Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen

2014 ◽  
Vol 463 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Hanna Tarhonskaya ◽  
Rasheduzzaman Chowdhury ◽  
Ivanhoe K. H. Leung ◽  
Nikita D. Loik ◽  
James S. O. McCullagh ◽  
...  
Keyword(s):  
Active Site ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Slow Reaction ◽  
Domain Protein ◽  
Prolyl Hydroxylase Domain

Studies of active site variants of the hypoxia-inducible factor (HIF) hydroxylase prolyl hydroxylase domain protein 2 (PHD2) reveal residues critical for binding Fe(II) and 2-oxoglutarate (2OG) and show that the Fe-chelating residue Asp315 has a role in the relatively slow reaction of PHD2 with O2.

scholarly journals Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3

2018 ◽  
Vol 315 (5) ◽  
pp. H1148-H1158 ◽  
Author(s):  
Jan Neckář ◽  
Anna Hsu ◽  
Md. Abdul Hye Khan ◽  
Garrett J. Gross ◽  
Kasem Nithipatikom ◽  
...  
Keyword(s):  
Infarct Size ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Male Rats ◽  
Epoxyeicosatrienoic Acid ◽  
Degrading Enzyme ◽  
Hypoxia Inducible Factor 1Α ◽  
Acid Analog ◽  
Domain Protein ◽  
Prolyl Hydroxylase Domain

Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.

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

2016 ◽  
Vol 36 (18) ◽  
pp. 2328-2343 ◽  
Author(s):  
Patrick R. Arsenault ◽  
Daisheng Song ◽  
Yu Jin Chung ◽  
Tejvir S. Khurana ◽  
Frank S. Lee
Keyword(s):  
High Altitude ◽  
Zinc Finger ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Loss Of Function ◽  
Domain Protein ◽  
Factor Α ◽  
Prolyl Hydroxylase Domain

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 bothin vitroandin vivo. We show thatin 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/C42SEgln1knock-in mutation results in upregulation of theerythropoietingene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks ofEgln1loss 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-functionEGLN1allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.

scholarly journals Enhancement of Angiogenesis Through Stabilization of Hypoxia-inducible Factor-1 by Silencing Prolyl Hydroxylase Domain-2 Gene

2008 ◽  
Vol 16 (7) ◽  
pp. 1227-1234 ◽  
Author(s):  
Shourong Wu ◽  
Nobuhiro Nishiyama ◽  
Mitsunobu R Kano ◽  
Yasuyuki Morishita ◽  
Kohei Miyazono ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Hypoxia Inducible Factor 1 ◽  
Prolyl Hydroxylase Domain

Abstract 396: Inhibition of Prolyl Hydroxylase Domain-containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Toshihiro Ichiki
Keyword(s):  
Cellular Response ◽  
Target Genes ◽  
Interstitial Fibrosis ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Renin Angiotensin System ◽  
Prolyl Hydroxylase ◽  
Ang Ii ◽  
Prolyl Hydroxylase Domain

Background: Prolyl hydroxylase domain-containing protein (PHD) mediates hydroxylation of hypoxia-inducible factor (HIF)-1α and thereby induces proteasomal degradation of HIF-1α. Inhibition of PHD by hypoxia or hypoxia mimetics such as cobalt chloride (CoCl2) stabilizes HIF-1 and increases the expression of target genes such as vascular endothelial growth factor (VEGF). Although hypoxia activates the systemic renin angiotensin system (RAS), the role of PHD in regulating RAS remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin (Ang) II type 1 receptor (AT1R) and its signaling. Methods and Results: Hypoxia (1% O2), CoCl2 (100-300 μmol/L), and dimethyloxalylglycine (0.25-1.0 mmol/L), all known to inhibit PHD, reduced AT1R expression by 37.7±7.6, 39.6±8.4-69.7±9.9, and 13.4±6.1-25.2±7.0%, respectively (p<0.01) in cultured vascular smooth muscle cell. The same stimuli increased the expression of nuclear HIF-1α and VEGF (p<0.05), suggesting that PHD activity is inhibited. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT1R expression by 55.3±6.0% (p<0.01). CoCl2 decreased AT1R mRNA through transcriptional and posttranscriptional mechanisms (p<0.01 and <0.05, respectively). CoCl2 and PHD2 knockdown diminished Ang II-induced ERK phosphorylation (P<0.01). Over-expression of the constitutively active HIF-1α did not impact the AT1R gene promoter activity. Oral administration of CoCl2 (14 mg/kg/day) to C57BL/6J mice receiving Ang II infusion (490 ng/kg/min) for 4 weeks significantly reduced the expression of AT1R in the aorta by 60.9±11.3% (p<0.05) and attenuated coronary perivascular fibrosis by 85% (p<0.01) without affecting blood pressure. However, CoCl2 did not affect Ang II-induced renal interstitial fibrosis. Conclusion: PHD inhibition downregulates AT1R expression independently of HIF-1α, reduces the cellular response to Ang II, and attenuates profibrotic effect of Ang II on the coronary arteries. PHD inhibition may be beneficial for the treatment of cardiovascular diseases, in which activation of RAS plays a critical role.

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