Regulation of HIF: Prolyl Hydroxylases

The model of the placental barrier based on the human choriocarcinoma cell line BeWo b30 allows studying the effect of hypoxia on trophoblast cells. The effect of the oxyquinoline derivative inhibiting HIF-prolyl hydroxylases was studied on this model. Inhibition of these enzymes leads to an increase in the HIF-1α subunit in the cytoplasm, mimicking the cell response to hypoxia. Incubation of the cells with the drug at a concentration of 10 uM for 24 h did not affect the paracellular transport, but reduced the transport of glucose through the cell barrier. The transcriptome analysis after the exposure with oxyquinoline derivative revealed a decreased expression of the KIAA1217 gene and its intronic gene MIR603, which encodes microRNA hsa-miR-603. The expression of the target gene of this miRNA, CCND1 encoding cyclin D1, after oxyquinoline derivative exposition increased significantly, which may indicate a potential microRNA-mRNA regulatory mechanism in the response of trophoblast cells to hypoxia. BeWo b30, placenta, hypoxia, oxyquinoline, barrier, microRNA, cyclin The study was performed with the equipment of the «Postgenomic and Metabolomic Methods of Study in Molecular Biology» Common Use Center (BioClinicum Scientific and Technical Center). The study was supported by the Ministry of Education and Science of the Russian Federation in the framework of the Federal Targeted Program for Research and Development in Priority Areas of Advancement of the Russian Scientific and Technological Complex for 2014-2020 (Project no. RFMEFI58817X0007).

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Managing Anemia across the Stages of Kidney Disease in Those Hyporesponsive to Erythropoiesis-Stimulating Agents

American Journal of Nephrology ◽

10.1159/000516901 ◽

2021 ◽

Vol 52 (6) ◽

pp. 450-466

Author(s):

Matthew R. Weir

Keyword(s):

Drug Targets ◽

Iron Status ◽

Therapeutic Option ◽

Attractive Potential ◽

Dialysis Modality ◽

Erythroid Progenitors ◽

Prolyl Hydroxylases ◽

Erythropoiesis Stimulating Agents ◽

Sustained Effects ◽

Concomitant Medications

Background: Patients with CKD frequently have anemia that results from iron-restricted erythropoiesis and inflammation. Anemia of CKD is currently managed with iron supplements and erythropoiesis-stimulating agents (ESAs) to promote erythropoiesis and with RBC transfusion in severe cases. Hyporesponse to ESAs, or the need for larger than usual doses to attain a given hemoglobin (Hb) level, is associated with increased morbidity and mortality and presents a pressing clinical challenge, particularly for patients on dialysis. This paper reviews ESA hyporesponse and potential new therapeutic options in the management of anemia of CKD. Summary: The most common causes of ESA hyporesponse include iron deficiency and inflammation, and to a lesser degree, secondary hyperparathyroidism, inadequate dialysis, malnutrition, and concomitant medications. Management of ESA hyporesponse is multipronged and involves treating low level infections, ensuring adequate nutrition, and optimizing iron status and dialysis modality, although some patients can remain refractory. Inflammation directly increases production and secretion of hepcidin, contributes to an impaired response to hypoxia, and suppresses proliferation of erythroid progenitors. Coordination of renal and hepatic erythropoietin (EPO) production and iron metabolism is under the control of hypoxia-inducible factors (HIF), which are in turn regulated by HIF-prolyl hydroxylases (HIF-PHs). HIF-PHs and hepcidin are therefore attractive potential drug targets particularly in patients with ESA hyporesponse. Several oral HIF-PH inhibitors have been evaluated in patients with anemia of CKD and have been shown to increase Hb and reduce hepcidin regardless of inflammation, iron status, or dialysis modality. These sustained effects are achieved through more modest increases in endogenous EPO compared with ESAs. Key Messages: Treatments that address ESA hyporesponse remain a significant unmet clinical need in patients with anemia of CKD. New therapies such as HIF-PH inhibitors have the potential to address fundamental aspects of ESA hyporesponse and provide a new therapeutic option in these patients.

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Roles of HIF and 2-Oxoglutarate-Dependent Dioxygenases in Controlling Gene Expression in Hypoxia

Cancers ◽

10.3390/cancers13020350 ◽

2021 ◽

Vol 13 (2) ◽

pp. 350

Author(s):

Julianty Frost ◽

Mark Frost ◽

Michael Batie ◽

Hao Jiang ◽

Sonia Rocha

Keyword(s):

Gene Expression ◽

Hypoxia Inducible Factor ◽

Transcriptional Regulator ◽

Hydroxylase Activity ◽

Control Of Gene Expression ◽

Prolyl Hydroxylases ◽

Chromatin Organisation ◽

Sense And Respond ◽

Almost All ◽

And Function

Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.

