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)

Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria.
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PMID:The N-terminal region of mature mitochondrial aspartate aminotransferase can direct cytosolic dihydrofolate reductase into mitochondria in vitro. 802 46

Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state. Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter. We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters. In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings. In some cases, two gold densities were observed in the same groEL complex. We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings. In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein.
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PMID:A polypeptide bound by the chaperonin groEL is localized within a central cavity. 809 82

The protooncogene product Bcl-2 is an integral membrane protein that functions as a suppressor of programmed cell death. It contains a single predicted transmembrane segment located at its COOH terminus. Here, we show that the transmembrane domain of human Bcl-2 functions as a mitochondrial signal anchor sequence that targets and inserts the protein into the outer membrane in an Ncyto-C(in) orientation, leaving the bulk of the polypeptide facing the cytosol. Deletion of the COOH-terminal 22 amino acids of Bcl-2 abrogated protein targeting, whereas fusion of this domain to the COOH terminus of dihydrofolate reductase resulted in targeting and insertion of the hybrid protein into the outer membrane in a manner similar to that of Bcl-2. The sequence of the hydrophobic core of the Bcl-2 signal anchor is similar to the corresponding region of the NH2-terminal signal anchor of the mitochondrial outer membrane protein in yeast, Mas70p. A synthetic peptide comprising the Mas70p signal anchor sequence effectively competed for insertion of Bcl-2 into the outer membrane but had no effect on the comparatively low association that Bcl-2 makes with endoplasmic reticulum microsomes. Insertion of Bcl-2 into the mitochondrial outer membrane is mechanistically different than its association with microsomes.
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PMID:Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence. 824 56

X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive. This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant. We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant. We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B.
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PMID:Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase. 826 22

The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly overexpressed in Escherichia coli, with most of the recombinant protein occurring in a soluble and an active form.
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PMID:Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923. 836 65

A cDNA clone encoding a cognate 70-kDa heat shock protein from the spinach chloroplast outer envelope (SCE70) was recently characterized (Ko, K., Bornemisza, O., Kourtz, L., Ko, Z. W., Plaxton, W. C., and Cashmore, A. R. (1992) J. Biol. Chem. 267, 2986-2993). Initial studies revealed that SCE70 is targeted to the chloroplast outer envelope membrane without further processing. To determine whether SCE70 possesses a "targeting domain," we tested the targeting ability of SCE70 proteins with various carboxyl- and amino-terminal deletions. Carboxyl-terminal deletions of up to 60% of the protein had no apparent effect on the targeting ability of SCE70. Amino-terminal deletions abolished targeting to the chloroplast except when the extreme NH2-terminal 48-amino acid sequence was retained. We further assessed the chloroplast-targeting ability of the NH2-terminal 48 amino acids by fusing to the foreign protein, mouse dihydrofolate reductase (DHFR). The resulting fusion protein, SCE70-DHFR, was localized to the outer envelope membrane of isolated chloroplasts. SCE70-DHFR exhibited targeting characteristics similar to native SCE70. The targeting of SCE70-DHFR was inhibited effectively by anti-SCE70 antibodies. Immunoprecipitation and chemical cross-linking experiments revealed that SCE70-DHFR is targeted to the same complex as SCE70 in the chloroplast envelope. These results suggest that the extreme NH2 terminus of SCE70 is required for directing SCE70 to a destination in the chloroplast outer envelope membrane, possibly through assembling the polypeptide into a protein complex.
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PMID:Identification of an uncleavable targeting signal in the 70-kilodalton spinach chloroplast outer envelope membrane protein. 836 85

Yeast tRNA ligase possesses multiple activities which are required for the joining of tRNA halves during the tRNA splicing process: cyclic phosphodiesterase, kinase, adenylylate synthetase, and ligase. A deletion polypeptide of a dihydrofolate reductase-ligase fusion protein, designated DAC, was previously shown to join tRNA halves although ATP-dependent kinase activity was not measurable in the assay used. We describe here a characterization of the mechanism of joining used by DAC and the structure of the tRNA product. DAC produces a joined tRNA and a splice junction with a structure identical to that produced by DAKC, the full-length dihydrofolate reductase-ligase fusion. Furthermore, DAC can use GTP as the sole cofactor in the joining reaction, in contrast to DAKC, which can only complete splicing in the presence of ATP. Both enzymes exhibit GTP-dependent kinase activity at 100-fold greater efficiency than with ATP. These results suggest that a potential function for the center domain of tRNA ligase (missing in DAC) is to provide structural integrity and aid in substrate interactions and specificity. They also support the hypothesis that ligase may prefer to use two different cofactors during tRNA splicing.
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PMID:Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing. 842 18

Ricin A chain is a polypeptide of 267 amino acids containing a hydrophobic region near its carboxyl-terminus (residues 245-256) which has been implicated in the membrane translocation step necessary for this catalytically active toxin to reach its intracellular substrate. DNA fusions were constructed that encoded hybrid proteins consisting of carboxyl-terminal residues 233-267 or residues 238-267 of ricin A chain preceding mouse dihydrofolate reductase. When in vitro transcripts prepared from these constructs were translated in cell-free systems, the ricin A chain-derived sequences functioned as efficient signal peptides which directed dihydrofolate reductase into microsomes or into proteoliposomes containing microsomal membrane components.
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PMID:A hydrophobic region of ricin A chain which may have a role in membrane translocation can function as an efficient noncleaved signal peptide. 852 41

The chaperonin GroEL is able to mediate protein folding in its central cavity. GroEL-bound dihydrofolate reductase assumes its native conformation when the GroES cofactor caps one end of the GroEL cylinder, thereby discharging the unfolded polypeptide into an enclosed cage. Folded dihydrofolate reductase emerges upon ATP-dependent GroES release. Other proteins, such as rhodanese, may leave GroEL after having attained a conformation that is committed to fold. Incompletely folded polypeptide rebinds to GroEL, resulting in structural rearrangement for another folding trial in the chaperonin cavity.
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PMID:Protein folding in the central cavity of the GroEL-GroES chaperonin complex. 855 46

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.
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PMID:Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR). 857 79


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