Evaluation of cytochrome c affinity to anionic phospholipids by means of surface plasmon resonance

We attempted to evaluate the affinity of the anionic phospholipids to cytochrome c by means of surface plasmon resonance (SPR) technique and to correlate it with the cytochrome c active site alterations and peroxidase activity. Our experiments showed a strong interdependence between the phospholipid fatty acid saturation degree, the active site structure alterations and peroxidase activity of the cytochrome c phospholipid complex. Cytochrome c peroxidase activity and Trp59 fluorescence increase in the sequence of phosphatidyl choline (PC) → phosphatidylserine (PS) → cardiolipin (CL) → phosphatidic acid (PA). The association constant (K_a) increased in the sequence PC → PA → PS → CL. The SPR spectroscopy data shows that K_a is independent of lipid saturation degree, but correlates with phospholipid negative charge value.     

Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size

The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G). However, the properties and nature of Bax/Bak apoptotic pores remain enigmatic. Here, we performed a detailed analysis of the membrane permeabilizing activity of Bax and Bak at the single vesicle level. We directly visualized that cBid-activated Bax and BakΔC21 can form membrane pores large enough to release not only cytochrome c, but also allophycocyanine, a protein of 104 kDa. Interestingly, the size of Bax and BakΔC21 pores is not constant, as typically observed in purely proteinaceous channels, but evolves with time and depends on protein concentration. We found that Bax and BakΔC21 formed long-lived pores, whose areas changed with the amount of Bax/BakΔC21 but not with cardiolipin concentration. Altogether, our results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores.     

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X_L protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.     

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.     

Electron flow into cytochrome c coupled with reactive oxygen species from the electron transport chain converts cytochrome c to a cardiolipin peroxidase: role during ischemia–reperfusion

Background

Cytochrome c (Cyt c) is a mobile component of the electron transport chain (ETC.) which contains a tightly coordinated heme iron. In pathologic settings, a key ligand of the cyt c's heme iron, methionine (Met₈₀), is oxidized allowing cyt c to participate in reactions as a peroxidase with cardiolipin as a target. Myocardial ischemia (ISC) results in ETC. blockade and increased production of reactive oxygen species (ROS). We hypothesized that during ischemia–reperfusion (ISC-REP); ROS generation coupled with electron flow into cyt c would oxidize Met₈₀ and contribute to mitochondrial-mediated ETC. damage.

Methods

Mitochondria were incubated with specific substrates and inhibitors to test the contributions of ROS and electron flow into cyt c. Subsequently, cyt c and cardiolipin were analyzed. To test the pathophysiologic relevance, mouse hearts that underwent ISC-REP were tested for methionine oxidation in cyt c.

Results

The combination of substrate/inhibitor showed that ROS production and electron flux through cyt c are essential for the oxidation of methionine residues that lead to cardiolipin depletion. The content of cyt c methionine oxidation increases following ISC-REP in the intact heart.

Conclusions

Increase in intra-mitochondrial ROS coupled with electron flow into cyt c, oxidizes cyt c followed by depletion of cardiolipin. ISC-REP increases methionine oxidation, supporting that cyt c peroxidase activity can form in the intact heart.

General significance

This study identifies a new site in the ETC. that is damaged during cardiac ISC-REP. Generation of a neoperoxidase activity of cyt c favors the formation of a defective ETC. that activates signaling for cell death.     

Molecular mechanisms of apoptosis. Structure of cytochrome c-cardiolipin complex

One of the functions of cytochrome c in living cells is the initiation of apoptosis by catalyzing lipid peroxidation in the inner mitochondrial membrane, which involves cytochrome c bound with acidic lipids, especially cardiolipin. In this paper the results of studies of cytochrome c-cardiolipin complex structure carried out by different authors mainly on unilamellar cardiolipin-containing phospholipid liposomes are critically analyzed. The principal conclusion from the published papers is that cytochrome c-cardiolipin complex is formed by attachment of a cytochrome c molecule to the membrane surface via electrostatic interactions and the subsequent penetration of one of the fatty-acid cardiolipin chains into the protein globule, this being associated with hydrophobic interactions that break the >Fe…S(Met80) coordinate bond and giving rise to appearance of cytochrome c peroxidase activity. Nevertheless, according to data obtained in our laboratory, cytochrome c and cardiolipin form spherical nanoparticles in which protein is surrounded by a monolayer of cardiolipin molecules. Under the action of cooperative forces, the protein in the globule expands greatly in volume, its conformation is modified, and the protein becomes a peroxidase. In extended membranes, such as giant monolayer liposomes, and very likely in biological membranes, the formation of nanospheres of cytochrome c-cardiolipin complex causes fusion of membrane sections and dramatic chaotization of the whole membrane structure. The subsequent disintegration of the outer mitochondrial membrane is accompanied by cytochrome c release from the mitochondria and triggering of a cascade of programmed cell death reactions.     

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca²⁺-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca²⁺-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca²⁺-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca²⁺ overload.     

