Can estrogenic radicals, generated by lactoperoxidase, be involved in the molecular mechanism of breast carcinogenesis?

Mutations of regulatory genes, which perturb the mechanism of cell replication resulting in abnormal cell proliferation, are the main cause of cancer. Many endogenous and exogenous chemicals (including estrogenic hormones) are known to represent a major carcinogenic risk for humans. 2-OH- and 4-OH-derivatives of estrogenic molecules have been shown to form stable adducts with purine DNA bases and act as 'depurinating' agents, thus altering gene transcription (Cavalieri EL, Stack DE, Devanesan PD et al. Proc Natl Acad Sci USA 1997; 94: 10937-10942). Lactoperoxidase (LPO), which is produced by mammary glands, is likely to be involved in breast carcinogenesis, because of its ability to interact with estrogenic hormones and oxidise them through two one-electron reaction steps. We investigated the reactivity of LPO towards five molecules: 17-beta-estradiol (a natural hormone), diethylstilbestrol (a synthetic drug, supplied to pregnant women for preventing spontaneous abortion), exestrol (a synthetic antigonadotropic estrogen), 2-OH- and 4-OH-estradiol (catabolic products of estradiol). Enzymatically generated radical derivatives of such molecules were stabilized by spin-trapping or by chelation of a diamagnetic metal ion and characterized with EPR spectroscopy. A kinetic study of the oxidation process was carried out using EPR and UV-visible spectroscopy.

Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV-visible combined approach.

The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.

Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase.

The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N3-, CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (KD increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N3-, and CN- (that bind at the iron site). KD values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as KD; KM for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket.

The interaction of the cell-penetrating peptide penetratin with heparin, heparansulfates and phospholipid vesicles investigated by ESR spectroscopy.

An ESR investigation of the interaction of spin-labelled penetratin with heparin, heparansulfates and several phospholipid vesicle formulations is reported. Penetratin is a 16-aa peptide corresponding to the third helix of the Antennapedia homeodomain and belonging to the cell-penetrating peptide family. The present study shows that ESR spectroscopy can provide specific and reliable information about the mechanism of interaction of penetratin with polysaccharides and lipids, at a molecular level. The study showed that: (i) heparin and heparansulfates specifically interact with spin-labelled penetratin and promote peptide aggregation and concentration on their molecular surface; (ii) penetratin does not interact with neutral lipids, whereas it enters negatively charged lipid bilayers; (iii) cholesterol plays a negative effect on the insertion of penetratin into the lipid membrane; (iv) the interaction of penetratin with lipid vesicles is strongly dependent on lipid concentration. In a low lipid regime, penetratin associates with the polar heads of phospholipids and aggregates on the membrane surface; once the lipid concentration attains a threshold, the peptide enters the lipid bilayer. This step is characterized by reduced peptide mobility and partial disaggregation.It has been shown that ESR spectroscopy is a valuable investigation tool in studies related to the still unclear mechanism of the internalization process.

Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.

Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase.

In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed.

Migration of natural killer cells across endothelial cell monolayers.

Under certain conditions, NK cells accumulate rapidly at extrahematic sites. In an effort to define the mechanisms underlying recruitment of NK cells in tissues, we investigated their ability to migrate across endothelial cell (EC) monolayers. A considerable proportion of NK cells adhered to EC and about 30 to 40% of the adherent NK cells migrated across EC. NK cells were two to three times more efficient than resting unseparated T cells but were considerably less efficient than monocytes. Exposure of NK cells to IL-2 or of EC to IL-1, TNF, or IFN-gamma augmented transendothelial migration. mAb directed against CD18 and CD11a inhibited binding and migration of NK cells across resting or IL-1-activated EC, whereas anti-CD11b and c mAb did not. Thus, the LFA-1 pathway is a crucial determinant of the adhesive and migratory interactions of NK cells with vascular endothelium. Using IL-1-activated EC, We found that anti-VLA-4 and anti-VCAM-1 mAb, utilized in concert with anti-CD18, significantly reduced adhesion and transmigration. The CS-1 peptide of fibronectin had no effect on binding and migration but, when used in concert with anti-CD18 and anti-VCAM-1 (but not anti-VLA-4), caused a small, but significant, increase in inhibition. The capacity to bind and migrate across endothelial monolayers underlies the recruitment of NK cells in tissues under certain physiologic and pathologic conditions.

Conformational polymorphism of the amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein.

