Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis.

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Structures of escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity.

BACKGROUND: . Nucleoside monophosphate kinases (NMP kinases) catalyze the reversible transfer of a phosphoryl group from a nucleoside triphosphate to a nucleoside monophosphate. Among them, cytidine monophosphate kinase from Escherichia coli has a striking particularity: it is specific for CMP, whereas in eukaryotes a unique UMP/CMP kinase phosphorylates both CMP and UMP with similar efficiency. RESULTS: . The crystal structure of the CMP kinase apoenzyme from E. coli was solved by single isomorphous replacement and refined at 1.75 A resolution. The structure of the enzyme in complex with CDP was determined at 2.0 A resolution. Like other NMP kinases, the protein contains a central parallel beta sheet, the strands of which are connected by alpha helices. The enzyme differs from other NMP kinases in the presence of a 40-residue insert situated in the NMP-binding (NMPbind) domain. This insert contains two domains: one comprising a three-stranded antiparallel beta sheet, the other comprising two alpha helices. CONCLUSIONS: . Two features of the CMP kinase from E. coli have no equivalent in other NMP kinases of known structure. Firstly, the large NMPbind insert undergoes a CDP-induced rearrangement: its beta-sheet domain moves away from the substrate, whereas its helical domain comes closer to it in a motion likely to improve the protection of the active site. Secondly, residues involved in CDP recognition are conserved in CMP kinases and have no counterpart in other NMP kinases. The structures presented here are the first of a new family of NMP kinases specific for CMP.

In vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells.

It has been proposed that proteases secreted by cancer cells facilitate metastasis by degrading extracellular matrix. Estrogen receptor-positive breast cancer cells secrete a Mr 52,000 pro-cath-D under estrogen stimulation, whereas this protease is produced constitutively by estrogen receptor-negative cancer cells. We report on the degradation in vitro of extracellular matrix by purified Mr 52,000 cathepsin D (cath-D) and by conditioned media prepared from different cell lines. The purified Mr 52,000 pro-cath-D was autoactivated at pH 4.5 into a Mr 51,000 cath-D and found to digest the extracellular matrix of endothelial bovine corneal cells labeled with [3H]proline or [35S]methionine. Culture medium conditioned by estrogen-treated MCF7 cells had a similar effect at pH 4.5 but not at pH 7.4. Matrix degradation was totally inhibited by pepstatin. Other breast cancer cells (BT20, MDA-MB231, T47D cells, etc.) and other cancer cells also secreted a pepstatin-sensitive proteinase able to degrade extracellular matrix. By contrast, the U2 variant of MCF7 cells, which lacks the Mr 52,000 cath-D gene, and the nontumoral epithelial mammary cells secreted a negligible amount of this proteinase. In all conditioned media, the pepstatin-dependent extracellular matrix degrading activity was highly correlated to the Mr 52,000 cath-D concentration measured by immunoenzymatic assay. We conclude that the Mr 52,000 cath-D is the major acidic protease secreted by mammary cancer cells. We suggest that this protease may degrade basement membrane and consequently facilitate tumor invasion when it is released in an acidic microenvironment.

Structure, function, regulation and clinical significance of the 52K pro-cathepsin D secreted by breast cancer cells.

In estrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75-1), estrogens specifically increase the secretion into the culture medium of a 52,000 Da (52K) glycoprotein and stimulate cell proliferation. The 52K protein has been purified to homogeneity using monoclonal antibodies and identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals. The secreted precursor 52K protein is mitogenic in vitro in estrogen-deprived MCF7 cells, can be taken up by these cells via mannose-6-phosphate receptors, and can degrade extracellular matrix and proteoglycans following its auto-activation. The protease is also produced constitutively by ER-negative cell lines, and is inducible by tamoxifen in some antiestrogen-resistant variants. The corresponding cDNA has been cloned using N-terminal sequencing of the protein and monoclonal antibodies. Its complete sequencing indicates a strong homology with pro-cathepsin D of normal tissues. Using a cDNA probe, the regulation of 52K cathepsin D mRNA by estrogens and antiestrogens has been studied and chromosome localization determined by in situ hybridization. Clinical studies using both immunohistochemistry and immunoenzymatic assay of breast cancer cytosol have shown that the concentration of total cellular cathepsin D (52K + 48K + 34K) is related to the proliferation of mammary ducts and to the prognosis of breast cancer. Its cytosolic concentration in primary tumors of postmenopausal patients is correlated slightly with lymph node invasion and significantly with shorter disease-free intervals in a 6-year retrospective study with the Danish Breast Cancer Groups and Finsen Institute (S. Thorpe et al.).(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral administration of picrotoxin and bicuculline stimulates in vivo somatostatin release from rat median eminence.

The gamma-Aminobutyric acid-A (GABAA) antagonist picrotoxin and bicuculline were administered to male rats to determine their effects on somatostatin (SRIF) release, measured in unanesthetized animals stereotaxically implanted with push-pull cannula in the median eminence (ME). I.p. injection (3 mg/kg) of picrotoxin (n = 5) or bicuculline (n = 6) significantly increased (35.4 +/- 10.8 vs 13.7 +/- 4.3 pg/15 min; P less than 0.03 and 38 +/- 3.5 vs 14 +/- 1.8 pg/15 min; P less than 0.001, respectively) SRIF release from the ME compared to baseline levels measured in the same animals. In contrast, with local perfusion of picrotoxin, (10(-4) to 10(-6) M) SRIF release from the ME was not affected. These data suggest a physiological endogenous inhibitory tone of GABA on SRIF release.

