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)

Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.
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PMID:A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes. 821 67

The main line of defense now available against parasitic protozoa--which are responsible for major diseases of humans and domestic animals--is chemotherapy. This defense is being eroded by drug resistance and, with few new drugs in the pipeline, prevention and circumvention of resistance are medical and veterinary priorities. Although studies of resistance mechanisms in parasites have lagged behind similar studies in bacteria and cancer cells, the tools to tackle this problem are rapidly improving. Transformation with exogenous DNA is now possible with all major parasitic protozoa of humans. Hence, putative resistance genes can be tested in sensitive protozoa, allowing an unambiguous reconstruction of resistance mechanisms. Gene cloning, the polymerase chain reaction, and monoclonal antibodies against resistance-related proteins have made it possible to analyze potential resistance mechanisms in the few parasites that can be obtained from infected people. Hence, the prospect of applying new knowledge about resistance mechanisms to parasites in patients is good, even though today virtually all knowledge pertains to parasites selected for resistance in the laboratory. Resistance mechanisms highlighted in this review include: 1. Decrease of drug uptake because of the loss of a transporter required for uptake. This decrease contributes to resistance to arsenicals and diamidines in African trypanosomes. 2. The export of drugs from the parasite by P-glycoproteins and other traffic ATPases. This export could potentially be an important mechanism of resistance, as these proteins are richly represented in the few protozoa analyzed. There are indications that such transmembrane transporters can be involved in resistance to emetine in Entamoeba spp., to mefloquine in Plasmodium spp., and to antimonials in Leishmania spp. 3. The possible involvement of the P-glycoprotein encoded by the Plasmodium falciparum pfmdr1 gene in chloroquine resistance. We present the available data that lead to the conclusion that overproduction of the wild-type version of this protein results in chloroquine hypersensitivity rather than resistance. 4. The involvement of the PgpA P-glycoprotein of Leishmania spp. in low-level resistance to arsenite and antimonials. We raise the possibility that this protein transports glutathione conjugates of arsenite and antimonials rather than the compounds themselves. 5. Loss of drug activation as the main mechanism of metronidazole resistance in Trichomonas and Giardia spp. Recent evidence indicates that a decrease of the proximal cellular electron donor for metronidazole activation, ferredoxin, is the main cause of resistance in Trichomonas. 6. Resistance arising through alteration of drug targets. The amino acid substitutions in the dihydrofolate reductase-thymidylate synthase of Plasmodium spp. are good examples of this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:New mechanisms of drug resistance in parasitic protozoa. 856 67

The sequence and spacing requirements of the archaeal "distal promoter element' (DPE) were examined by randomizing positions -19 to -32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 4(14) entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high-activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: (Formula: see text) (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)-long transcript, and very resistant clones contained both the 700nt-long transcript and a second, more abundant, 500nt-long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3'-untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.
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PMID:Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection. 873 Aug 68

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.
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PMID:Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria. 1044 70