Prion-related encephalopathies are characterized by cerebral accumulation of a post-translationally modified form of the cellular prion protein (PrPC), designated PrPSc. Evidence suggests that the conversion from PrPC to PrPSc involves changes in the secondary structure leading to an increase in beta-sheet content. We have previously shown that a synthetic peptide homologous to residues 106-126 of human PrP, belonging to a predicted alpha-helical domain, exhibits a beta-sheet conformation, forms amyloid-like fibrils, and is neurotoxic in vitro. The present study investigated how different chemicophysical conditions such as pH and ionic strength or a membrane-like environment influenced the secondary structure of this peptide. PrP 106-126 exhibited a predominantly beta-sheet structure in 200 mM phosphate buffer, pH 5.0, but a combination of beta-sheet and random coil structure in 200 mM phosphate buffer, pH 7.0, or in deionized water. The addition of trifluoroethanol (50% final concentration) to solutions of peptide in deionized water induced the appearance of an alpha-helical secondary structure, but did not modify the beta-sheet conformation of the peptide dissolved in 200 mM phosphate buffer, pH 5.0. In the presence of micelles formed by a 5% solution of sodium dodecyl sulfate, PrP 106-126 showed a high content of alpha-helix. When the peptide was dissolved in 5 mM phosphate buffer, pH 7.4, and incubated with liposomes, it changed from a prevalently random coil structure to a beta-sheet conformation. The environment-dependent conformational polymorphism of PrP 106-126 and its marked tendency to form stable beta-sheet structures at acidic pH could account for the shift from alpha-helix to beta-sheet associated with the conversion of PrPC to PrPSc, which occurs most likely in the endosomal-lysosomal compartment.

Catechol(amine)s as probes of lactoperoxidase catalytic site structure: spectroscopic and modeling studies.

Binding affinities to lactoperoxidase (LPO) of a homologous series of substituted catechol(amine)s [such as catechol, 4-methylcatechol, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid; dopamine, noradrenaline, adrenaline; L-3,4-dihydroxyphenylalanine] were studied by UV-visible spectroscopy and docking simulations. Dissociation constant (Kd) values were calculated by direct fitting of the experimental data and fall in a range of 3-95 mM. Thermodynamic parameters are comparable with those reported for the interaction of LPO with p-substituted phenols, suggesting a similar general mode of binding. Furthermore, the relative contributions to binding energy, described by the unimolecular constant Ku, show that interaction between protein and ligands originates from a relatively large number of groups. Docking and molecular dynamics simulations, in agreement with experimental evidence, predict that the substrate is localized into the access channel in the vicinity of heme distal pocket. This channel is characterized by a hydrophobic patch (six Phe residues) and by a charged contribution (two Glu and one His residues). All of the substrates, except caffeic acid, may approach the protein active site. Positively charged Arg372 acts as a gate above the heme distal pocket and seems to address substrate orientation in relation to the side-chain terminal group.

Clustering of vitronectin and RGD peptides on microspheres leads to engagement of integrins on the luminal aspect of endothelial cell membrane.

In previous work (Conforti et al, Blood 80:437, 1992), we have shown that integrins in endothelial cells (EC) are not polarized to the basal cell membrane, but are also exposed on the apical cell surface, in contact with blood. Therefore, endothelial integrins might be available for binding circulating plasma proteins. However soluble plasma vitronectin (vn) bound very poorly to EC apical surface and this interaction was unaffected by Arg-Gly-Asp (RGD) peptides or an anti-alpha v beta 3 serum. In contrast, beads (diameter, 4.5 microns) coupled with plasma vn associated to EC apical surface in a time- and concentration-dependent way. Addition of antibodies directed to vn, alpha v beta 3, and RGD-containing peptides blocked the interaction of vn beads with EC. In contrast, heparin and antibodies directed to alpha v beta 5 and beta 1 integrin chain had no effect. Beads coupled with Gly-Arg-Gly-Asp-Ser-Pro bound to the EC surface, but not those coupled with Gly-Arg-Gly-Glu-Ser-Pro. This interaction was blocked by alpha v beta 3 antibodies and RGD peptides, but not by alpha v beta 5 antibody. Overall, these results indicate that luminal alpha v beta 3 retains its binding capacity for surface-linked vn and RGD-containing ligands, but binding is observed only when the ligand is offered in a clustered, multivalent form. We propose that when vn or RGD-containing proteins are bound to circulating cells, they can act as bridging molecules by promoting adhesion of the cells to the endothelium via apical integrins.

Synthetic peptides homologous to prion protein residues 106-147 form amyloid-like fibrils in vitro.

Gerstmann-Sträussler-Scheinker disease (GSS) is a prion-related encephalopathy pathologically characterized by massive deposition of prion protein (PrP) amyloid in the central nervous system. The major component of amyloid fibrils isolated from patients of the Indiana kindred of GSS (GSS-Ik) is an 11-kDa fragment of PrP spanning residues 58 to approximately 150. These patients carry a missense mutation of the PRNP gene, causing a Phe-->Ser substitution at codon 198. We investigated fibrillogenesis in vitro by using synthetic peptides homologous to consecutive segments of GSS-Ik amyloid protein (residues 57-64, 89-106, 106-126, and 127-147) as well as peptides from the PrP region with the GSS-Ik mutation (residues 191-205 and 181-205, both wild type and mutant). Peptide PrP-(106-126) formed straight fibrils similar to those extracted from GSS brains, whereas peptide PrP-(127-147) formed twisted fibrils resembling scrapie-associated fibrils isolated from subjects with transmissible spongiform encephalopathies. Congo red staining and x-ray fibril diffraction showed that both straight and twisted fibrils had tinctorial and conformational properties of native amyloid. Conversely, the other peptides did not form amyloid-like fibrils under similar conditions. These findings suggest that the sequence spanning residues 106-147 of PrP is central to amyloid fibril formation in GSS and related encephalopathies.