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Collagen metabolism as a regulator of proline dehydrogenase/proline oxidase-dependent apoptosis/autophagy

Amino Acids ◽

10.1007/s00726-021-02968-y ◽

2021 ◽

Author(s):

Jerzy Palka ◽

Ilona Oscilowska ◽

Lukasz Szoka

Keyword(s):

Cell Metabolism ◽

Underlying Mechanism ◽

Collagen Biosynthesis ◽

Proline Metabolism ◽

Proline Dehydrogenase ◽

Histone Demethylases ◽

Proline Oxidase ◽

Prolyl Hydroxylases ◽

Transport Chain ◽

And Control

AbstractRecent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.

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Hypoxia-inducible factor prolyl hydroxylases as targets for neuroprotection by “antioxidant” metal chelators: From ferroptosis to stroke

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2013.01.026 ◽

2013 ◽

Vol 62 ◽

pp. 26-36 ◽

Cited By ~ 53

Author(s):

Rachel E. Speer ◽

Saravanan S. Karuppagounder ◽

Manuela Basso ◽

Sama F. Sleiman ◽

Amit Kumar ◽

...

Keyword(s):

Hypoxia Inducible Factor ◽

Metal Chelators ◽

Prolyl Hydroxylases

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

Biochemical Journal ◽

10.1042/bj20101201 ◽

2011 ◽

Vol 436 (2) ◽

pp. 363-369 ◽

Cited By ~ 25

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.

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Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates

eLife ◽

10.7554/elife.46490 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 22

Author(s):

Matthew E Cockman ◽

Kerstin Lippl ◽

Ya-Min Tian ◽

Hamish B Pegg ◽

William D Figg ◽

...

Keyword(s):

Oxygen Sensors ◽

Prolyl Hydroxylase ◽

Hydroxylase Activity ◽

Recombinant Enzymes ◽

Animal Cells ◽

Prolyl Hydroxylases ◽

Biological Responses ◽

Wide Range ◽

Prolyl Hydroxylation ◽

Hif Prolyl Hydroxylase

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported.

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Abstract P466: Sqstm1/p62 Regulates Hypoxia Inducible Factor 1 Alpha Stabilization And Protects The Heart From Hypoxic Injury

Circulation Research ◽

10.1161/res.129.suppl_1.p466 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Rajeshwary Ghosh ◽

Nima Fatahian ◽

Vinod Vishaka ◽

J D Symons ◽

Sihem Boudina

Keyword(s):

Cell Death ◽

Transcriptional Activation ◽

Cardiac Tissue ◽

Hypoxia Inducible Factor ◽

Ubiquitin Proteasome System ◽

Hypoxia Tolerance ◽

H9c2 Cells ◽

Prolyl Hydroxylases ◽

Hypoxic Injury ◽

Cardiac Myoblasts

Ischemic heart disease (IHD) is characterized by cardiac tissue hypoxia, dysregulatedmetabolism, and cell death. Hypoxia inducible factor 1a (HIF1α) is a major component of thehypoxia pathway that regulates metabolic and angiogenic genes. Under normoxia, HIF1α getshydroxylated by the prolyl hydroxylases (PHDs), followed by its ubiquitination, and degradationby the Ubiquitin Proteasome System (UPS). Given the short half-life of HIF1α (>5mins), PHDinhibitors are employed to stabilize HIF1α and improve cardiac function in animal models ofischemia/infarction. Because PHD inhibitors exert off-target effects, alternative strategies tostabilize HIF1α are needed. In cancer cells, p62 stabilizes HIF1α by binding PHD3. Wehypothesized that p62 would stabilize HIF1α and provides cardioprotection from hypoxia. Wegenerated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (cip62KO mice)and exposed them to 7% O2 for 6h. Compared to wild-type (WT) littermates, cip62KO micedisplayed increased oxidative stress and enhanced cell death. Hypoxia caused contractiledysfunction in cip62KO vs. WT mice. To gain mechanistic insights as to how lack of p62exacerbated hypoxic injury, cultured H9c2 cardiac myoblasts were exposed to 21% (normoxia)or 1% (hypoxia) O2 for 24h. Hypoxia increased HIF1α and p62 protein expression in H9c2 cells(p<0.05). To determine whether hypoxia-induced p62 accumulation is required for HIF1αstabilization, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, andexposed to hypoxia. Compared to ctrl siRNA cells, hypoxia-induced HIF1α protein accumulationwas reduced (p<0.05) after p62 knockdown. Additionally, hypoxia increased expression ofHIF1α downstream targets: Egln1 , Vegfa , Bnip3 and Hmox1 mRNA, in ctrl siRNA cells, but theresponse was blunted (p<0.05) after p62 knockdown. These data indicate that p62 contributes tohypoxia-induced HIF1α stabilization and transcriptional activation. Defining how p62contributes to HIF1α stabilization and hypoxia tolerance is relevant clinically and could identifyp62 as a therapeutic target for treating IHD.

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