Assembly of the Mitochondrial Apoptosis-induced Channel, MAC

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane α-helices in Bax and Bak, mature MAC pores would typically contain ∼9 monomers and have diameters of 5.5–6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.Permeabilization of the mitochondrial outer membrane is the commitment step in intrinsic apoptosis. This process is tightly regulated by Bcl-2 family proteins that control formation of the megachannel mitochondrial apoptosis-induced channel (MAC)² in this membrane. MAC formation correlates with release of pro-apoptotic factors, including cytochrome c from the intermembrane space into the cytosol, and initiates apoptosis (1–7).MAC is absent from normal mitochondria but forms in the outer membrane early in apoptosis, reaching peak conductances of 1.5–5 nS. This channel is formed in the presence of the multidomain pro-apoptotic proteins Bax and/or Bak (8–13), and may be composed of these proteins along with other components (14, 15). Unlike Bax, Bak is normally a resident of the mitochondrial outer membrane and is bound to VDAC2, another outer membrane protein (16). However, Bak is not available for oligomerization until another pro-apoptotic protein, like t-Bid, disrupts the interaction of Bak with VDAC2. In contrast, most Bax is located in the cytoplasm until an apoptotic signal induces the translocation of Bax to the outer membrane of mitochondria and eventual Bax oligomerization in this same membrane (14, 17).Bax and Bak have multiple putative transmembrane domains; the amphipathic helices 5 and 6 of Bax are predicted to form, at least in part, the pore of the cytochrome c release channel (18). Bax lacking helices 5 and 6 does not translocate to mitochondria nor cause cytochrome c release (19, 20). Given the structural similarities between Bax and Bak, the same helices may be important in formation of the MAC pore by both proteins (21). Although Bax and Bak are certainly involved in MAC formation, the exact molecular composition of this channel remains unknown.In this study we report that Bax and Bak are functionally redundant with regard to MAC formation and cytochrome c release in mouse embryonic fibroblasts (MEF). This is true despite the fact that Bak normally resides in the outer membrane, whereas Bax is generally translocated to this membrane to induce MAC formation. Our experimental design bypasses Bax translocation and any underlying autocatalytic mechanism that might be involved (22). Instead, it focuses on formation of the MAC pore. Early MAC-associated conductance increments are relatively small, suggesting that Bax-dependent formation of the cytochrome c-permeable pore does not occur prior to membrane insertion of Bax. Mathematical modeling of the conductance changes indicates that, if MAC is a circular pore assembled by sequential addition of helices 5 and 6 from Bax and/or Bak monomers, the mature, cytochrome c transport-competent pore is likely a 9–10-mer of these proteins.     

Pseudomonas aeruginosa homoserine lactone triggers apoptosis and Bak/Bax‐independent release of mitochondrial cytochrome C in fibroblasts

Pseudomonas aeruginosa use N‐(3‐oxododecanoyl)‐homoserine lactone (C12) as a quorum‐sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12‐triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild‐type (WT) and Bax^?/?/Bak^?/? (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψ_mito), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax‐dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψ_mito and release of cytochrome C and cell death. Re‐expression of Bax or Bak in DKO MEF did not alter the WT‐like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax‐independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψ_mito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.     

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ_m) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.     

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.     

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.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Antioxidant responses of citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), exposed to thermal stress

Relatively low or high temperatures are responsible for a variety of physiological stress responses in insects and mites. Induced thermal stress was recently associated with increased reactive oxygen species (ROS) generation, which caused oxidative damage. In this study, we examined the time-related effect of the relatively low (0, 5, 10, and 15 °C) or high (32, 35, 38, and 41 °C) temperatures on the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), and glutathione-S-transferase (GST), and the total antioxidant capacity (TEAC) of the citrus red mite, Panonychus citri (McGregor). The malondialdehyde (MDA) concentration, as a marker of lipid peroxidation in organisms, was also measured in the citrus red mite under thermal stress conditions. Results showed that SOD and GST activities were significantly increased and play an important role in the process of antioxidant response to thermal stress. Lipid peroxidation levels increased significantly (P < 0.001) and changed in a time-dependent manner. CAT and POX activity, as well as TEAC, did not vary significantly and play a minor role to remove the ROS generation. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play an important role in reducing oxidative damage in the citrus red mite.     

The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis

While many studies have focused on cytochrome c release from mitochondria, little attention has been given to the specific interaction between cardiolipin (CL) and cytochrome c, the breaching of which likely represents a critical event in the initiation of mitochondrially mediated apoptosis. Mounting evidence suggests that a decrease in the level of CL affects cytochrome c binding to the inner membrane, thus leading to higher levels of soluble cytochrome c in the mitochondrial intermembrane space. Among the factors known to affect CL levels are thyroid status, plasma concentrations of free fatty acids, Ca²⁺ dysregulation, and reactive oxygen species (ROS). These factors, especially Ca²⁺ and ROS, have long been recognized as triggers of cell death and, more recently, as modulators of mitochondrially mediated apoptosis. In this review, we discuss the significance of the disruption of the CL-cytochrome c interaction for cytochrome c release and apoptosis.     

Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt c’s conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria—its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition—are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.