Antimitotics induce cardiolipin cluster formation. Possible role in mitochondrial enzyme inactivation

We demonstrate here that drugs which inactivate cytochrome c oxidase are able to segregate cardiolipin essential for the enzyme activity, in a separate phase inaccessible for the enzyme. A molecular explanation of the drug-induced aggregation process is proposed.     

Functional role of cardiolipin in mitochondrial bioenergetics

Cardiolipin is a unique phospholipid which is almost exclusively located in the inner mitochondrial membrane where it is biosynthesized. Considerable progress has recently been made in understanding the role of cardiolipin in mitochondrial function and bioenergetics. This phospholipid is associated with membranes designed to generate an electrochemical gradient that is used to produce ATP, such as bacterial plasma membranes and inner mitochondrial membrane. This ubiquitous and intimate association between cardiolipin and energy transducing membranes indicates an important role for cardiolipin in mitochondrial bioenergetic processes. Cardiolipin has been shown to interact with a number of proteins, including the respiratory chain complexes and substrate carrier proteins. Over the past decade, the significance of cardiolipin in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Moreover, cardiolipin is involved in different stages of the mitochondrial apoptotic process, as well as in mitochondrial membrane stability and dynamics. This review discusses the current understanding of the functional role that cardiolipin plays in several reactions and processes involved in mitochondrial bioenergetics. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

The role of cardiolipin in the structural organization of mitochondrial membranes

Considerable progress has recently been made in understanding the role of cardiolipin in mitochondria. In this brief review, we discuss new data that show how cardiolipin specifically contributes to the lateral organization of mitochondrial membranes. We argue that the function of cardiolipin has to be understood in the context of dynamic membrane assembly rather than static membrane structure, and we propose that remodeling of cardiolipin, i.e. the formation of uniformly substituted molecular species, may reduce the energy barrier of the assembly process.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

Effect of modulation of enzyme inactivation on temperature optimization for reactor operation with chitin-immobilized lactase

Temperature is a critical variable to be optimized in any enzymatic process, producing opposite effects on enzyme activity and inactivation rate. Temperature functions for all kinetic and inactivation parameters were validated for chitin-immobilized yeast lactase (CIL). Enzyme inactivation was described by a two-stage series mechanism. The effect of galactose and lactose on inactivation was determined in terms of modulation factors that were positive for galactose and negative for lactose. Modulation factors were mild functions of temperature in the first stage and strong functions in the second stage of enzyme inactivation, where galactose positive modulation factors increase while lactose negative modulation factors decrease with temperature. Temperature-explicit functions for kinetic and inactivation parameters were incorporated into a scheme to optimize temperature in the simulation of a continuous packed-bed reactor operation with chitin-immobilized lactase, based on an annual cost objective function. Optimum temperature was 20°C at enzyme replacement of 25% residual activity, and increased only slightly at higher replacement frequencies. The effect of modulation factors on reactor design and temperature optimization is presented and discussed. Software for temperature optimization that allows the introduction of variations in all parameters and operational criteria to perform sensitivity analysis was developed.     

Deficiency of cardiolipin synthase causes abnormal mitochondrial function and morphology in germ cells of Caenorhabditis elegans

Cardiolipin (CL) is a major membrane phospholipid specifically localized in mitochondria. At the cellular level, CL has been shown to have a role in mitochondrial energy production, mitochondrial membrane dynamics, and the triggering of apoptosis. However, the in vivo role of CL in multicellular organisms is largely unknown. In this study, by analyzing deletion mutants of a CL synthase gene (crls-1) in Caenorhabditis elegans, we demonstrated that CL depletion selectively caused abnormal mitochondrial function and morphology in germ cells but not in somatic cell types such as muscle cells. crls-1 mutants reached adulthood but were sterile with reduced germ cell proliferation and impaired oogenesis. In the gonad of crls-1 mutants, mitochondrial membrane potential was significantly decreased, and the structure of the mitochondrial cristae was disrupted. Contrary to the abnormalities in the gonad, somatic tissues in crls-1 mutants appeared normal with respect to cell proliferation, mitochondrial function, and mitochondrial morphology. Increased susceptibility to CL depletion in germ cells was also observed in mutants of phosphatidylglycerophosphate synthase, an enzyme responsible for producing phosphatidylglycerol, a precursor phospholipid of CL. We propose that the contribution of CL to mitochondrial function and morphology is different among the cell types in C. elegans.     