[Update on the mechanism of action of synthetic antiestrogens]. / Le point sur le mécanisme d'action des antioestrogènes de synthèse.

Synthetic antiestrogens are drugs commonly used in the endocrine therapy of breast cancer. Their pharmacology is complex, most of them presenting a partial estrogenic effect. Their action via the estrogen receptor (ER) has been better understood through the use of cellular models. The antihormone-ER complex binds to estrogen responsive elements on DNA, but is unable to mimic exactly the transcriptional activation induced by the estradiol-ER complex. The antiproliferative effect of these antihormones, mainly cystostatic but also cytotoxic, is the result of the competitive inhibition of estrogens, and might also involve interaction with other cellular mediators (stimulation of inhibitory factors, and growth factors inhibition). The term antiestrogen seems therefore to be too restrictive and it may be more accurate to name them estrogen-receptor mediated drugs inhibiting cell proliferation.

Adenosine 5'-diphosphate binding and the active site of nucleoside diphosphate kinase.

The X-ray structure of nucleoside diphosphate kinase (NDP kinase) from the slime mold Dictyostelium discoideum has been determined to 2.2-A resolution and refined to an R-factor of 0.19 with and without bound ADP-Mg2+. The nucleotide binds near His 122, a residue which becomes phosphorylated during the catalytic cycle. The mode of binding is different from that observed in other phosphokinases, and it involves no glycine-rich sequence. The adenine base makes only nonpolar contacts with the protein. It points outside, explaining the lack of specificity of NDP kinase toward the base. The ribose 2'- and 3'-hydroxyls and the pyrophosphate moiety are H-bonded to polar side chains. A Mg2+ ion bridges the alpha- to the beta-phosphate which approaches the imidazole group of His 122 from the N delta side. The geometry at the active site in the ADP-Mg2+ complex suggests a mechanism for catalysis whereby the gamma-phosphate of a nucleoside triphosphate can be transferred onto His 122 with a minimum of atomic motion.

MCF7 mammary cancer cells respond to bFGF and internalize it following its release from extracellular matrix: a permissive role of cathepsin D.

High and low affinity receptors for basic fibroblast growth factor (bFGF) were detected by binding experiments on MCF7 breast cancer cells. These cells were stimulated for growth by physiological concentrations of bFGF. However, in contrast to endothelial cells, these MCF7 cells did not produce detectable amounts of biologically active bFGF or related heparin-binding growth factor(s) of the FGF family. In vitro, the cathepsin D (cath-D) secreted by MCF7 cells was able to digest extracellular matrix (ECM) and to release ECM-bound 125I-bFGF. When MCF7 cells were cultured on ECM containing bound bFGF, they internalized bFGF, which was slowly and partially proteolyzed in the cells. Processing occurred in acidic compartments and was inhibited by leupeptin. Pepstatin A, an inhibitor of aspartyl proteases, had no effect on the processing but reduced internalization of matrix-bound bFGF by MCF7 cells. Taken together, these results suggest a cooperation between cath-D and bFGF, by which the protease could facilitate the release of bFGF from ECM and its subsequent use by breast cancer cells and/or adjacent cells involved in angiogenesis.

Crystallization and preliminary X-ray diffraction studies of C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C-1027 from Streptomyces globisporus.

C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C- 1027, isolated from Streptomyces globisporus, was crystallized by the vapor-diffusion procedure using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell dimensions a = 55.1, b = 61.3 and c = 79.1 A. Assuming that the asymmetric unit contains two or three molecules, the V(m) value is calculated as 3.2 or 2.1 A(3) Da(-1), respectively. A total of 7630 independent reflections was obtained up to 2.5 A resolution with synchrotron radiation, the merging R factor being 0.077 for 24 713 measurements.

Substrate-induced fit of the ATP binding site of cytidine monophosphate kinase from Escherichia coli: time-resolved fluorescence of 3'-anthraniloyl-2'-deoxy-ADP and molecular modeling.

The conformation and dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichia coli (CMPK(coli)), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the enzyme. The spectroscopic properties of the bound fluorescent nucleotide change strongly with respect to those in aqueous solution. These changes (red shift of the absorption and excitation spectra, large increase of the excited state lifetime) are compared to those observed in different solvents. These data, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected from the aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP. The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completely blocked by CMP binding. The energy-minimized models of CMPK(coli) complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes and, respectively) were compared to the crystal structure of UMP/CMP kinase from Dictyostelium discoideum complexed with substrates (PDB code ). The key residues for ATP/ADP binding to CMPK(coli) were identified as R157 and I209, their side chains sandwiching the adenine ring. Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins. In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LID conformation remained unchanged, by contrast to the fluorescence data. These results provide the first experimental arguments about an ATP-mediated induced-fit of the LID in CMPK(coli) modulated by CMP, leading to a closed conformation of the active site, protected from water.

Regulation, clinical and biological significance of cathepsin D in breast cancer.

The lysosomal protease, pro-cathepsin D, is overexpressed and secreted by human breast cancers. In estrogen-responsive breast cancer cell lines, estrogens and growth factors stimulate cathepsin D expression through distinct mechanisms. Clinical studies indicate that high cathepsin D concentration in primary breast cancers is correlated with an increased risk of metastasis and particularly useful to orientate node-negative tumors towards an adjuvant therapy.

CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.