scholarly journals Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the β-carbon of asparagine-803

2002 ◽  
Vol 367 (3) ◽  
pp. 571-575 ◽  
Author(s):  
Luke A. McNEILL ◽  
Kirsty S. HEWITSON ◽  
Timothy D. CLARIDGE ◽  
Jürgen F. SEIBEL ◽  
Louise E. HORSFALL ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Hypoxia Inducible Factor ◽  
Transactivation Domain ◽  
Α Subunit ◽  
Asparaginyl Hydroxylase ◽  
Threo Isomer

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 α-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1α/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the β-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.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites

2011 ◽  
Vol 436 (2) ◽  
pp. 363-369 ◽  
Author(s):  
Melissa B. Pappalardi ◽  
Dean E. McNulty ◽  
John D. Martin ◽  
Kelly E. Fisher ◽  
Yong Jiang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Transactivation Domain ◽  
Prolyl Hydroxylases ◽  
Proline Residue ◽  
Protein Substrates ◽  
Steady State Kinetic ◽  
Asparaginyl Hydroxylase

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1–3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography–tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis

Blood ◽  
2009 ◽  
Vol 114 (10) ◽  
pp. 2015-2019 ◽  
Author(s):  
Gregg L. Semenza
Keyword(s):  
Blood Cells ◽  
Tissue Oxygenation ◽  
Hypoxia Inducible Factor ◽  
Α Subunit ◽  
Vhl Gene ◽  
Von Hippel Lindau ◽  
Hypoxia Inducible Factor 1 ◽  
Oxygen Homeostasis ◽  
Genes Encoding ◽  
Asparaginyl Hydroxylase

Abstract Red blood cells deliver O2 from the lungs to every cell in the human body. Reduced tissue oxygenation triggers increased production of erythropoietin by hypoxia-inducible factor 1 (HIF-1), which is a transcriptional activator composed of an O2-regulated α subunit and a constitutively expressed β subunit. Hydroxylation of HIF-1α or HIF-2α by the asparaginyl hydroxylase FIH-1 blocks coactivator binding and transactivation. Hydroxylation of HIF-1α or HIF-2α by the prolyl hydroxylase PHD2 is required for binding of the von Hippel-Lindau protein (VHL), leading to ubiquitination and proteasomal degradation. Mutations in the genes encoding VHL, PHD2, and HIF-2α have been identified in patients with familial erythrocytosis. Patients with Chuvash polycythemia, who are homozygous for a missense mutation in the VHL gene, have multisystem pathology attributable to dysregulated oxygen homeostasis. Intense efforts are under way to identify small molecule hydroxylase inhibitors that can be administered chronically to selectively induce erythropoiesis without undesirable side effects.

scholarly journals Functions of the Per/ARNT/Sim Domains of the Hypoxia-inducible Factor

2005 ◽  
Vol 280 (43) ◽  
pp. 36047-36054 ◽  
Author(s):  
Jinsong Yang ◽  
Lei Zhang ◽  
Paul J. A. Erbel ◽  
Kevin H. Gardner ◽  
Kan Ding ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Hypoxia Inducible Factor ◽  
Transactivation Domain ◽  
Structural Elements ◽  
Α Subunit ◽  
Solution Structures ◽  
Starting Point ◽  
Tremendous Progress ◽  
Attractive Therapeutic Target ◽  
Made In

The heterodimeric transcription factor hypoxia-inducible factor (HIF) plays an important role in the progression of a number of processes in which O2 availability is compromised and, as such, has become an increasingly attractive therapeutic target. Although tremendous progress has been made in recent years in unraveling the mechanisms underlying O2-dependent regulation of HIF through its O2-dependent degradation domain and C-terminal transactivation domain, our understanding of the contributions of other structural elements, particularly the Per/ARNT/Sim (PAS)-A and PAS-B domains, to the activity of HIF is incomplete. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point, we have explored the function(s) of the HIF-2α PAS domains via mutational analysis. In contrast to recent models, our data reveal that both PAS domains of the HIF-α subunit are necessary for heterodimer formation but are not required to mediate other HIF functions in which PAS domains have been implicated. Because disruption of individual PAS domains compromise HIF function independent of the mechanism of HIF induction, these data demonstrate the potential utility of targeting these domains for therapeutic applications.

scholarly journals Ginsenoside 20(S)-Rg3 suppresses ovarian cancer migration via hypoxia-inducible factor 1 alpha and nuclear factor-kappa B signals

