scholarly journals Characterization of different isoforms of the HIF prolyl hydroxylase PHD1 generated by alternative initiation

2006 ◽  
Vol 397 (1) ◽  
pp. 179-186 ◽  
Author(s):  
Ya-Min Tian ◽  
David R. Mole ◽  
Peter J. Ratcliffe ◽  
Jonathan M. Gleadle
Keyword(s):  
Regulation Of Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Biologically Active ◽  
Genetic Studies ◽  
Translational Initiation ◽  
Von Hippel Lindau ◽  
Seven In Absentia ◽  
Hif Prolyl Hydroxylase

The heterodimeric transcription factor HIF (hypoxia-inducible factor) is central to the regulation of gene expression by oxygen. Three oxygen-dependent prolyl hydroxylase enzymes [PHD1 (prolyl hydroxylase domain 1), PHD2 and PHD3] control the abundance of HIF. In the presence of oxygen, they hydroxylate specific proline residues in HIF-α, allowing recognition by pVHL (von Hippel-Lindau protein) and subsequent ubiquitylation and proteasomal destruction. The precise roles and regulation of these enzymes are therefore of particular importance in understanding the physiological and pathological responses to hypoxia. In the present study, we define the existence of two species of PHD1 and provide evidence that they are generated by alternative translational initiation. We demonstrate that these alternative forms are both biologically active with similar HIF prolyl hydroxylase activity but that they differ in their responses to oestrogen, cell confluence and proteasomal inhibition. We show that the two PHD1 species are subject to proteolytic regulation but differ markedly in their protein stability. Though each isoform has the potential to interact with members of the Siah (seven in absentia homologue) ubiquitin ligase family, genetic studies indicated that other proteolytic mechanisms are responsible for control of stability under the conditions examined. The data define the existence of a further level of control in the pathway that regulates cellular responses to hypoxia.

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.

scholarly journals The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2

2018 ◽  
Author(s):  
Nina Kozlova ◽  
Daniela Mennerich ◽  
Anatoly Samoylenko ◽  
Elitsa Y. Dimova ◽  
Peppi Koivunen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Survival Function ◽  
Hypoxia Inducible Factor ◽  
Adaptor Protein ◽  
Prolyl Hydroxylase ◽  
Binding Partner ◽  
And Migration ◽  
Hif Prolyl Hydroxylase ◽  
Promote Breast Cancer

SummaryThe EGFR-adaptor protein CIN85 has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill-defined. Here, we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SH3 domains of CIN85 interact with the proline-arginine rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF-degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9 edited cells affected growth and migration properties as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2, and uncovered an essential survival function in tumor cells linking growth factor adaptors with hypoxia signaling.

scholarly journals Carcinogenicity Assessment of Daprodustat (GSK1278863), a Hypoxia-Inducible Factor (HIF)-Prolyl Hydroxylase Inhibitor

2019 ◽  
Vol 48 (2) ◽  
pp. 362-378 ◽  
Author(s):  
David F. Adams ◽  
Mark S. Watkins ◽  
Luc Durette ◽  
Josée Laliberté ◽  
Félix Goulet ◽  
...  
Keyword(s):  
Cell Mass ◽  
Hypoxia Inducible Factor ◽  
Carcinogenic Risk ◽  
Prolyl Hydroxylase ◽  
High Dose ◽  
Sprague Dawley ◽  
Time Curve ◽  
Clinical Exposure ◽  
High Doses ◽  
Hif Prolyl Hydroxylase

Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat’s biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat’s 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration–time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.

scholarly journals A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 2193-2196 ◽  
Author(s):  
Melanie J. Percy ◽  
Paul W. Furlow ◽  
Philip A. Beer ◽  
Terence R. J. Lappin ◽  
Mary Frances McMullin ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Prolyl Hydroxylase ◽  
Red Cell ◽  
Mutation Site ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Vhl Protein ◽  
Inhibitory Activities ◽  
Binding Groove ◽  
Hif Prolyl Hydroxylase

AbstractThe molecular basis of the erythrocytosis group of red cell disorders is incompletely defined. Some cases are due to dysregulation of erythropoietin (Epo) synthesis. The hypoxia inducible transcription factor (HIF) tightly regulates Epo synthesis. HIF in turn is regulated through its α subunit, which under normoxic conditions is hydroxylated on specific prolines and targeted for degradation by the von Hippel Lindau (VHL) protein. Several mutations in VHL have been reported in erythrocytosis, but only 1 mutation in the HIF prolyl hydroxylase PHD2 (prolyl hydroxylase domain protein 2) has been described. Here, we report a novel PHD2 mutation, Arg371His, which causes decreased HIF binding, HIF hydroxylase, and HIF inhibitory activities. In the tertiary structure of PHD2, Arg371 lies close to the previously described Pro317Arg mutation site. These findings substantiate PHD2 as a critical enzyme controlling HIF and therefore Epo in humans, and furthermore suggest the location of an active site groove in PHD2 that binds HIF.

scholarly journals Hypoxia-inducible factor (HIF)-prolyl hydroxylase 3 (PHD3) maintains high HIF2A mRNA levels in clear cell renal cell carcinoma

2019 ◽  
Vol 294 (10) ◽  
pp. 3760-3771 ◽  
Author(s):  
Petra Miikkulainen ◽  
Heidi Högel ◽  
Fatemeh Seyednasrollah ◽  
Krista Rantanen ◽  
Laura L. Elo ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Clear Cell ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Mrna Levels ◽  
Cell Renal Cell Carcinoma ◽  
Hif Prolyl Hydroxylase
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