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)

There is a need for a specific, sensitive, robust, and large-scale method for diagnosis of drug resistance genes in natural Plasmodium falciparum infections. Established polymerase chain reaction (PCR)-based methods may be compromised by the multiplicity of P. falciparum genotypes in natural infections. Here we adopt a dot-blot method to detect point mutations at nucleotide 323 (residue 108) in the P. falciparum dihydrofolate reductase (dhfr) gene using allele-specific oligonucleotide probes. Serine (Ser) or threonine (Thr) at this position are associated with sensitivity to pyrimethamine while asparagine (Asn) is associated with resistance. The method combines PCR amplification and hybridization of amplified products with radiolabeled allele-specific probes. This technique is specific and sensitive; it detects parasitemia of less than 100 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture. Analysis of P. falciparum isolates from Sudan, of known response to pyrimethamine, has demonstrated the sensitivity and specificity of the method and its ability to detect multiple genotypes in single infections. Furthermore, it has confirmed the association between pyrimethamine responses and dhfr alleles. The method has been successfully extended for analysis of other point mutations in dhfr at residues 51 and 59, which are associated with a high level of pyrimethamine resistance.
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PMID:Detection of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) gene by dot-blot hybridization. 1236 59

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an approximately 16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.
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PMID:Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition. 1238 95

The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) can be phosphorylated at serine 2 (Ser-2) and serine 5 (Ser-5) of the CTD heptad repeat YSPTSPS, and this phosphorylation is important in coupling transcription to RNA processing, including 5' capping, splicing, and polyadenylation. The mammalian endogenous dihydrofolate reductase and gamma-actin genes have been used to study the association of Pol II with different regions of transcribed genes (promoter-proximal compared to distal regions) and the phosphorylation status of its CTD. For both genes, Pol II is more concentrated in the promoter-proximal regions than in the interior regions. Moreover, different phosphorylation forms of Pol II are associated with distinct regions. Ser-5 phosphorylation of Pol II is concentrated near the promoter, while Ser-2 phosphorylation is observed throughout the gene. These results suggest that the accumulation of paused Pol II in promoter-proximal regions may be a common feature of gene regulation in mammalian cells.
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PMID:RNA polymerase II accumulation in the promoter-proximal region of the dihydrofolate reductase and gamma-actin genes. 1261 70

The resistance to pyrimethamine (PYR) of Plasmodium falciparum arising from mutation at position 108 of dihydrofolate reductase (pfDHFR) from serine to asparagine (S108N) is due to steric interaction between the bulky side chain of N108 and Cl atom of the 5-p-Cl aryl group of PYR, which consequently resulted in the reduction in binding affinity between the enzyme and inhibitor. Molecular modeling suggested that the flexible antifolate, such as trimethoprim (TMP) derivatives, could avoid this steric constraint and should be considered as new, potentially effective compounds. The hydrophobic interaction between the side chain of inhibitor and the active site of the enzyme around position 108 was enhanced by the introduction of a longer and more hydrophobic side chain on TMP's 5-benzyl moiety. The prepared compounds, especially those bearing aromatic substituents, exhibited better binding affinities to both wild type and mutant enzymes than the parent compound. Binding affinities of these compounds correlated well with their antimalarial activities against both wild type and resistant parasites. Molecular modeling of the binding of such compounds with pfDHFR also supported the experimental data and clearly showed that aromatic substituents play an important role in enhancing binding affinity. In addition, some compounds with 6-alkyl substituents showed relatively less decrease in binding constants with the mutant enzymes and relatively good antimalarial activities against the parasites bearing the mutant enzymes.
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PMID:Target guided synthesis of 5-benzyl-2,4-diamonopyrimidines: their antimalarial activities and binding affinities to wild type and mutant dihydrofolate reductases from Plasmodium falciparum. 1471 7

Glycosylation of proteins can have a dramatic effect on their physical, chemical, and biological properties. Analogues of dihydrofolate reductase and firefly luciferase containing glycosylated amino acids at single, predetermined sites have been elaborated. Misacylated suppressor tRNAs activated with glycosylated serine and tyrosine derivatives were used for suppression of the nonsense codons in a cell-free protein biosynthesizing system, thereby permitting the preparation of the desired glycosylated proteins. In this fashion, it was possible to obtain proteins containing both mono- and diglycosylated amino acids, including glycosylated serine and tyrosine moieties. For the modified firefly luciferases, the effect of these substitutions on the wavelength of the light emitted by firefly luciferase was investigated. The maximum wavelength for mutants containing peracetylated glycosylated serine derivatives at position 284 showed a red shift in the emission spectra. For mutants containing glycosylated tyrosines, the red shift was observed only when the carbohydrate moiety was fully deacetylated.
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PMID:Site-specific incorporation of glycosylated serine and tyrosine derivatives into proteins. 1733 22