Tumor Biology ◽  
2017 ◽  
Vol 39 (5) ◽  
pp. 101042831769222 ◽  
Author(s):  
Ting Liu ◽  
Le Zhao ◽  
Huilian Hou ◽  
Lu Ding ◽  
Wei Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Hypoxia Inducible Factor ◽  
Nuclear Factor ◽  
Strong Inhibitor ◽  
Α Subunit ◽  
Hypoxia Inducible Factor 1Α ◽  
Oxygen Levels ◽  
Hypoxia Inducible Factor 1 ◽  
Cancer Migration

Hypoxia-inducible factor 1 is believed to play a prominent role in the survival and developing progress of cancers. As a result, inhibiting α subunit of hypoxia-inducible factor 1 represents an attractive strategy against tumor. Although hypoxia-inducible factor 1α is a hypoxia-regulated subunit, increasing evidence indicates that hypoxia-inducible factor 1α could stable expression under normoxic conditions, regulated by non-hypoxia-mediated mechanisms. However, there are few strategies to target hypoxia-inducible factor 1α under normoxic conditions. Here, we report that ginsenoside 20(S)-Rg3, one of the main active ingredients in red ginseng, restrains hypoxia-inducible factor 1α expression under normal oxygen levels in human ovarian cancer cell lines, SKOV3 and 3AO, which leads to potently inhibits migration of ovarian cancer in vitro and in vivo. 20(S)-Rg3 could decrease the expression of hypoxia-inducible factor 1α by upregulation of prolyl hydroxylase domain protein 1 to promoting hypoxia-inducible factor 1α ubiquitin–proteasome degradation under normal oxygen levels. Furthermore, 20(S)-Rg3 could attenuate the expression of nuclear factor-κ B, which may be another possible mechanism for 20(S)-Rg3 to block ovarian cancer migration. Taken together, our study suggests that 20(S)-Rg3 is a strong inhibitor of hypoxia-inducible factor 1α, which may provide a novel agent for future treatments for ovarian cancer.

scholarly journals c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription

2002 ◽  
Vol 22 (1) ◽  
pp. 12-22 ◽  
Author(s):  
Arántzazu Alfranca ◽  
M. Dolores Gutiérrez ◽  
Alicia Vara ◽  
Julián Aragonés ◽  
Felipe Vidal ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Cooperative Effect ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Transactivation Domain ◽  
Low Oxygen ◽  
Jnk Pathway ◽  
Hypoxia Inducible Factor 1

ABSTRACT Under low-oxygen conditions, cells develop an adaptive program that leads to the induction of several genes, which are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1). On the other hand, there are other factors which modulate the HIF-1-mediated induction of some genes by binding to cis-acting motifs present in their promoters. Here, we show that c-Jun functionally cooperates with HIF-1 transcriptional activity in different cell types. Interestingly, a dominant-negative mutant of c-Jun which lacks its transactivation domain partially inhibits HIF-1-mediated transcription. This cooperative effect is not due to an increase in the nuclear amount of the HIF-1α subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1α are able to associate in vivo but not in vitro, suggesting that this interaction involves the participation of additional proteins and/or a posttranslational modification of these factors. In this context, hypoxia induces phosphorylation of c-Jun at Ser63 in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser63 and Ser73 impair its functional cooperation with HIF-1. The functional interplay between c-Jun and HIF-1 provides a novel insight into the regulation of some genes, such as the one for VEGF, which is a key regulator of tumor angiogenesis.

scholarly journals A Feedback Loop Involving the Phd3 Prolyl Hydroxylase Tunes the Mammalian Hypoxic Response In Vivo

2009 ◽  
Vol 29 (21) ◽  
pp. 5729-5741 ◽  
Author(s):  
Yoji Andrew Minamishima ◽  
Javid Moslehi ◽  
Robert F. Padera ◽  
Roderick T. Bronson ◽  
Ronglih Liao ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Premature Mortality ◽  
Prolyl Hydroxylase ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Regulatory Feedback Loop