Role of mitochondrial cardiolipin peroxidation in apoptotic photokilling of 5-aminolevulinate-treated tumor cells

In 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), ALA taken up by tumor cells is metabolized to protoporphyrin IX (PpIX), which sensitizes photodamage leading to apoptotic or necrotic cell death. Since lipophilic PpIX originates in mitochondria, we postulated that photoperoxidation of highly unsaturated cardiolipin (CL), which anchors cytochrome c (cyt c) to the inner membrane, is an early proapoptotic event. As initial evidence, PpIX-sensitized photooxidation of liposomal CL to hydroperoxide (CLOOH) species precluded cyt c binding, but this could be reinstated by GSH/selenoperoxidase (GPX4) treatment. Further support derived from site-specific effects observed using (i) a mitochondrial GPX4-overexpressing clone (7G4) of COH-BR1 tumor cells, and (ii) an ALA treatment protocol in which most cellular PpIX is either inside (Pr-1) or outside (Pr-2) mitochondria. Sensitized cells were exposed to a lethal light dose, and then analyzed for death mechanism and lipid hydroperoxide (LOOH) levels. Irradiated Pr-1 vector control (VC) cells died apoptotically following cyt c release and caspase-3 activation, whereas 7G4 cells were highly resistant. Irradiated Pr-2 VC and 7G4 cells showed negligible cyt c release or caspase-3 activation, and both types died via necrosis. CLOOH (detected long before cyt c release) accumulated approximately 70% slower in Pr-1 7G4 cells than in Pr-1 VC, and this slowdown exceeded that of all other LOOHs. These and related findings support the hypothesis that CL is a key upstream target in mitochondria-dependent ALA-PDT-induced apoptosis.     

Role of cardiolipin peroxidation and Ca in mitochondrial dysfunction and disease

Cardiolipin is a unique phospholipid which is almost exclusively located at the level of the inner mitochondrial membrane where it is biosynthesized. This phospholipid is known to be intimately involved in several mitochondrial bioenergetic processes. In addition, cardiolipin also has active roles in several of the mitochondrial-dependent steps of apoptosis and in mitochondrial membrane dynamics. Alterations in cardiolipin structure, content and acyl chains composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including ischemia/reperfusion, different thyroid states, diabetes, aging and heart failure. Cardiolipin is particularly susceptible to ROS attack due to its high content of unsaturated fatty acids. Oxidative damage to cardiolipin would negatively impact the biochemical function of the mitochondrial membranes altering membrane fluidity, ion permeability, structure and function of components of the mitochondrial electron transport chain, resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to cardiolipin peroxidation are described. Ca²⁺, particularly at high concentrations, appears to have several negative effects on mitochondrial function, some of these effects being linked to CL peroxidation. Cardiolipin peroxidation has been shown to participate, together with Ca²⁺, in mitochondrial permeability transition. In this review, we provide an overview of the role of CL peroxidation and Ca²⁺ in mitochondrial dysfunction and disease.     

The role of cardiolipin in promoting the membrane pore-forming activity of BAX oligomers

BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5–α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.     

Lysozyme inactivation and aggregation in stirred-reactor

Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme.

The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation.

During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates.

Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased.

This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.     

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.     

Kinetic study in the transient phase of the suicide inactivation of frog epidermis tyrosinase

This paper deals with the kinetic study of a multisubstrate mechanism with enzyme inactivation induced by a suicide substrate. A transient phase approach has been developed that enables the deduction of explicit equations of product concentration vs. time. From these equations kinetic constants which characterize the suicide substrate can be obtained. This study with tyrosinase enzyme, which acts on L-dopa and catechol allowed us to determine the corresponding kinetic parameters, indicating that catechol is about 8-times more powerful as a suicide substrate than is L-dopa.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria Role of cardiolipin in fluidity of mitochondrial membranes

