Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.
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PMID:Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase. 170 64

Previously we had demonstrated by photoaffinity labeling that a 57-kDa protein of the endoplasmic reticulum can bind and become covalently linked to glycosylatable photoreactive peptides containing the sequence-Asn-Xaa-Ser/Thr-. Subsequently, it was found that this protein, called glycosylation site-binding protein, was a multifunctional protein, i.e. it was identical to protein disulfide isomerase (PDI), the beta-subunit of prolyl hydroxylase and thyroid hormone-binding protein. In this study, the peptide specificity for binding to this 57-kDa protein, hereafter called PDI, has been investigated in more detail using photoaffinity probes. The results reveal that although N-glycosylation by oligosaccharyl transferase in the endoplasmic reticulum has an absolute requirement for an hydroxyamino acid in the third amino acid residue of the glycosylation site sequence, no such specificity is observed in the binding of such peptides to PDI. In addition to the lack of specificity for an hydroxyamino acid in the third residue position, no specificity was observed for the asparagine residue in the first position. Thus, binding is not restricted to peptides containing N-glycosylation sites. We have investigated the discrepancy between this apparent lack of sequence specificity and earlier results indicating that binding of peptides to PDI was specific for N-glycosylation site sequences. We now demonstrate that PDI in the lumen of microsomes is more efficiently labeled by peptides containing photoreactive-Asn-Xaa-Ser/Thr- sequences than by nonacceptor site sequences because the former become glycosylated. This increased labeling does not occur because the glycosylated form of the probes are preferentially recognized by PDI. Rather, it appears that increased polarity of the affinity probe after attachment of the oligosaccharide chain prevents its exit from the sealed microsomes, in effect concentrating it within the lumen of the microsome. These results, coupled with other studies on the multifunctional nature of PDI, suggest that the observed peptide binding may be a manifestation of the ability of PDI to recognize the backbone of polypeptides in the lumen of the endoplasmic reticulum.
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PMID:Peptide binding by protein disulfide isomerase, a resident protein of the endoplasmic reticulum lumen. 191 71

The protein or cDNA sequencing revealed that the membrane-associated 3,5,3'-triiodo-thyronine binding protein (T3BP) acts as a multifunctional protein:protein disulfide isomerase (PDI) catalyzing isomerization of intra- and inter-molecular disulfide bridge in the proteins, beta-subunit of prolyl 4-hydroxylase catalyzing the formation of 4-hydroxyproline in collagen molecules, glycosylation site binding protein which is a component of oligosaccharyl transferase transferring oligosaccharide chains to the asparagine residues of Asn-X-Ser/Thr site in nascent polypeptide, and a component of triglyceride transfer protein complex involved in the transfer unit of triglyceride, cholesteryl ester and phosphatidylcholine between biomembranes. The functions of 55 k-T3BP/PDI, mainly involved in important post-translational modifications, are discussed in relation to the domain structure of the molecule.
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PMID:[Molecular cloning and multifunctions of membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with protein disulfide isomerase activity]. 819 76

Sensing of ambient dioxygen levels and appropriate feedback mechanisms are essential processes for all multicellular organisms. In animals, moderate hypoxia causes an increase in the transcription levels of specific genes, including those encoding vascular endothelial growth factor and erythropoietin. The hypoxic response is mediated by hypoxia-inducible factor (HIF), an alphabeta heterodimeric transcription factor in which both the HIF subunits are members of the basic helix-loop-helix PAS (PER-ARNT-SIM) domain family. Under hypoxic conditions, levels of HIFalpha rise, allowing dimerization with HIFbeta and initiating transcriptional activation. Two types of dioxygen-dependent modification to HIFalpha have been identified, both of which inhibit the transcriptional response. Firstly, HIFalpha undergoes trans -4-hydroxylation at two conserved proline residues that enable its recognition by the von Hippel-Lindau tumour-suppressor protein. Subsequent ubiquitinylation, mediated by an ubiquitin ligase complex, targets HIFalpha for degradation. Secondly, hydroxylation of an asparagine residue in the C-terminal transactivation domain of HIFalpha directly prevents its interaction with the co-activator p300. Hydroxylation of HIFalpha is catalysed by enzymes of the iron(II)- and 2-oxoglutarate-dependent dioxygenase family. In humans, three prolyl hydroxylase isoenzymes (PHD1-3) and an asparagine hydroxylase [factor inhibiting HIF (FIH)] have been identified. The role of 2-oxoglutarate oxygenases in the hypoxic and other signalling pathways is discussed.
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PMID:The role of iron and 2-oxoglutarate oxygenases in signalling. 1277 46

The activity of hypoxia-inducible transcription factor HIF, an alphabeta heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-alpha subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1alpha sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2alpha peptides was far less efficient than that of the corresponding HIF-1alpha peptides. The K(m) of FIH for O(2) was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For example, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response.
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PMID:Catalytic properties of the asparaginyl hydroxylase (FIH) in the oxygen sensing pathway are distinct from those of its prolyl 4-hydroxylases. 1470 57

