Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.
 ...
PMID:Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability. 935 52

A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein-protein interactions in vivo.
 ...
PMID:Development of a yeast protein fragment complementation assay (PCA) system using dihydrofolate reductase (DHFR) with specific additives. 1867 Jul 70

Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system.
 ...
PMID:Towards the application of Tc toxins as a universal protein translocation system. 3174 51