Alexander M. Firsov

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex.

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer

Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e6 (the conjugate of chlorin e6 with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e6 against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e6 is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e6. Unlike chlorin e6, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters.

Single channel activity of OmpF-like porin from Yersinia pseudotuberculosis.

To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.

A mitochondria‐targeted antioxidant can inhibit peroxidase activity of cytochrome c by detachment of the protein from liposomes

Interaction of cytochrome c with cardiolipin converts this respiratory chain electron‐transfer protein into a peroxidase, supposedly involved in mitochondria‐mediated apoptosis initiation. Liposome membrane permeabilization provoked by peroxidase activity of the cytochrome c/cardiolipin complex has been previously shown to be suppressed by conventional antioxidants. Here, the mitochondria‐targeted antioxidant SkQ1 (plastoquinonyl‐decyl‐triphenylphosphonium) was found to strongly inhibit both cytochrome c/cardiolipin peroxidase activity and the permeabilization of liposomes composed of phosphatidylcholine and cardiolipin. A number of binding assays revealed a significant inhibiting effect of SkQ1 on cytochrome c binding to liposomes, thus suggesting that SkQ1‐mediated protection of liposomes from the cytochrome c/H2O2‐induced permeabilization involved distortion of the cytochrome c‐membrane binding. It is suggested that antioxidant and antiapoptotic effects of alkyltriphenylphosphonium cations can be related to the prevention of cytochrome c/cardiolipin interaction.

pH-Dependent properties of ion channels formed by N-terminally glutamate substituted gramicidin A in planar lipid bilayers

The N-terminally glutamate substituted analogue of the pentadecapeptide gramicidin A [Glu1]gA has been previously described as a low-toxic uncoupler of mitochondrial oxidative phosphorylation and neuroprotector. Here, we studied ion channel-forming activity of this peptide in planar bilayer lipid membranes (BLMs). [Glu1]gA exhibited an ability to induce both macroscopic current and single channels in a broad pH range, albeit with a lower potency than the parent gramicidin A (gA). Single-channel recordings in 1M KCl at pH about 4 showed channel openings of one type with the conductance (about 26pS), similar to that of gA, and the lifetime (40ms), much shorter than that of gA. By contrast, two populations of channels were found at pH9, one of which had much longer duration (several seconds) and lower conductance (3.5-10pS). Autocorrelation function of the current noise of [Glu1]gA revealed a marked shift towards longer correlation times upon alkalinization. The sensitized photoinactivation technique also revealed substantial differences in [Glu1]gA conducting properties at alkaline and acidic pH, in particular deceleration of the photoinactivation kinetics and a sharp decrease in its amplitude upon alkalinization. A double-logarithmic plot of the concentration dependence of [Glu1]gA-induced BLM conductance had the slope of about 3, which pointed to peptide aggregation in the membrane. The data were discussed in relation to pH-dependent aggregation of [Glu1]gA, resulting from deprotonation of the glutamate side chain at alkaline pH.

Alkyl-substituted phenylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole as uncouplers of oxidative phosphorylation and antibacterial agents: involvement of membrane proteins in the uncoupling action

In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work (Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines (NBD-Cn) were shown to exhibit uncoupling activity. It was concluded that despite a pKa value being about 10, the expected hindering of the uncoupling activity could be overcome by insertion of an alkyl chain. There is evidence in the literature that the introduction of an aryl substituent in the 4-amino NBD group shifts the pKa to neutral values. Here we report the data on the properties of a number of 4-arylamino derivatives of NBD, namely, alkylphenyl-amino-NBD (Cn-phenyl-NBD) with varying alkyl chain Cn. By measuring the electrical current across planar bilayer lipid membrane, the protonophoric activity of Cn-phenyl-NBD at neutral pH grew monotonously from C1- to C6-phenyl-NBD. All of these compounds increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Importantly, the uncoupling action of C6- and C4-phenyl-NBD was partially reversed by glutamate, diethyl pyrocarbonate (DEPC), 6-ketocholestanol, and carboxyatractyloside, thus pointing to the involvement of membrane proteins in the uncoupling activity of Cn-phenyl-NBD in mitochondria. The pronounced recoupling effect of DEPC, an inhibitor of an aspartate-glutamate carrier (AGC), and that of its substrates for the first time highlighted AGC participation in the action of potent uncouplers on mitochondria. C6-phenyl-NBD produced strong antimicrobial effect on Bacillus subtilis, which manifested itself in cell membrane depolarization and suppression of bacterial growth at submicromolar concentrations.

