Regulation of hypoxia-inducible factor 1 by prolyl and asparaginyl hydroxylases

Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of oxygen homeostasis by mediating a wide range of cellular and systemic adaptive physiological responses to reduced oxygen availability. In this review, we will summarize recent progress in elucidating the molecular mechanisms of HIF-1 activation, focusing on the role of oxygen-dependent prolyl and asparaginyl hydroxylases in hypoxia signal transduction.     

Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation.     

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely β2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.     

Analogues of dealanylalahopcin are inhibitors of human HIF prolyl hydroxylases

Analogues of the naturally occurring cyclic hydroxamate dealanylalahopcin, which is an inhibitor of procollagen prolyl-4-hydroxylase, were synthesised and shown to be inhibitors of the human hypoxia-inducible factor prolyl hydroxylases.     

Expression of prolyl hydroxylases 2 and 3 in chick embryos

Hypoxic cellular response is crucial for normal development as well as in pathological conditions in order to tolerate low oxygen. The response is mediated by Hypoxia Inducible Factors (HIFs), where the α-subunit of HIF is stabilised and able to function only in low oxygen. Prolyl hydroxylases (PHDs) are oxygen dependent dioxygenase enzymes that hydroxylate HIF-α leading to HIF degradation. Thus PHDs function as an oxygen sensor for the function of HIFs. Here we describe the mRNA expression pattern of PHDs in chick embryos. Up to embryonic day 2, PHDs are weak without specific localisation, whereas from day 3 localised expression was observed in the eye, branchial arches and dermomyotome. Later in the limb development PHDs were expressed in the perichondral mesenchyme, excluded from the developing limb cartilages.     

Siah proteins, HIF prolyl hydroxylases, and the physiological response to hypoxia

New evidence suggests that at least two members of the family of hypoxia-inducible factor (HIF) prolyl hydroxylases that regulate HIF stability in response to oxygen (O2) availability are also targeted for proteosome-dependent degradation by the E3 ubiquitin ligases Siah1a and Siah2. This preview examines cellular responses to O2 deprivation (hypoxia) and the complexity of the regulation of the HIF O2 sensing pathway in mammals.     

Mechanism-based in vivo inactivation of lauric acid hydroxylases

The hepatic cytochrome P-450 isozymes that catalyze omega- and (omega - 1)-hydroxylation of lauric acid are specifically inactivated in vitro but not in vivo by 10-undecynoic acid. The lack of in vivo activity may result from rapid degradation of the inhibitor by beta-oxidation. Strategies for the construction of fatty acid analogues that retain the ability to inactivate fatty acid hydroxylases but are resistant to metabolic degradation have therefore been sought. Fatty acid analogues in which the carboxylic acid group is replaced by a sulfate moiety, or in which two methyl groups are placed vicinal to the carboxylic acid group, have been found to inactivate lauric acid hydroxylases in vitro and in vivo without causing time-dependent inhibition of ethoxycoumarin O-deethylation or N-methyl-p-chloroaniline N-demethylation.     

Reversible inactivation of superoxide-sensitive aconitase in Abeta1-42-treated neuronal cell lines

The activity of the superoxide-sensitive enzyme aconitase was monitored to evaluate the generation of superoxide in neuronal cell lines treated with beta-amyloid (Abeta) peptide 1-42. Treatment of differentiated and undifferentiated rat PC12 and human neuroblastoma SK-N-SH cells with soluble Abeta1-42 (Abeta-derived diffusible ligands) or fibrillar Abeta1-42 caused a 35% reversible inactivation of aconitase, which preceded loss of viability and was correlated with altered cellular function. Aconitase was reactivated upon incubation of cellular extracts with iron and sulfur, suggesting that Abeta causes the release of iron from 4Fe-4S clusters. Abeta neurotoxicity was partially blocked by the iron chelator deferoxamine. These data suggest that increased superoxide generation and the release of iron from 4Fe-4S clusters are early events in Abeta1-42 neurotoxicity.     

Design and synthesis of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors

Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series with consistent activity against EGLN-1.     

Design and synthesis of a series of novel pyrazolopyridines as HIF-1alpha prolyl hydroxylase inhibitors

Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.