During temperature acclimation of Tetrahymena pyriformis, the changes in fluidity and composition of total lipids from three membrane fractions, mitochondria, pellicles and microsomes were studied by a spin-label technique using a stearate probe and thin-layer and gas-liquid chromatography. The increase of fluidity observed in microsomal and pellicular lipids following the temperature shift from 39 to 15°C corresponds with the increase of the ratio of total unsaturated to saturated fatty acid content. However, despite the increase of this ratio, the fluidity of mitochondrial lipids was found to be constant up to 10 h after the temperature shift. The fluidity of total lipids of mitochondria isolated from Tetrahymena cells grown at 39°C was not changed by removal of cardiolipin, whereas cardiolipin-depleted lipids of mitochondria from 15°C-acclimated cells showed a decrease in fluidity. The re-addition of cardiolipin to the mitochondrial lipids depleted of cardiolipin restored the fluidity to the initial leve, thereby confirming the rigidifying effect of cardiolipin in cold-acclimated cells. These results suggest that cardiolipin may be implicated in maintaining consistent fluidity of mitochondrial membranes against change in thermal environment.     

Lysozyme inactivation under mechanical stirring: effect of physical and molecular interfaces

This article focuses on the role of interfaces on lysozyme inactivation and aggregation process in stirred reactor. The first order inactivation constant of this process has found to be proportional not only to the power imparted by the impeller but also to the area of glass-liquid, air-liquid and PTFE-liquid interfaces in three reactors. Both area and type of interfaces act on inactivation: PTFE and air are four more efficient than glass to promote lysozyme inactivation because of their hydrophobicity. As well as physical interfaces, molecular surfaces of inactivated enzymes -more hydrophobic than native enzymes- enhance lysozyme inactivation and aggregation. This enhancement has been found to be correlated with the properties of aggregates of inactivated enzymes, especially their number. Then, under mechanical stirring, inactivation-aggregation process is induced by physical interfaces and self-catalyzed by increasing hydrophobic surfaces of inactivated enzymes.     

Changes in catalytic activity and association state of pyruvate carboxylase which are dependent on enzyme concentration

The specific activity of chicken liver pyruvate carboxylase has been shown to decrease with decreasing enzyme concentration, even at 100 microM, which is close to the estimated physiological concentration. The kinetics of the loss of enzyme specific activity following dilution were biphasic. Incubation of dilution-inactivated enzyme with ATP, acetyl CoA, Mg2+ + ATP or, to a lesser degree, with Mg2+ alone resulted in a high degree of reactivation, while no reactivation occurred in the presence of pyruvate. The association state of the enzyme before, during, and after dilution inactivation has been assessed by gel filtration chromatography. These studies indicate that on dilution, there is dissociation of the catalytically active tetrameric enzyme species into inactive dimers. Reactivation of the enzyme resulted in reassociation of enzymic dimers into tetramers. The enzyme was shown to form high molecular weight aggregates at high enzyme concentrations.     

Allantoinase from Pseudomonas aeruginosa Purification,properties and immunochemical characterization of its in vivo inactivation

The catabolic enzyme allantoinase is rapidly inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. This process is irreversible since the protein synthesis inhibitor chloramphenicol completely blocked the reappearance of allantoinase activity that is observed when allantoin is added to stationary cells. Purified allantoinase appeared to be a protein composed of four identical subunits with a molecular weight of 38 000. With antibodies raised against purified allantoinase it was found that allantoinase inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggest that allantoinase inactivation is caused or followed by rapid proteolysis.     

Role of oxyradicals in the inactivation of catalase by ozone

The antioxidant enzymes, catalase and superoxide dismutase, are inactivated upon exposure to ozone. In this study, the mechanism of this inactivation was examined using catalase as a model system. The data show that the inactivation of catalase is dependent on ozone concentration, time of exposure, and pH. Loss of catalase activity is accompanied with loss of the heme spectra. Tiron, desferal-Mn, trolox-c, and pyruvate protect the enzyme against ozone inactivation. SOD is less effective due to its inactivation by ozone. On the other hand, alcohols do not provide significant protection. The data suggest the possible involvement of superoxide radicals in the inactivation of catalase by ozone.     

Molecular aspects of the bilayer stabilization induced by poly(l-lysines) of varying size in cardiolipin liposomes

The interaction between poly(l-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and ³¹P- and ¹³C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca²⁺ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca²⁺, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca²⁺ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca²⁺, the lipid was organized in the hexagonal H_II phase in the presence of Ca²⁺. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.     

Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation

The ‘high ammonia pathway’ enzyme glutamate dehydrogenase (NADP⁺) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth in reached. Purified glutamate dehydrogenase (NADP⁺) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP⁺) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP⁺) inactivation is caused or followed by rapid proteolysis.