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:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated a protein that exhibits a potent differentiation-inducing activity toward mouse Friend erythroleukemia (MEL) cells and human K-562 cells. The protein, designated erythroid differentiation factor (EDF), was found in the culture fluid of human THP-1 cells that had been treated with phorbol 12-myristate 13-acetate. EDF is a homodimer with a Mr of 25,000; the Mr of the monomer is 15,500. cDNA clones encoding the Mr 15,500 subunit of EDF from THP-1 libraries were isolated and sequenced. Surprisingly, the sequence of EDF mRNA is identical to that for the beta A subunit of inhibin, a gonadal protein that suppresses the secretion of pituitary follicle-stimulating hormone. Southern blot analysis indicates that only one gene for EDF/inhibin beta A exists in the human genome. When the EDF subunit cDNA was linked to a simian virus 40 expression vector containing the dihydrofolate reductase gene and transfected into Chinese hamster ovary dihydrofolate reductase negative cells, the transformants began to secrete EDF, demonstrating that the cDNA actually encoded the EDF subunit.
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PMID:Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin beta A chain. 326 9

We expressed human macrophage colony-stimulating factor (M-CSF) in Chinese hamster ovary (CHO) cells by introducing an expression plasmid coding for a 554-amino-acid M-CSF precursor and dihydrofolate reductase (DHFR) gene, and by amplifying the sequence. A cell line was obtained that secreted approximately 200,000 units/ml after 6 days in culture. The expressed recombinant human M-CSF (rhM-CSF) primarily consisted of two molecular species, a main 80-90 kD M-CSF as a homodimer and a molecular form higher than 150 kD. Purification of a main rhM-CSF gave an apparently homogeneous protein disulfide-bonded from 42-kD subunits, but one of the purified rhM-CSFs was composed of two subunit species with molecular masses of 44 and 42 kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4 x 10(7) units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trifluoromethanesulfonic acid) and enzymatic methods found a terminal neuraminic acid in addition to N- and O-glycosylation, but the two subunit species did not coalesce into a single molecule.
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PMID:Purification and characterization of recombinant human macrophage colony-stimulating factor expressed in Chinese hamster ovary cells. 776 77

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-alpha-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-alpha-NH-protein extensions, including ubiquitin-alpha-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-alpha-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through epsilon-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.
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PMID:New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle. 923 Jan 10

In Plasmodium falciparum, dihydrofolate reductase and thymidylate synthase activities are conferred by a single 70-kDa bifunctional polypeptide (DHFR-TS, dihydrofolate reductase-thymidylate synthase) which assembles into a functional 140-kDa homodimer. In mammals, the two enzymes are smaller distinct molecules encoded on different genes. A 27-kDa amino domain of malarial DHFR-TS is sufficient to provide DHFR activity, but the structural requirements for TS function have not been established. Although the 3'-end of DHFR-TS has high homology to TS sequences from other species, expression of this protein fragment failed to yield active TS enzyme, and it failed to complement TS(-) Escherichia coli. Unexpectedly, even partial 5'-deletion of full-length DHFR-TS gene abolished TS function on the 3'-end. Thus, it was hypothesized that the amino end of the bifunctional parasite protein plays an important role in TS function. When the 27-kDa amino domain (DHFR) was provided in trans, a previously inactive 40-kDa carboxyl-domain from malarial DHFR-TS regained its TS function. Physical characterization of the "split enzymes" revealed that the 27- and the 40-kDa fragments of DHFR-TS had reassembled into a 140-kDa hybrid complex. Thus, in malarial DHFR-TS, there are physical interactions between the DHFR domain and the TS domain, and these interactions are necessary to obtain a catalytically active TS. Interference with these essential protein-protein interactions could lead to new selective strategies to treat malaria resistant to traditional DHFR-TS inhibitors.
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PMID:Essential protein-protein interactions between Plasmodium falciparum thymidylate synthase and dihydrofolate reductase domains. 1060 39

