EC:1.14.11.2 - FACTA Search

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Query: EC:1.14.11.2 (prolyl hydroxylase)
1,814 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.
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PMID:Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen. 215 80

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. 2,7,8-Trihydroxyanthraquinone (THA) is an effective inhibitor of prolyl 4-hydroxylase by virtue of its ability to compete with the co-substrate 2-oxoglutarate (Ki = 40.3 microM). Using a simple and reproducible assay for collagen hydroxylation, we show that THA inhibits the hydroxylation of collagen in embryonic-chick tendon cells in short-term culture, with an IC50 value (concn. giving 50% inhibition) of 32 microM. In comparison, the ethyl ester of 3,4-dihydroxybenzoic acid has an IC50 value of 0.1 mM against collagen hydroxylation by chick tendon cells, whereas its Ki versus isolated prolyl 4-hydroxylase is 49 microM.
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PMID:Inhibition of collagen hydroxylation by 2,7,8-trihydroxyanthraquinone in embryonic-chick tendon cells. 254 62

The enzymatically catalyzed formation of 4-hydroxyproline plays a key role in the intracellular biosynthesis of collagen, since a critical number of 4-hydroxyprolyl residues is required for synthesis and secretion of triple-helical procollagen molecules under physiologic conditions. The enzyme catalyzing the conversion of prolyl residues to 4-hydroxyproline, prolyl 4-hydroxylase, requires ferrous ion, alpha-ketoglutarate, and ascorbate for its activity. 3,4-Dihydroxybenzoic acid has been known to act as potent competitive inhibitor of purified prolyl 4-hydroxylase with respect to one or several of the cofactors or cosubstrates of the enzyme. 3,4-Dihydroxybenzoic acid, however, is a poor inhibitor of prolyl hydroxylation in intact cells, probably due to its polarity not allowing it to enter the cells. In this study, several hydrophobic modifications of 3,4-dihydroxybenzoic acid were tested in human skin fibroblast cultures for their efficacy to inhibit the synthesis of 4-hydroxyproline. The results indicated that the ethyl ester of 3,4-dihydroxybenzoic acid was an efficient inhibitor of prolyl hydroxylation in fibroblast cultures, with Ki of approximately 0.4 mM. Ethyl 3,4-dihydroxybenzoate had little, if any, effect on the hydroxylation of lysyl residues, and it did not affect total protein synthesis or DNA replication in these cells. To test the hypothesis that ethyl 3,4-dihydroxybenzoate might serve as a potential antifibrotic agent, its efficacy in inhibiting prolyl hydroxylation in scleroderma fibroblasts was also tested. The results indicated that the synthesis of 4-hydroxyproline in scleroderma cell cultures was similarly reduced by ethyl 3,4-dihydroxybenzoate. Thus, structural analogs of the cofactors or cosubstrates of prolyl 4-hydroxylase, such as ethyl 3,4-dihydroxybenzoate tested here or its further modifications, may serve as inhibitors of posttranslational hydroxylation of prolyl residues also in vivo. These compounds could potentially provide a novel means of reducing collagen deposition in tissues in fibrotic diseases, such as scleroderma.
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PMID:Inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures by ethyl 3,4-dihydroxybenzoate. 282 18

Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.
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PMID:The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes. 283 64

Collagen biosynthesis involves many unique post-translational events. Inhibition of some of these will lead either to decreased formation of the extracellular collagen fibres or to an accumulation of fibres with altered functional properties. The events that would seem most suitable targets for chemical regulation are triple helix formation, the cleavage of propeptides from the procollagen molecules and cross-link formation. Attempts have recently been made to develop inhibitors of prolyl 4-hydroxylase in particular, as inhibition of this enzyme will prevent triple helix formation and thus lead to a non-functional protein. Prolyl 4-hydroxylase is inhibited competitively with respect to ferrous ion by several bivalent cations, especially zinc, with respect to 2-oxoglutarate by pyridine 2,5-dicarboxylate, pyridine 2,4-dicarboxylate, 3,4-dihydroxybenzoate and many related compounds, with respect to oxygen by superoxide dismutase-active copper chelates and with respect to the peptide substrate by a number of peptides. Triple helix formation can also be inhibited by administering certain proline analogues such as cis-4-hydroxyproline and L-azetidine-2-carboxylic acid, which are incorporated into proteins in place of proline. Only preliminary data are available on the possibilities for using any of these substances to inhibit collagen accumulation in fibrotic processes.
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PMID:Synthesis of collagen: chemical regulation of post-translational events. 299 12

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.
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PMID:Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action. 303 58