An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the alpha prime (alpha') subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of endothelial NO synthase, increased endothelial cell superoxide production, and prevented the increase in NO production caused by shear. Thus, shear stress not only increases endothelial NO synthase levels but also stimulates production of H4B by markedly enhancing GTPCH-1 activity via casein kinase 2-dependent phosphorylation on serine 81. These findings illustrate a new function of casein kinase 2 in the endothelium and provide insight into regulation of GTPCH-1 activity.
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PMID:Regulation of tetrahydrobiopterin biosynthesis by shear stress. 1793 32

Chemotherapy for non-small cell lung cancer (NSCLC) can prolong survival and improve quality of life, but the majority of advanced stage patients succumb to disease within 2 years, meaning that there is room for improvement. The standard chemotherapy for NSCLC involves one of a number of chemotherapy doublets that have been shown to improve survival when compared with single agents or best supportive care. These doublets are generally comparable in terms of efficacy, differing primarily in their toxicity profiles. However, encouraging new options may be approaching, including therapies targeted to specific patient subpopulations, and the use of combinations of current and new drugs to produce synergistic effects. Targeted therapies include the anti-epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib, EGFR monoclonal antibody cetuximab, and vascular endothelial growth factor (VEGF) inhibitors such as sorafenib, a small molecule TKI, and bevacizumab, a recombinant monoclonal VEGF antibody. Most attempts to combine EGFR-targeted therapies with standard chemotherapy in NSCLC have produced poor results, possibly as a result of antagonism between EGFR TKIs and chemotherapy. Positive results with bevacizumab suggest that VEGF-rather than EGFR-targeted therapies may produce better results when combined with chemotherapy. Other new drugs being tested include enzastaurin, an oral serine threonine kinase inhibitor; vinflunine, a vinca alkaloid; dihydrofolate reductase inhibitors; and thymidylate synthase inhibitors. Combinations of therapies, especially those acting via different mechanisms, hold promise for improvements in survival, but careful testing is required to determine optimum combinations of available drugs and where new drugs fit into the armamentarium.
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PMID:Systemic treatment for advanced (stage IIIb/IV) non-small cell lung cancer: more treatment options; more things to consider. Conclusion. 1826 73

Trimethoprim sulfamethoxazole (cotrimoxazole, CTX) is used frequently as part of standard medical care for people living with HIV/AIDS in Africa. The mechanisms of resistance to sulfonamides and trimethoprim in commensal streptococci from Uganda were determined and compared to S. pneumoniae. Commensal streptococci showing high-level resistance to cotrimoxazole were cultured and analysed for species identity and polymorphisms in the genes coding for dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). Seven isolates of S. pneumoniae from blood and cerebrospinal fluid (CSF) were similarly examined. There was considerable polymorphism in both DHPS and DHFR. In DHFR, the mutations E20D and I100L were present in all sequenced isolates. Other mutations such as L135F, and different substitutions in D92, were frequent. The most common DHPS variants had 2 serine residues added after amino acid 60, or arginine and proline added after amino acid 59. In addition, 3 new insertions/substitutions were found. There were no obvious differences between the mutation patterns in S. pneumoniae and commensal streptococci, suggesting that the chromosomal mutations have been spread by transformational interchanges of DNA among related organisms.
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PMID:Cotrimoxazole resistance of Streptococcus pneumoniae and commensal streptococci from Kampala, Uganda. 1914 88

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.
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PMID:Tetrahydrobiopterin recycling, a key determinant of endothelial nitric-oxide synthase-dependent signaling pathways in cultured vascular endothelial cells. 1928 67

The three enzymes that constitute the de novo thymidylate synthesis pathway in mammals, cytoplasmic serine hydroxymethyltransferase (SHMT1), thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR) undergo sumoylation and nuclear import during S-phase. In this study, we demonstrate that purified intact mouse liver nuclei convert dUMP to dTMP in the presence of NADPH and serine. Neither nuclear extracts nor intact nuclei exposed to aminomethylphosphonate, a SHMT inhibitor, exhibit thymidylate synthesis activity. Nuclei isolated from Shmt1(-/-) mouse livers retained 25% of thymidylate synthesis activity exhibited by nuclei isolated from wild type mice. This residual activity was due to the presence of a cytoplasmic/nuclear isozyme of SHMT encoded by Shmt2. Shmt2 is shown to encode two transcripts, one which encodes a protein that localizes exclusively to the mitochondria (SHMT2), and a second transcript that lacks exon 1 and encodes a protein that localizes to the cytoplasm and nucleus during S-phase (SHMT2alpha). The ability of Shmt2 to encode a cytoplasmic isozyme of SHMT may account for the viability of Shmt1(-/-) mice and provide redundancy that permitted the expansion of the human SHMT1 L474F polymorphism that impairs SHMT1 sumoylation and nuclear translocation.
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PMID:SHMT1 and SHMT2 are functionally redundant in nuclear de novo thymidylate biosynthesis. 1951 16


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