Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5–1 μM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.

Electrostatic interaction between helical macromolecules in dense aggregates: An impetus for DNA poly- and meso-morphism

DNA exhibits a surprising multiplicity of structures when it is packed into dense aggregates. It undergoes various polymorphous transitions (e.g., from the B to A form) and mesomorphous transformations (from hexagonal to orthorhombic or monoclinic packing, changes in the mutual alignment of nearest neighbors, etc). In this report we show that such phenomena may have their origin in the specific helical symmetry of the charge distribution on DNA surface. Electrostatic interaction between neighboring DNA molecules exhibits strong dependence on the patterns of molecular surface groups and adsorbed counter-ions. As a result, it is affected by such structural parameters as the helical pitch, groove width, the number of base pairs per helical turn, etc. We derive expressions which relate the energy of electrostatic interaction with these parameters and with the packing variables characterizing the axial and azimuthal alignment between neighboring macromolecules. We show, in particular, that the structural changes upon the B-to-A transition reduce the electrostatic energy by ≈kcal/mol per base pair, at a random adsorption of counter ions. Ion binding into the narrow groove weakens or inverts this effect, stabilizing B-DNA, as it is presumably the case in Li+-DNA assemblies. The packing symmetry and molecular alignment in DNA aggregates are shown to be affected by the patterns of ion binding.

Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG.

Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light.

Peroxynitrite rapidly permeates phospholipid membranes

Peroxynitrite (ONOO−) is a potent oxidant implicated in a number of pathophysiological processes. The activity of ONOO− is related to its accessibility to biological targets before its spontaneous decomposition (t1/2 ≈ 1 s at pH 7.4, 37°C). Using model phospholipid vesicular systems and manganese porphyrins as reporter molecules, we demonstrated that ONOO− freely crosses phospholipid membranes. The calculated permeability coefficient for ONOO− is ≈8.0 × 10−4 cm⋅s−1, which compares well with that of water and is ≈400 times greater than that of superoxide. We suggest that ONOO− is a significant biological effector molecule not only because of its reactivity but also because of its high diffusibility.

Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis

Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes’ peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.

Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-α-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately −100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.

Signals and signs in the nervous system: The dynamic anatomy of electrical activity is probably information-rich

The dichotomy between two groups of workers on neuroelectrical activity is retarding progress. To study the interrelations between neuronal unit spike activity and compound field potentials of cell populations is both unfashionable and technically challenging. Neither of the mutual disparagements is justified: that spikes are to higher functions as the alphabet is to Shakespeare and that slow field potentials are irrelevant epiphenomena. Spikes are not the basis of the neural code but of multiple codes that coexist with nonspike codes. Field potentials are mainly information-rich signs of underlying processes, but sometimes they are also signals for neighboring cells, that is, they exert influence. This paper concerns opportunities for new research with many channels of wide-band (spike and slow wave) recording. A wealth of structure in time and three-dimensional space is different at each scale—micro-, meso-, and macroactivity. The depth of our ignorance is emphasized to underline the opportunities for uncovering new principles. We cannot currently estimate the relative importance of spikes and synaptic communication vs. extrasynaptic graded signals. In spite of a preponderance of literature on the former, we must consider the latter as probably important. We are in a primitive stage of looking at the time series of wide-band voltages in the compound, local field, potentials and of choosing descriptors that discriminate appropriately among brain loci, states (functions), stages (ontogeny, senescence), and taxa (evolution). This is not surprising, since the brains in higher species are surely the most complex systems known. They must be the greatest reservoir of new discoveries in nature. The complexity should not deter us, but a dose of humility can stimulate the flow of imaginative juices.

Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site.

Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region. The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.