Photodynamic inactivation of gramicidin channels in bilayer lipid membranes: Protective efficacy of singlet oxygen quenchers depends on photosensitizer location

The impact of double bonds in fatty acyl tails of unsaturated lipids on the photodynamic inactivation of ion channels formed by the pentadecapeptide gramicidin A in a planar bilayer lipid membrane was studied. The presence of unsaturated acyl tails protected gramicidin A against photodynamic inactivation, with efficacy depending on the depth of a photosensitizer in the membrane. The protective effect of double bonds was maximal with membrane-embedded chlorin e6-monoethylenediamine monoamide dimethyl ester, and minimal — in the case of water-soluble tri-sulfonated aluminum phthalocyanine (AlPcS3) known to reside at the membrane surface. By contrast, the protective effect of the hydrophilic singlet oxygen scavenger ascorbate was maximal for AlPcS3 and minimal for amide of chlorin e6 dimethyl ester. The depth of photosensitizer position in the lipid bilayer was estimated from the quenching of photosensitizer fluorescence by iodide. Thus, the protective effect of a singlet oxygen scavenger against photodynamic inactivation of the membrane-inserted peptide is enhanced upon location of the photosensitizer and scavenger molecules in close vicinity to each other.

Effect of Site-Specific Intermolecular Lysine–Tryptophan Interactions on the Aggregation of Gramicidin-Based Peptides Leading to Pore Formation in Lipid Membranes

In contrast to the parent pentadecapeptide gramicidin A (gA), some of its cationic analogs have been shown previously to form large-diameter pores in lipid membranes. These pores are permeable to fluorescent dyes, which allows one to monitor pore formation by using the fluorescence de-quenching assay. According to the previously proposed model, the gA analog with lysine substituted for alanine at position 3, [Lys3]gA, forms pores by a homopentameric assembly of gramicidin double-stranded β-helical dimers. Here, we studied the newly synthesized analogs of [Lys3]gA with single, double and triple substitutions of isoleucines for tryptophans at positions 9, 11, 13, and 15. Replacement of any of the tryptophans of [Lys3]gA with isoleucine resulted in suppression of the pore-forming activity of the peptide, the effect being significantly dependent on the position of tryptophans. In particular, the peptide with a single substitution of tryptophan 13 showed much lower activity than the analogs with single substitutions at positions 9, 11, or 15. Of the peptides with double substitutions, the strongest suppression of the leakage was observed with tryptophans 13 and 15. In the case of triple substitutions, only the peptide retaining tryptophan 11 exhibited noticeable activity. It is concluded that tryptophans 11 and 13 contribute most to pore stabilization in the membrane, whereas tryptophan 9 is not so important for pore formation. Cation–π interactions between the lysine and tryptophan residues of the peptide are suggested to be crucial for the formation of the [Lys3]gA pore.

Calcein leakage as a robust assay for cytochrome c/H2O2–mediated liposome permeabilization

Membrane-permeabilizing activity of cytochrome c (cyt c) in the presence of hydrogen peroxide associated with its functioning as peroxidase is considered relevant to initiation of the mitochondrial pathway of apoptosis. Here, we present evidence that the choice of a fluorescent dye for measuring cyt c/H2O2-induced dye leakage from liposomes by fluorescence de-quenching is of major importance. The popular fluorescent marker 5(6)-carboxyfluorescein appeared highly susceptible to cyt c-mediated peroxidative destruction and therefore unsuitable for the leakage assay with cyt c/H2O2. On the contrary, calcein, another conventional marker, proved resistant to oxidative stress and thus perfectly suitable for the assay. Based on the concentration dependences of the cyt c/H2O2-induced calcein leakage, the optimal conditions for the assay were found.