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)

Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.
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PMID:The gene for a novel protein, a member of the protein disulphide isomerase/form I phosphoinositide-specific phospholipase C family, is amplified in hydroxyurea-resistant cells. 131 Nov 71

With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.
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PMID:Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation. 184 36

The mussel foot secretes a variety of unusual hydroxyproline-containing collagenous and noncollagenous proteins. Prolyl 4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (alpha 2 beta 2) composed of two subunits with Mr of 60,000 (alpha) and 57,000 (beta) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20 degrees C. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzyme-substrate i.e. (Pro-Pro-Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.
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PMID:Prolyl 4-hydroxylase in the foot of the marine mussel Mytilus edulis L.: purification and characterization. 283 10

In 1979 it was proposed that prolyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) recognizes the beta-turn conformation in nascent procollagen chains and that the hydroxylation process involves a conformational change resulting in "straightening" of the beta-turn segments into the linear triple-helical conformation of native collagen. We present experimental data that verify both these postulates. The following peptides were synthesized and studied for their conformation and interaction with prolyl hydroxylase: tBoc-Pro-Gly-Ala-OH, tBoc-Pro-Gly-Val-OH, tBoc-Gly-Val-Pro-Gly-Val-OH, and tBoc-Pro-DAla-Ala-OH. Spectral data showed that these peptides preferred a beta-turn conformation. All of them acted as inhibitors of the enzyme; the pentapeptide also acted as a substrate. To mimic the biosynthetic event, a collagen model polypeptide, (Pro-Pro-Gly)10, was incubated at 37 degrees C with purified prolyl hydroxylase and the necessary cosubstrates and cofactors at pH 7.8. A progressive change from the initially nonhelical to the triple-helical conformation, as monitored by CD spectra and gel filtration, occurred during the course of proline hydroxylation. In addition to leading to increased thermal stability of the triple-helical conformation in (Pro-Pro-Gly)10 and (Pro-Pro-Gly)5, the enzymatic incorporation of the hydroxyproline residues was found to enable these polypeptides to fold into this conformation faster than the unhydroxylated counterparts. These conformational implications of proline hydroxylation in collagen may also be of use in the study of the complement subcomponent Clq and of acetylcholine esterase which contain collagen-like regions in them.
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PMID:Conformational implications of enzymatic proline hydroxylation in collagen. 629 23

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.
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PMID:Structural aspects of hydroxyproline-containing proteins. 640 Nov 22

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.
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PMID:Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates. 1042 73

The mechanism by which hypoxia induces gene transcription involves the inhibition of hypoxia-inducible factor (HIF)-1alpha prolyl hydroxylase activity, which prevents von Hippel-Lindau (vHL)-dependent targeting of HIF-1alpha to the ubiquitin-proteasome pathway. HIF-1alpha is stabilized, translocates to the nucleus, interacts with hypoxia-responsive elements, and promotes the activation of target genes. This report shows that cyclosporin A (CsA) interferes with the hypoxic signaling cascade in C6 glioma cells. CsA inhibits hypoxia-dependent gene transcription in a reporter gene assay and prevents the hypoxic accumulation of HIF-1alpha. Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1alpha to the green fluorescent protein (GFP) destabilized the protein in an oxygen-dependent manner. CsA prevented the hypoxic stabilization of an ODD.GFP fusion protein. An assay for 2-oxoglutarate-dependent dioxygenases was developed using a light mitochondrial kidney fraction as a source of enzyme. It uses the capacity of specific peptides to stimulate the degradation of [(14)C]2-oxoglutarate. CsA stimulated the enzymatic activity in the presence of a peptide that mimicked the 557-576 sequence of HIF-1alpha. The enzyme promoted [(35)S]vHL binding to glutathione S-transferase (GST).ODD fusion protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in the HIF-1alpha sequence was replaced by a hydroxyproline or an alanine residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We conclude that CsA destabilizes HIF-1alpha by promoting hydroxylation of Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic adaptation during CsA-induced nephrotoxicity and contribute to the adverse effects of this drug.
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PMID:Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation. 1258 29

Hypoxia-inducible factor (HIF)-1alpha, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF-1alpha is constantly degraded via the ubiquitin-proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1alpha through specific recognition of hydroxylated Pro-402 or Pro-564, both of which are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1alpha for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu-574 of human HIF-1alpha--10 residues downstream of Pro-564--is essential for VHL recognition. We show here that the role of Leu-574 is to recruit PHD2/HPH2 for Pro-564 hydroxylation. An antibody specific for hydroxylated Pro-564 has been used to determine the hydroxylation status; mutation or deletion of Leu-574 results in a significant decrease in the ratio of the hydroxylated HIF-1alpha to the total amount. The nine-residue spacing between Pro-564 and Leu-574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu-574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen-dependent proteolysis of HIF-1alpha. Hence, our findings indicate that Leu-574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF-1alpha activity.
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PMID:Leu-574 of human HIF-1alpha is a molecular determinant of prolyl hydroxylation. 1508 14

Hypoxia inducible factor 1 (HIF-1) is a key regulator of the genes involved in the cellular response to hypoxia. HIF consists of alpha and beta subunits, with the alpha subunit being degraded under normoxic conditions and stabilized under hypoxia. We investigated C1772T and G1790A polymorphisms in exon 12 of the HIF gene, which result in an amino acid change from proline 582 to serine and from alanine 588 to threonine, respectively. These polymorphisms are found within the oxygen-dependent degradation domain of the HIF-1alpha protein and may be important in the oxygen regulation of the protein via hydroxylation of the proline residue at position 564 (P564) by HIF-alpha prolyl hydroxylase (HIF-PH). The frequency of these polymorphisms was studied in 160 nontumor DNA samples from patients with renal cell carcinoma (RCC). There was a highly significant increase in the frequency of both the G/A1790 (45.9 vs. 13.5%, P < 0.00001) and C/C1772 (10 vs. 0.7%, P=0.0004) genotypes in patients with RCC compared with normal healthy controls. A decrease was seen for the GG (44.5 vs. 83%, P < 0.00001) and CT (33.8 vs. 55.5%, P=0.0001) genotypes in patients compared with controls. There was a marked increase in the T-A haplotype (22.8 vs. 9.5%, P=0.00008) and an increase in the C-A haplotype (4.9 vs. 1.1%, P=0.02) in patients compared with controls, and a decrease in the T-G haplotype (53.4 vs. 65.1%, P=0.01). No statistical difference was found for the other haplotypes. These findings show that polymorphisms of the HIF1A gene may confer susceptibility to RCC.
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PMID:Polymorphisms in the hypoxia inducible factor-1alpha gene (HIF1A) are associated with the renal cell carcinoma phenotype. 1535 Mar 1

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.
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PMID:Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected]. 1653 14


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