Molecular characteristics of a protease-resistant, amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein.

In the prion-related encephalopathies the prion protein is converted to an altered form, known as PrPSc, that is partially resistant to protease digestion. This abnormal isoform accumulates in the brain and its protease-resistant core aggregates extracellularly into amyloid fibrils. We have investigated the conformational properties, aggregation behaviour and sensitivity to protease digestion of a synthetic peptide homologous to residues 106-126 of human PrP, which was previously found to form amyloid-like fibrils in vitro and displayed neurotoxic activity toward primary cultures of rat hippocampal neurons. A scrambled sequence of peptide PrP 106-126 was used as a control. By circular dichroism, PrP 106-126 exhibited a secondary structure composed largely of beta-sheet, whereas the scrambled sequence of PrP 106-126 showed a random coil structure. The beta-sheet content of PrP 106-126 was much higher in 200 mM phosphate buffer at pH 5.0 than in the same buffer at pH 7.0. Laser light scattering analysis showed that PrP 106-126 aggregated immediately after dissolution in 20 mM or 200 mM phosphate buffer, pH 5.0 and 7.0, whereas scrambled PrP 106-126 did not. PrP 106-126 aggregates had an average hydrodinamic diameter of 100 nm and an average molecular weight of 12 x 10(6) +/- 30% Daltons, corresponding to the aggregation of 6000 +/- 30% molecules. Peptide PrP 106-126 showed partial resistance to digestion with Proteinase K and Pronase, whereas scrambled PrP 106-126 was completely degraded by incubation with the enzymes at 37 degrees C for 30 minutes.

Purification, cDNA cloning, and tissue distribution of bovine liver aldehyde oxidase.

Aldehyde oxidase was purified to homogeneity from bovine liver and primary structural information obtained by sequencing a series of cleavage peptides permitted the cloning of the corresponding cDNA. The cDNA is 4,630 base pairs long, and it consists of a 102-base pair 5'-untranslated region followed by a 4017-base pair coding region and a 511-base pair 3'-untranslated region. The open reading frame predicts a 1339-amino acid polypeptide with a calculated molecular weight of 147,441, which is consistent with the size of the aldehyde oxidase monomeric subunit. The aldehyde oxidase polypeptide contains consensus sequences for iron-sulfur centers and a molybdopterin binding site. The amino acid sequence deduced from the cDNA shows significant similarity with that of xanthine dehydrogenases from various sources. The primary structure of bovine aldehyde oxidase is remarkably similar (approximately 86%) to that of the translation product of a cDNA recently isolated by Wright et al. (Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S., and Repine, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10690-10694) and reported to represent human xanthine dehydrogenase. With the help of a monospecific antibody raised against the purified protein and the isolated cDNA, the tissue distribution of the bovine aldehyde oxidase protein and corresponding mRNA was determined. Aldehyde oxidase is expressed at high levels in liver, lung, and spleen, and, at a much lower level, in many other organs.

A neurotoxic prion protein fragment induces rat astroglial proliferation and hypertrophy.

Prion-related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrPSc) and the deposition of PrP amyloid in the brain. This process is accompanied by neuronal loss and astrogliosis. We recently showed that a synthetic peptide corresponding to residues 106-126 of human PrP is amyloidogenic and causes neuronal death by apoptosis in vitro. In the present study we investigated the effects of 1- and 14-day exposures of rat astroglial cultures to micromolar concentrations of this peptide as well as peptides homologous to other portions of PrP, a peptide corresponding to residues 25-35 of amyloid-beta protein, and a scrambled sequence of PrP 106-126. No significant changes were observed after 1-day exposure of cultures to any peptide. Conversely, 14-day treatment with PrP 106-126 (50 microM) resulted in a 5-fold increase in glial fibrillary acidic protein (GFAP) expression, as evaluated by Northern and Western blot analyses, and a 1.5-fold increment in cell number. Light and electron microscopy immunohistochemistry showed an enlargement in size and density of astroglial processes, and an increase in GFAP-immunoreactive intermediate filaments. These changes were not observed after 14-day treatment of cultures with the other peptides, including PrP 106-126 scrambled. The increase in GFAP expression of astroglial cultures exposed to PrP 106-126 was quantitatively similar to that found in scrapie-infected hamster brains.(ABSTRACT TRUNCATED AT 250 WORDS)