ABSTRACT Hypoxia-inducible factor (HIF), consisting of a labile α subunit and a stable β subunit, is a master regulator of hypoxia-responsive mRNAs. HIFα undergoes oxygen-dependent prolyl hydroxylation, which marks it for polyubiquitination by a complex containing the von Hippel-Lindau protein (pVHL). Among the three Phd family members, Phd2 appears to be the primary HIF prolyl hydroxylase. Phd3 is induced by HIF and, based on findings from in vitro studies, may participate in a HIF-regulatory feedback loop. Here, we report that Phd3 loss exacerbates the HIF activation, hepatic steatosis, dilated cardiomyopathy, and premature mortality observed in mice lacking Phd2 alone and produces a closer phenocopy of the changes seen in mice lacking pVHL than the loss of Phd2 alone. Importantly, the degree to which Phd3 can compensate for Phd2 loss and the degree to which the combined loss of Phd2 and Phd3 resembles pVHL loss appear to differ for different HIF-responsive genes and in different tissues. These findings highlight that the responses of different HIF target genes to changes in prolyl hydroxylase activity differ, quantitatively and qualitatively, in vivo and have implications for the development of paralog-specific prolyl hydroxylase inhibitors as therapeutic agents.

scholarly journals Vhlh Gene Deletion Induces Hif-1-Mediated Cell Death in Thymocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9038-9047 ◽  
Author(s):  
Mangatt P. Biju ◽  
Aaron K. Neumann ◽  
Steven J. Bensinger ◽  
Randall S. Johnson ◽  
Laurence A. Turka ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Germ Line ◽  
Hypoxia Inducible Factor ◽  
Caspase 8 ◽  
Thymocyte Development ◽  
Double Positive ◽  
Α Subunit ◽  
Von Hippel Lindau

ABSTRACT The von Hippel-Lindau gene product (pVHL) targets the α subunit of basic helix-loop-helix transcription factor hypoxia-inducible factor (HIF) for proteasomal degradation. Inactivation of pVhl in the mouse germ line results in embryonic lethality, indicating that tight control of Hif-mediated adaptive responses to hypoxia is required for normal development and tissue function. In order to investigate the role of pVhl in T-cell development, we generated mice with thymocyte-specific inactivation of Vhlh resulting in constitutive transcriptional activity of Hif-1, as well as mice with thymocyte-specific repression of Hif-1 in a wild-type and Vhlh-deficient background. Thymi from Vhlh-deficient mice were small due to a severe reduction in the total number of CD4/CD8-double-positive thymocytes which was associated with increased apoptosis in vivo and in vitro. Increased apoptosis was a result of enhanced caspase 8 activity, while Bcl-2 and Bcl-XL transgene expression had little effect on this phenotype. Inactivation of Hif-1 in Vhlh-deficient thymocytes restored thymic cellularity as well as thymocyte viability in vitro. Our data suggest that tight regulation of Hif-1 via pVhl is required for normal thymocyte development and viability and that an increase in Hif-1 transcriptional activity enhances caspase 8-mediated apoptosis in thymocytes.

scholarly journals A small molecule HIF-1α stabilizer that accelerates diabetic wound healing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guodong Li ◽  
Chung-Nga Ko ◽  
Dan Li ◽  
Chao Yang ◽  
Wanhe Wang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Small Molecule ◽  
Hypoxia Inducible Factor ◽  
Diabetic Patients ◽  
Diabetic Mice ◽  
Impaired Wound Healing ◽  
Von Hippel Lindau ◽  
Design And Synthesis

AbstractImpaired wound healing and ulcer complications are a leading cause of death in diabetic patients. In this study, we report the design and synthesis of a cyclometalated iridium(III) metal complex 1a as a stabilizer of hypoxia-inducible factor-1α (HIF-1α). In vitro biophysical and cellular analyses demonstrate that this compound binds to Von Hippel-Lindau (VHL) and inhibits the VHL–HIF-1α interaction. Furthermore, the compound accumulates HIF-1α levels in cellulo and activates HIF-1α mediated gene expression, including VEGF, GLUT1, and EPO. In in vivo mouse models, the compound significantly accelerates wound closure in both normal and diabetic mice, with a greater effect being observed in the diabetic group. We also demonstrate that HIF-1α driven genes related to wound healing (i.e. HSP-90, VEGFR-1, SDF-1, SCF, and Tie-2) are increased in the wound tissue of 1a-treated diabetic mice (including, db/db, HFD/STZ and STZ models). Our study demonstrates a small molecule stabilizer of HIF-1α as a promising therapeutic agent for wound healing, and, more importantly, validates the feasibility of treating diabetic wounds by blocking the VHL and HIF-1α interaction.

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