsequent increase in pulmonary microvascular eNOS and ET(B) receptor levels and the onset of HPS. Selective ET(B) receptor inhibition in vivo significantly decreased pulmonary eNOS and ET(B) receptor levels and ameliorated HPS. CBDL pulmonary artery segments had markedly increased ET(B) receptor mediated, nitric oxide dependent vasodilatory responses to ET-1 compared with controls and ET-1 triggered an ET(B) receptor dependent stimulation of eNOS in RPMVECs. Pulmonary intravascular macrophages also accumulated after CBDL and expressed HO-1 and iNOS at 3 weeks. Selective ET(B) receptor blockade also decreased macrophage accumulation and iNOS production. In conclusion, ET-1 plays a central role in modulating pulmonary micovascular tone in experimental HPS.