Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes

The present study investigated the effects of Delta I and Delta pH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. Delta pH generated by lowered bulk pH (pH(out)) of PCPECL liposomes, with an internal pH (pH(in)) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK (a) similar to 6.9). Conversely, Delta pH generated by enhanced pH(in) of PCPECL at a pH(out) of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when Delta pH was generated by lowered pH(in) at a pH(out) of 8.0. At bulk acidic pH, Delta I generated by Na+ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH(out), the presence of Delta pH and Delta I did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. the effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the Delta I-M blocks inner mitochondrial membrane fusion during apoptosis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed ABC, CCNH, BR-09210580 Santo Andre, SP, BrazilUniversidade Federal de São Paulo, Dept Biol Mol, São Paulo, BrazilUniv São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Mol, São Paulo, BrazilWeb of Scienc

Organotellurane-promoted mitochondrial permeability transition concomitant with membrane lipid protection against oxidation

Organotelluranes exhibit potent antioxidant properties as well as the ability to react with protein thiol groups and, thereby, they are good models to study the mechanism of the mitochondrial permeability transition (MPT). We evaluated the effects of the concentration of organotelluranes, namely RT-03 and RT-04, on rat liver mitochondria. At the concentration range of 0.25-1.0 mu M, organotelluranes did not cause any mitochondrial dysfunction. At the concentration range of 5-10 mu M, RT-03 and RT-04 caused the Ca2+-depenent opening of the (MPT) pore, regulated by Cyclosporin A. At the concentration range of 15-30 mu M the swelling was not inhibited by Cyclosporin A and in the absence of Ca2+, a significant decrease of respiratory control ratio was observed due to concomitant phosphorylation impairment and uncoupling, transmembrane potential disruption, depletion of mitochondrial reduced thiol groups, and alterations in the bilayer fluidity. Above 100 mu M, the organotelluranes caused complete inhibition of respiratory chain. Over the whole studied concentration range, RT-03 and RT-04 did not induce mitochondrial oxidative stress assessed by using the reactive oxygen and nitrogen species indicator 2',7'-dichlorodihydrofluorescein diacetate. Further, the organotelluranes also exhibited protective effect against t-butyl hydroperoxide-induced oxidative stress as well as against Fe (2+)/citrate-induced peroxidation of mitochondrial membranes and PCPECL liposomes. These results point out that MPT pore opening can involve damage exclusively to mitochondrial membrane proteins. the exclusive antioxidant activity observed at nanomolar range is also an interesting new finding described in this work.Univ Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniv São Paulo, Inst Quim, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilWeb of Scienc

Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics

Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity

Specific effects of reactive thiol drugs on mitochondrial bioenergetics

In this minireview, the more recent findings about the effects of peculiar reactive thiol drugs on mitochondria are presented. These include the following compounds: metallo meso-tetrakis porphyrins, palladacycles, telluranes and phenothiazines. Metallo meso-tetrakis porphyrins can exhibit both beneficial and deleterious effects on mitochodria that are modulated by the central metal, cell location, and availability of axial ligands. Therefore, these compounds have the versatility to be used for cell and mitochondria protection and death. the antioxidant activity of manganese porphyrins is related to a glutathione peroxidase-like activity. By attacking exclusively the membrane protein thiol groups without glutathione depletion, palladacycles are able to induce mitochondrial permeability transition (MPT) and cytochrome c release in the absence of oxidative stress. in hepatoma cells, the mitochondrial action of palladacycles was able to induce apoptotic death. As opposed to palladacycles, telluranes and phenothiazines are able to conjugate the capacity to promote the MPT in a dose-dependent manner in association with efficient antioxidant activity toward lipids. These studies demonstrated that the action of drugs on mitochondrial bioenergetics can be modulated by peculiar reactivity with thiol groups. Therefore, they contribute to studies of toxicity as well as the design of new drugs.Univ Fed ABC, Ctr Ciencias Nat & Humanas, BR-09210170 Santo Andre, SP, BrazilUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Dept Biol Mol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Mol, BR-04044020 São Paulo, BrazilWeb of Scienc

Interatoma of rat Cygb with hydrogen peroxide.

<p>The network shows, in each node, a protein predicted to have functional links with Cygb and hydrogen peroxide. Inside the figure the abbreviations are SOD1 (superoxide dismutase [Cu-Zn]), Hmox2 (heme oxygenase 2 [HO-2]), Mb (myoglobin), Mpo (myeloperoxidase), cat (catalase), Cygb (cytoglobin), Prdx1 (peroxyredoxin-1), Prdx5 (peroxyredoxin-5) and Srxn1 (Ab2-390). In the figure light green, cyan and magenta lines correspond, respectively, to textmining, databases and experiments supporting the relationship among the proteins and hydrogen peroxide.</p

Formation of Cygb amyloid structure after challenge by peroxides.

<p>A), B) and C) show, respectively the epifluorescence images of Cygb control, control plus GSH and challenged by hydrogen peroxide obtained immediately (left panels) 24 h (right panels) after incubation and staining by thioflavine-T. For the low-vacuum SEM experiments, it was used 7 μmol.L^-1 cygb solution with 70 μmol.L^-1 peroxide solutions. For the epifluorescence experiments 70 μmol.L^-1 protein solution was incubated for 1 h with 700 μmol.L^-1 peroxide solution in the presence of thioflavin-T. For FTIR measurements, 7 μmol.L^-1 protein solution was incubated with 70 μmol.L^-1 peroxide solutions for 1 h. The results are representative of three independent experiments.</p

Spectroscopic characteristics of Cygb.

<p>The upper panel shows the EA spectrum of Cygb and the respective inset the corresponding far-UV CD spectrum. The lower panel shows the CD (light gray line) and the MCD spectra of Cygb obtained by addition and subtraction of the original spectra obtained at positive and negative magnetic fields. MCD is shown at increasing magnetic fields and the respective inset shows the linear increase of Soret band intensity promoted by increasing the magnetic field. The experiments of EA spectroscopy were performed with 65 μmol.L^-1 Cygb using 0.1 cm optical length. The experiments of CD and MCD were performed using 20 μmol.L^-1 protein solution in 20 mmol.L^-1 phosphate buffer, pH 7.4. These results are representative of three independent replicates.</p