To determine the regulatory role of prolyl hydroxylation in intracellular cardiac procollagen turnover, we examined the effects of prolyl 4-hydroxylase inhibitors (alpha, alpha-dipyridil, 3,4-dihydroxybenzoic acid ethyl ester, pyridine 2,4-dicarboxylic acid ethyl ester) and ascorbic acid on procollagen metabolism by cultured, neonatal rat cardiac fibroblasts. Ascorbate-deficient fibroblasts showed decreased rates of prolyl hydroxylation and total collagen accumulation without a significant reduction in alpha 1(I) and alpha 1(III) mRNA levels. The fraction of newly synthesized procollagens degraded intracellularly was also substantially increased in ascorbate-deficient cells (50 +/- 7 v 30 +/- 3% in ascorbate-deficient v control fibroblasts; P < 0.05). These findings were associated with increased intracellular accumulation of Type I procollagen, enhanced secretion of "underhydroxylated" pro alpha 1 (I) polypeptide into the cell culture medium, and decreased extracellular Type I collagen deposition. Similar results were obtained by treating cells with alpha, alpha-dipyridil (300 microns), and 3,4-dihydroxybenzoic acid ethyl ester (400 microM) in the presence of ascorbate. A major portion of the enhanced degradation of newly synthesized procollagens occurred within acidic intracellular compartments as indicated by the inhibition of procollagen degradation by chloroquine (25 microM). Inhibition of procollagen secretion by colchicine (0.5 micrograms/ml) enhanced the diversion to, and subsequent intracellular degradation of underhydroxylated procollagens in cardiac fibroblast lysosomes. We conclude that inactivation of prolyl 4-hydroxylase increases intracellular accumulation and intralysosomal degradation of newly synthesized cardiac procollagen polypeptides. These observations suggest that procollagen prolyl hydroxylation may be important in the regulation of collagen accumulation by cardiac interstitial cells during fibrotic processes in vivo
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PMID:Prolyl hydroxylation regulates intracellular procollagen degradation in cultured rat cardiac fibroblasts. 852 10

We examined the effect of a prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate (E-DHB), on collagen production, proliferation, and spreading by cultured rabbit keratocytes and subconjunctival fibroblasts. Proliferating cells were incubated with or without E-DHB, and were examined by phase-contrast microscopy and proliferation assays. In addition, wounds were made in confluent cultures and were allowed to heal with or without exposure to E-DHB. Confluent cells were also observed by transmission electron microscopy after exposure to E-DHB (0.2-0.4 mM). Finally, confluent cells were incubated in the presence or absence of E-DHB, after which the concentration of soluble collagen in the culture medium was determined. E-DHB (0.2-0.4 mM) inhibited the growth of rabbit keratocytes and subconjunctival fibroblasts and decreased collagen production without inducing cytotoxicity.. Wounds in the control culture were initially healed by individual migrating cells and later by spreading monolayer sheets and proliferating cells, while E-DHB inhibited cell migration into the wound. Electron microscopy revealed large irregular cisternae of endoplasmic reticulum in E-DHB-treated cells, possibly due to protein accumulation. These findings suggest that collagen production may be closely related to various cellular activities.
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PMID:Effect of a prolyl hydroxylase inhibitor on rabbit ocular fibroblasts. 892 6

Tissue homogenates of the deep sea annelids Alvinella caudata and Alvinella pompejana were found to contain enzyme activity resembling vertebrate prolyl 4-hydroxylase. The release of 3H2O from [3,4-(3)H]proline labeled, under-hydroxylated chicken protocollagen type I depended on the presence of the cofactors 2-oxoglutarate, ascorbate, Fe2+ and O2. The release of 3H2O could be inhibited by the prolyl 4-hydroxylase inhibitors zinc, 2,2'-dipyridyl, 3,4-dihydroxybenzoic acid and pyridine-2,4-dicarboxylate, as well as by the synthetic peptide (Pro-Pro-Gly)10. This synthetic peptide could also serve as substrate, because it enhanced the decarboxylation of 2-oxo[5-(14)C]glutarate. Alvinella prolyl hydroxylase appeared to be related to type II vertebrate enzyme because of its lack of affinity for poly (L-proline) and resistance to inactivation by an irreversible peptide inhibitor of chicken prolyl 4-hydroxylase. Maximal enzyme activity was observed in solutions with less than 10% oxygen saturation. By contrast, chicken enzyme was most active at saturating oxygen concentrations. Further data suggest that the Alvinella enzymes are able to accept the 2-oxo acids pyruvate, oxaloacetate and 2-oxoadipinate as substitutes of the cosubstarate 2-oxoglutarate. The data explain the high hydroxylation of Alvinella collagens despite the low oxygen concentrations around hydrothermal vents.
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PMID:Prolyl hydroxylase activity in tissue homogenates of annelids from deep sea hydrothermal vents. 970 43

Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [(3)H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-d-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O(2), alpha-ketoglutarate, and ascorbate and was inhibited by CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.
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PMID:The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-alpha class of animal prolyl 4-hydroxylases. 1570 70

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