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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)

Precursor proteins are believed to have secondary and tertiary structure prior to translocation across the Escherichia coli plasma membrane. We now find that preprotein unfolding during translocation can be driven by the translocation event itself. At certain stages, translocation and unfolding can occur without exogenous energy input. To examine this unfolding reaction, we have prepared proOmpA-Dhfr, a fusion protein of the well studied cytosolic enzyme dihydrofolate reductase (Dhfr) connected to the C-terminus of proOmpA, the precursor form of outer membrane protein A. At an intermediate stage of its in vitro translocation, the N-terminal proOmpA domain has crossed the membrane while the folded Dhfr portion, stabilized by its ligands NADPH and methotrexate, has not. When the ligands are removed from this intermediate, translocation occurs by a two-step process. First, 20-30 amino acid residues of the fusion protein translocate concomitant with unfolding of the Dhfr domain. This reaction requires neither ATP, delta mu H+ nor the SecA subunit of translocase. Strikingly, this translocation accelerates the net unfolding of the Dhfr domain. In a second step, SecA and ATP hydrolysis drive the rapid completion of translocation. Thus energy derived from translocation can drive the unfolding of a substantial protein domain.
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PMID:Translocation can drive the unfolding of a preprotein domain. 842 82

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

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.
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PMID:Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites. 842 19

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

The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. Using two substrate proteins, dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46 degrees C, which is coupled with self-oligomerization of HSP90. While chemically denatured DHFR refolds spontaneously upon dilution from denaturant, oligomerized HSP90 bound DHFR during the process of refolding and prevented it from renaturation. DHFR was released from the complex with HSP90 by incubating with GroEL/ES complexes in an ATP-dependent manner and refolded into the native form. alpha-Casein inhibited the binding of DHFR to HSP90 and also chased DHFR from the complex with HSP90. These results suggest that HSP90 binds substrates to maintain them in a folding-competent structure. Furthermore, we found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates. This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages.
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PMID:Heat-induced chaperone activity of HSP90. 857 34

Ligand-induced conformational changes of GroEL alone and with bound rhodanese, citrate synthase, or dihydrofolate reductase were studied by limited proteolysis. Similar digestion patterns of GroEL, with or without bound substrate polypeptide, were obtained in the absence and presence of the chaperonin ligands, K+, Mg2+, or ATP. The rates of formation and degradation of the six produced proteolytic fragments were significantly different, however. Strikingly, only with Mg2+/ATP or K+/Mg2+/ATP an additional fragment of approximately 25 kDa was generated during digestion of GroEL alone or with bound rhodanese or dihydrofolate reductase, but not with bound citrate synthase. Most of the trypsin-sensitive sites in GroEL were localized in the flexible apical domain, which contains the putative polypeptide-binding region. Our data indicate that subtle structural changes in the trypsin-sensitive regions of GroEL occur as a result of the binding of the chaperonin ligands. However, these structural changes are influenced by the GroEL substrate polypeptides.
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PMID:Ligand-induced conformational changes of GroEL are dependent on the bound substrate polypeptide. 866 87

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 microM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 microM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuire et al., Adv. Exptl. Med. Biol. 163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (K(ii) = 0.9 microM; K(is) = 1.1 microM) and glutamic acid (K(ii) = 1.0 microM; K(is) = 5.2 microM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (K(ii) = 3.4 microM; K(is) = 0.35 microM), since the major effect is on its K(m). Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 microM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 microM; MTX, 48 microM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideout et al. (Int. J. Cancer 61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.
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PMID:Potent inhibition of human folylpolyglutamate synthetase by suramin. 891 44

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.
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PMID:Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling. 897 59

The interaction of GroEL with urea-unfolded dihydrofolate reductase (DHFR) has been studied in the presence of DHFR substrates by investigating the ability of GroES to release enzyme under conditions where a stable GroES-GroEL-DHFR ternary complex can be formed. In these circumstances, GroES could only partially discharge the DHFR if ADP was present in the solution and approximately half of the DHFR remained bound on the chaperonin. This bound DHFR could be rescued by addition of ATP and KCl into the refolding mixture. The stable ternary complex did not show any significant protection of bound DHFR against proteolysis by Proteinase K. These results are in contrast to those observed with the GroEL-DHFR complex formed by thermal inactivation of DHFR at 45 degrees C in which GroES addition leads to partial protection of bound DHFR. Thus, the method of presentation influences the properties of the bound intermediates. It is suggested that the ability of GroES to bind on the same side of the GroEL double toroid as the target protein and displace it into the central cavity depends on the way the protein-substrate is presented to the GroEL molecule. Therefore, the compact folding intermediate formed by thermal unfolding can be protected against proteolysis after GroES binds to form a ternary complex. In addition, structural changes within GroEL induced by the experimental conditions may contribute to differences in the properties of the complexes. The more open urea-unfolded DHFR binds on the surface of chaperonin and can be displaced into solution by the tighter binding GroES molecule. It is suggested that the state of the unfolded protein when it is presented to GroEL determines the detailed mechanism of its assisted refolding. It follows that individual proteins, having characteristic folding intermediates, can have different detailed mechanisms of chaperonin-assisted folding.
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PMID:Conditions of forming protein complexes with GroEL can influence the mechanism of chaperonin-assisted refolding. 899 21


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