Adaptations to change in oxygen availability are crucial for survival of multi-cellular organisms and are also implicated in several disease states. Such adaptations rely upon gene expression regulated by the heterodimeric transcription factors HIFs (hypoxia-inducible factors). Enzymes that link changes in oxygen tensions with the stability and transcriptional activity of HIFs are considered as oxygen sensors. These enzymes are oxygen-, iron- and 2-oxoglutarate-dependent dioxygenases that hydroxylate key proline and asparagine residues in HIFalpha subunits. The constitutive inhibitory action of these enzymes on HIFs is relieved by hypoxia and by agents that displace iron or 2-oxoglutarate. Two of the enzymes, HPH (HIF prolyl hydroxylase)-1 and HPH-2, are known to be inducible by hypoxia in a HIF-dependent manner. This suggests the existence of a novel feedback loop for adjusting hypoxia-regulated gene expression. We have recently shown that HIF-1alpha stability, HIF-1 nuclear translocation and HIF-mediated gene expression in human glioma cell lines can be stimulated by pyruvate independently of hypoxia. In the present study we show that the endogenous 2-oxoacid oxaloacetate can also activate HIF-mediated gene expression. Pyruvate and oxaloacetate treatment of cells also up-regulates HPH-1 and HPH-2, but not HPH-3 or the HIF asparaginyl hydroxylase FIH-1 (factor inhibiting HIF). Regulation of HIF-1 and the expression of HPH homologue genes can thus be influenced by specific glycolytic and tricarboxylic acid cycle metabolites. These findings may underlie important interactions between oxygen homoeostasis, glycolysis, the tricarboxylic acid cycle and gluconeogenesis.
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PMID:Endogenous 2-oxoacids differentially regulate expression of oxygen sensors. 1498 67

HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.
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PMID:Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity. 1523 70

The hypoxia-inducible factor-1alpha (HIF-1alpha) subunit is activated in response to lack of oxygen. HIF-1alpha-specific prolyl hydroxylase and factor inhibiting HIF-1alpha (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1alpha, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel-Lindau protein, leading to degradation of HIF-1alpha by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1alpha. We found that the Zn-specific chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1alpha-proline hydroxylase 2 but the level of HIF-1alpha protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1alpha, its presence leads to the accumulation of HIF-1alpha that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1alpha from interacting with CBP, so reducing expression of HIF-1alpha target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1alpha protein in both normoxia and hypoxia.
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PMID:The zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1alpha protein in normoxic cells. 1657 68

The function of the hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in cellular adaptation to hypoxia, is restricted to low oxygen tension (pO(2)). As such, this transcription factor is central in modulating the tumor microenvironment, sensing nutrient availability, and controlling anaerobic glycolysis, intracellular pH, and cell survival. Degradation and inhibition of the limiting HIF-1alpha subunit are intimately connected in normoxia. Hydroxylation of two proline residues by prolyl hydroxylase domain (PHD) 2 protein earmarks the protein for degradation, whereas hydroxylation of an asparagine residue by factor-inhibiting HIF-1 (FIH-1 or FIH) reduces its transcriptional activity. Indeed, silencing of either PHD2 or FIH in normoxia partially induced hypoxic genes, whereas combined PHD2/FIH silencing generated a full hypoxic gene response. Given the fact that HIF-1alpha possesses two transcriptional activation domains [TAD; NH(2)-terminal (N-TAD) and COOH-terminal (C-TAD)], we hypothesized on a possible bifunctional activity of HIF-1alpha that could be discriminated by FIH, an inhibitor of the C-TAD. In human cell lines engineered to overexpress or silence FIH in response to tetracycline, we show by quantitative reverse transcription-PCR that a set of hypoxic genes (ca9, phd3, pgk1, and bnip3) respond differently toward FIH expression. This finding, extended to 26 hypoxia-induced genes, indicates differential gene expression by the N-TAD and C-TAD in response to the hypoxic gradient. We propose that the oxygen-sensitive attenuator FIH, together with two distinct TADs, is central in setting the gene expression repertoire dictated by the cell pO(2).
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PMID:The oxygen sensor factor-inhibiting hypoxia-inducible factor-1 controls expression of distinct genes through the bifunctional transcriptional character of hypoxia-inducible factor-1alpha. 1658 95

The mechanisms controlling the expression of the gene encoding for the hormone erythropoietin (EPO) are exemplary for oxygen-regulated gene expression. In humans and other mammals, hypoxia modulates EPO levels by increasing expression of the EPO gene. An association between polycythaemia and people living at high altitudes was first reported more than 100 years ago. Since the identification of EPO as the humoral regulator of red blood cell production and the cloning of the EPO gene, considerable progress has been made in understanding the regulation of EPO gene expression. This has finally led to the identification of a widespread cellular oxygen-sensing mechanism. Central to this mechanism is the transcription factor complex hypoxia-inducible factor (HIF)-1. The abundance and activity of HIF-1, a heterodimer of an alpha- and beta-subunit, is predominantly regulated by oxygen-dependent post-translational hydroxylation of the alpha-subunit. Non-heme ferrous iron containing hydroxylases use dioxygen and 2-oxoglutarate to specifically target proline and an asparagine residue in HIF-1alpha. As such, the three prolyl hydroxylases (prolyl hydroxylase domain-containing protein (PHD) 1, PHD2 and PHD3) and the asparagyl hydroxylase (factor inhibiting HIF (FIH)-1) act as cellular oxygen sensors. In addition to erythropoiesis, HIF-1 regulates a broad range of physiologically relevant genes involved in angiogenesis, apoptosis, vasomotor control and energy metabolism. Therefore, the HIF system is implicated in the pathophysiology of many human diseases. In addition to the tight regulation by oxygen tension, temporal and tissue-specific signals limit expression of the EPO gene primarily to the fetal liver and the adult kidney.
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PMID:Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene expression. 1700 76


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