Two high-resolution structures have been obtained for dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima in its unliganded state, and in its ternary complex with the cofactor NADPH and the inhibitor, methotrexate. While the overall fold of the hyperthermophilic enzyme is closely similar to monomeric mesophilic dihydrofolate reductase molecules, its quaternary structure is exceptional, in that T. maritima dihydrofolate reductase forms a highly stable homodimer. Here, the molecular reasons for the high intrinsic stability of the enzyme are elaborated and put in context with the available data on the physical parameters governing the folding reaction. The molecule is extremely rigid, even with respect to structural changes during substrate binding and turnover. Subunit cooperativity can be excluded from structural and biochemical data. Major contributions to the high intrinsic stability of the enzyme result from the formation of the dimer. Within the monomer, only subtle stabilizing interactions are detectable, without clear evidence for any of the typical increments of thermal stabilization commonly reported for hyperthermophilic proteins. The docking of the subunits is optimized with respect to high packing density in the dimer interface, additional salt-bridges and beta-sheets. The enzyme does not show significant structural changes upon binding its coenzyme, NADPH, and the inhibitor, methotrexate. The active-site loop, which is known to play an important role in catalysis in mesophilic dihydrofolate reductase molecules, is rearranged, participating in the association of the subunits; it no longer participates in catalysis.
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PMID:The crystal structure of dihydrofolate reductase from Thermotoga maritima: molecular features of thermostability. 1073 19

By means of differential cDNA expression cloning, we earlier isolated a novel rat cDNA and its protein, named hepassocin, which is upregulated during liver regeneration. Using the rat cDNA as a probe, we have now isolated human hepassocin cDNA encoding a protein of 312 amino acids, which has 81.4% and 83.8% identity, respectively, to rat hepassocin before and after elimination of its signal peptide. Dot blot analysis revealed that hepassocin mRNA was strongly expressed in adult liver, fairly strongly in fetal liver, and weakly in pancreas, but not in other tissues. Recombinant human hepassocin produced in Chinese hamster ovary (CHO) cells by the dihydrofolate reductase-methotrexate (DHFR--MTX) gene amplification method is a homodimer (68 kDa) and has mitogenic activities in hepatocytes of various animal species including rat, mouse, rabbit and dog, and the activity was lost with 2-mercaptoethanol treatment. These results suggest that hepassocin is a potent regulator in liver cell growth not only in rats but also in humans. Computer searches revealed that human hepassocin as well as rat hepassocin has a characteristic disulfide structure close to that of fibrinogen-gamma. We assume that this newly identified growth factor exerts functions in association with an extracellular matrix such as fibrinogen.
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PMID:Molecular cloning and functional expression analysis of a cDNA for human hepassocin, a liver-specific protein with hepatocyte mitogenic activity. 1147 Jan 58

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.
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PMID:Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii. 1673 25

The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection.
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PMID:Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate. 1956 91

Dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) is unique among structurally characterized chromosomal DHFRs in that it forms a stable homodimer. Dimerization is believed to play a key role in the high thermal stability of TmDHFR, which is reflected in a melting temperature in excess of 85 degrees C. The dimer interface of TmDHFR is composed of a hydrophobic core with charged residues around the periphery. In particular, Lys129 of each subunit forms three-membered salt bridges with Glu136 and Glu138 of the other subunit. To probe the role of these salt bridges in the dimerization and thermal stability of TmDHFR, we generated a series of variants (TmDHFR-K129E, TmDHFR-E136K, TmDHFR-E138K, and TmDHFR-E136K/E138K) in which these residues were exchanged for residues whose side chains bear the opposite charge. Our results indicate that these salt bridges are key for the high thermal stability of TmDHFR but are not a requirement for dimerization. Although the rate of dihydrofolate reduction by TmDHFR is not significantly affected by the loss of the K129-E136-E138 salt bridges, changes to the temperature dependence of the kinetic isotope effect on hydride transfer are observed. These changes are in agreement with the proposal that DHFR catalysis may be affected by changes to the conformational ensemble of the enzyme rather than only to the coupling of protein motions to the reaction coordinate.
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PMID:The temperature dependence of the kinetic isotope effects of dihydrofolate reductase from Thermotoga maritima is influenced by intersubunit interactions. 2051 24

Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among immunocompromised patients, especially patients infected with HIV, but it has also been isolated from immunocompetent persons. This article discusses the application of multilocus genotyping analysis to the study of the genetic diversity of P. jirovecii and its epidemiological and clinical parameters, and the important concepts achieved to date with these approaches. The multilocus typing studies performed until now have shown that there is an important genetic diversity of stable and ubiquitous P. jirovecii genotypes; infection with P. jirovecii is not necessarily clonal, recombination between some P. jirovecii multilocus genotypes has been suggested. P. jirovecii-specific multilocus genotypes can be associated with severity of PcP. Patients infected with P. jirovecii, regardless of the form of infection they present with, are part of a common human reservoir for future infections. The CYB, DHFR, DHPS, mtLSU rRNA, SOD and the ITS loci are suitable genetic targets to be used in further epidemiological studies focused on the identification and characterization of P. jirovecii haplotypes correlated with drug resistance and PcP outcome.
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PMID:Pneumocystis jirovecii multilocus gene sequencing: findings and implications. 2072 2


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