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.     

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.     

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.     

Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel

The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.     

The HIF prolyl hydroxylase PHD3 is a potential substrate of the TRiC chaperonin

Hypoxia-inducible factor-1 (HIF) is regulated by oxygen-dependent prolyl hydroxylation. Of the three HIF prolyl hydroxylases (PHD1, 2 and 3) identified, PHD3 exhibits restricted substrate specificity in vitro and is induced in different cell types by diverse stimuli. PHD3 may therefore provide an interface between oxygen sensing and other signalling pathways. We have used co-purification and mass spectrometry to identify proteins that interact with PHD3. The cytosolic chaperonin TRiC was found to copurify with PHD3 in extracts from several cell types. Our results indicate that PHD3 is a TRiC substrate, providing another step at which PHD3 activity may be regulated.     

Radiommunoassay for human and chick prolyl hydroxylases.

A radioimmunoassay is reported for measuring prolyl hydroxylase. The assay is based on the displacement of radioactively-labelled prolyl hydroxylase from its antibody by the non-labelled enzyme, and on the subsequent precipitation of the enzyme-antibody complex by a cellulose-bound second antibody. Pure prolyl hydroxylase was isolated from foetal human or chick embryo tissues by an affinity column procedure usingpoly(L-proline). The enzyme was labelled with tritium using a technique of reductive alkylation with formaldehyde and sodium [3H]borohydride. No conversion of the enzyme tetramer to its monomers was found to take place during the tritiation reaction. Experiments on the dissociation of the non-labelled enzyme indicated that the degree of displacement of the labelled enzyme was similar regardless of whether the non-labelled enzyme was in the tetramer form or in that of the subunit monomers. The sensitivity of the radioimmunoassay is of the order of 5 -- 10 ng immunoreactive prolyl hydroxylase. The concentrations of the immunoreactive prolyl hydroxylase assayed with the present method in human serum and skin and in several chick embryo tissues are reported.     

Investigation of the dimer interface and substrate specificity of prolyl dipeptidase DPP8

DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.     

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.     

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.     

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.     

Observations on the substrate specificity of prostaglandin hydroxylases of monkey seminal vesicles and sheep vesicular glands

Prostaglandin (PG) 19-hydroxylase of monkey seminal vesicles metabolizes PGE1 and PGE2 to their 19-hydroxy metabolites, while PGE2 20-hydroxylase of ram vesicular glands metabolizes PGE2 to 20-hydroxy-PGE2. The purpose of the present study was determine whether PGF2 alpha is a substrate of these enzymes. Deuterated 20-hydroxy-PGF2 alpha was employed as an internal standard to study the hydroxylation of PGF2 alpha (0.2 mM) by microsomes of monkey (Macaca fascicularis) seminal vesicles in the presence of NADPH, and the biosynthesis was compared with the hydroxylation of PGE2 under identical conditions. 19-Hydroxy-PGF2 alpha was formed at a rate of 3.5% of the formation of 19-hydroxy-PGE2. Microsomes of ram vesicular glands also hydroxylated PGE2 more efficiently than PGF2 alpha, which was converted to both 20-hydroxy-PGF2 alpha and 19-hydroxy-PGF2 alpha at a combined rate of 5% of the biosynthesis of 20-hydroxy-PGE2 under the same conditions. 20-Hydroxy-PGF2 alpha was demonstrated in ram semen, but the concentration was low (0.1 microM) in comparison with 20-hydroxy-PGE2 (24 microM). The two genital PG hydroxylases thus metabolize PGF2 alpha much less efficiently than PGE2. This finding may suggest that 19-hydroxy- and 20-hydroxy-PGF2 alpha in seminal fluids also could be formed by other mechanisms, e.g., from 19-hydroxy- and 20-hydroxy-PGE2 by the PGE 9-keto reductase enzyme.     

Structure, mechanism, and substrate specificity of kynureninase

The kynurenine pathway is the major route for tryptophan catabolism in animals and some fungi and bacteria. The procaryotic enzyme preferentially reacts with l-kynurenine, while eucaryotic kynureninases exhibit higher activity with 3-hydroxy-l-kynurenine. Crystallography of kynureninases from Pseudomonas fluorescens (PfKyn) and Homo sapiens (HsKyn) shows that the active sites are nearly identical, except that His-102, Asn-333, and Ser-332 in HsKyn are replaced by Trp-64, Thr-282, and Gly-281 in PfKyn. Site-directed mutagenesis of HsKyn shows that these residues are, at least in part, responsible for the differences in substrate specificity since the H102W/S332G/N333T triple mutant shows activity with kynurenine but not 3-hydroxykynurenine. PfKyn is strongly inhibited by analogs of a proposed gem-diolate intermediate, dihydrokynurenine, and S-(2-aminophenyl)-l-cysteine S,S-dioxide, with K(i) values in the low nanomolar range. Stopped-flow kinetic experiments show that a transient quinonoid intermediate is formed on mixing, which decays to a ketimine at 740 s(-1). Quench experiments show that anthranilate, the first product, is formed in a stoichiometric burst at 50 s(-1) and thus the rate-determining step in the steady-state is the release of the second product, l-Ala. β-Benzoylalanine is also a good substrate for PfKyn but does not show a burst of benzoate formation, indicating that the rate-determining step for this substrate is benzoate release. A Hammett plot of rate constants for substituted β-benzoylalanines is non-linear, suggesting that carbonyl hydration is rate-determining for electron-donating groups, but C(β)-C(γ) cleavage is rate-determining for electron-withdrawing groups. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.