Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.     

Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.     

Lagging-strand, early-labelling, and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin

5-Acetoxyacetylimino-4-methyl-delta2-1,3,4,-thiadiazoline -2-sulfonamide (compound (1)) is an ester prodrug that lowered intraocular pressure (IOP) in albino New Zealand rabbits, but was found to be inactive in pigmented Dutch Belt rabbits. In order to explain the differences in pharmacological activity for the two rabbit species, metabolism and melanin binding were studied. Depending on the initial concentration, the binding of compound (1) to natural melanin (Sepia officinalis) was 20-60%. The binding constant, K, at 37 degrees C was 4.32 x 10(5) M(-1) and the maximum moles bound to melanin, r(max), was 4.5 x 10(-7) mol/mg of melanin. From a determination of binding at temperatures between 25 degrees C and 47 degrees C, a van't Hoff plot was constructed to determine enthalpy and entropy changes accompanying the binding process, deltaH and deltaS, respectively. Values calculated from the plot were -12.7 and -15.4 kcal/(mol deg), respectively. Negative values for these parameters are consistent with charge transfer interactions and therefore suggest that this may be an operative mechanism between compound (1) and melanin. The in vitro incubation of compound (1) was also studied with various ocular tissues from both albino and pigmented rabbits which were iris-ciliary body, intact cornea, stroma/endothelium and aqueous humor. A major metabolite, MET 1, was identified and also observed from in vivo analyses of the same tissues following topical application. The metabolite was isolated and subjected to mass spectroscopy and proton nuclear magnetic resonance spectroscopy analysis. From these analyses, it was hypothesized that the formation of MET 1 involved a GSH conjugation mechanism which displaced the sufonamide (-SO2NH2) group. The metabolism was found to be less extensive in the pigmented rabbit than in the albino rabbit and suggested that the binding affinity of compound (1) for melanin was a better explanation for the lack of IOP activity in the pigmented rabbit than differences in metabolism.     

Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

A three-dimensional structure model of the dihydrofolate reductase (DHFR) domain of the bifunctional DHFR-thymidylate synthase of Plasmodium falciparum was used as a basis for computational screening of commercially available compounds for candidate inhibitors. Compounds which can stably dock to the model with strong ionic hydrogen bonds via protonation by an aspartic acid residue at the bottom of the active site were identified through docking simulation. Among compounds thus identified, 21 were assayed for inhibitory activity towards the recombinant DHFR domain. Two compounds, 2-amino-1,4-dihydro-4,4,7,8-tetramethyl-s-triazino(1,2-a)benzimida zole and Trp-P-2, inhibited the recombinant P. falciparum DHFR domain with Ki values of 0.54 and 8.7 microM, respectively. Kinetic analysis showed that these compounds competitively inhibited the enzyme with respect to the substrate dihydrofolate. These findings support the validity of both the modeled structure and the docking results. Furthermore, these compounds serve as leads for developing new DHFR inhibitors, since their skeletal structures are different from any of known DHFR inhibitors. This paper also reveals a new biological activity of Trp-P-2, a potent mutagen.     

Cortactin associates with the cell-cell junction protein ZO-1 in both Drosophila and mouse

Cortactin is an actin filament-binding protein localizing at cortical regions of cells and a prominent substrate for Src family protein-tyrosine kinases in response to multiple extracellular stimuli. Human cortactin has been identified as a protein product of a putative oncogene, EMS1. In this report, we describe the identification of a Drosophila homolog of cortactin as a molecule that interacts with Drosophila ZO-1 using yeast two-hybrid screening. Drosophila cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. Deletion and substitution mutant analyses revealed that the SH3 domain of Drosophila cortactin binds to a PXXP motif in the proline-rich domain of Drosophila ZO-1. Colocalization of these proteins at cell-cell junction sites was evident under a confocal laser-scanning microscope. In vivo association was confirmed by coimmunoprecipitation of cortactin and ZO-1 from Drosophila embryo lysates. We also demonstrate an association for each of the murine homologs by immunoprecipitation analyses of mouse tissue lysates. Our previous work has demonstrated the involvement of ZO-1 in a signaling pathway that regulates expression of the emc gene in Drosophila. The potential roles of the cortactin.ZO-1 complex in cell adhesion and cell signaling are discussed.     

Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.     

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.     

Cathepsin D associates with lysosomal membranous protein

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.     

Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL

Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state. In contrast, murine DHFR shows a strong interaction with GroEL. Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E. coli protein. Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL. On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E. coli DHFR. In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E. coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q. In the second mutant (EcDHFR-i(7)136), residues 136-139 (V-F-S-E) of E. coli DHFR were replaced with the seven amino acid sequence L-P-E-Y-P-G-V. Both E. coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR. The binding was specific since the presence of MgATP caused the release of the proteins from GroEL. As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL. Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins. A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples. The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer. Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM.     

Novel T cell antigen 4-1BB associates with the protein tyrosine kinase p56lck1

4-1BB is a 30-kDa inducible T cell Ag, and is expressed predominantly as a 55-kDa dimer on both CD4+ and CD8+ T lymphocytes. The cytoplasmic tail of 4-1BB contains the sequence Cys-Arg-Cys-Pro, which is similar to the sequence Cys-X-Cys-Pro, which mediates the binding of the CD4 and CD8 molecules to the protein tyrosine kinase p56lck. An anti-4-1BB mAb, 53A2, was used to determine whether 4-1BB may associate with p56lck. The 53A2 mAb specifically recognized 4-1BB on a CD8+ T cell line, CTLL-2, and coimmunoprecipitated a 56-kDa protein along with 4-1BB. Peptide mapping indicated that the 56-kDa phosphoprotein was identical to p56lck. The coimmunoprecipitation of p56lck with 4-1BB also occurred in nonlymphoid cells such as insect (Sf-21) and HeLa cells when the two recombinant proteins were coexpressed. Analysis of mutant p56lck recombinant proteins showed that two cysteine residues critical for p56lck-CD4 (or -CD8) complex formation are also required for the p56lck-4-1BB interaction. These studies establish that 4-1BB physically associates with p56lck.     

Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells

Cervid species represent a growing livestock enterprise in the United States of America (USA). The zoonotic threat of bovine tuberculosis (Mycobacterium bovis) is the only significant public health risk posed by this alternative livestock industry. This paper examines the potential sources of tuberculosis exposure as related to public health and compares and contrasts the status of tuberculosis in Cervidae with the situation in the cattle industry in the USA. Based on the existing prevalence of the disease and the limited potential of human exposure to infected meat or meat products, bovine tuberculosis in Cervidae poses a minimal threat to public health. The only significant public health concern is exposure to infected free-ranging cervids of hunters who field-dress carcasses and may unknowingly incise tuberculous lesions. This risk is mitigated only by the small size of the cervid population at risk when compared to the general population of cervids hunted yearly.     

Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate.     

An amplified mosquito dihydrofolate reductase gene: amplicon size and chromosomal distribution

Correct interpretation of conspicuous blood flow velocity waveforms cannot rely solely on the evaluation of uteroplacental vascular Doppler flow patterns by means of angle-independent indices such as the resistance or pulsatility index. In addition to the degree of pulsatility, the waveform shape between the systolic and diastolic peak values is of considerable consequence. A subdivision of the total flow waveform into orthogonal polynomial components allows both pulsatility evaluation and notching to be registered, providing a higher sensitivity in identification of pathological vascular resistance. Accurate recording and assessment of the flow waveform is therefore an important qualitative criterion for the classification of Doppler flow patterns in pregnancies with reduced uteroplacental perfusion.