Mitochondrial Membrane System Research Articles

Investigation of the structural and dynamical properties of human uncoupling protein 2 through molecular dynamics simulations

Uncoupling protein 2 (UCP2) is an integral membrane protein that belongs to the family of mitochondrial anion carrier proteins. The absence of human UCP2 structure, lack of understanding of Cl− ion transport mechanism in the UCP2 and the associated biological functions motivated us to model the protein and investigate its structural and dynamical properties in a realistic mitochondrial lipid membrane system. The lipid-protein and protein-protein interactions were probed since they were found to be responsible for the conformational changes of the transmembrane (TM) helices which are involved in facilitating Cl− ion transport. Here, we employed multiscale molecular dynamics simulations including unbiased and biased MD for the investigation of the transport pathway in hUCP2 and interactions of the ion with TM helices within a membrane environment. We initially validated the hUCP2 model in the lipid membrane and then explored the transport pathway of Cl− ion and its interaction with positive residues of TM2 helix that have been reported to play a major role in the Cl− ion transport along with other TM helices of the protein. The simulation results suggest that the TM2 helix plays an important role in the formation of a stable ion channel due to the presence of arginine residues, in particular Arg88 which was found to be a key residue to maintain the channel pore through which the movement of Cl− ions occurs. Based on the results, it can be said that the study provides an atomic-level description of the Cl− ion transport mechanism in hUCP2 embedded in the mitochondrial lipid membrane.

Mitochondrial Membranes Involved in Apoptosis - the Bcl-2 Proteins

Removal of cells via programmed cell death (apoptosis) is an essential mechanism in life. Upon internal stress clearance of undesired cells is executed via intrinsic (mitochondrial) apoptosis, a pathway tightly controlled by the Bcl-2 protein family. Its pro- and anti-apoptotic members meet at the mitochondrial surface, more precisely at its outer membrane system, where they arbitrate a life or death (membrane permeabilization) decision. Failures cause pathological disorders including abnormal embryogenic development and cancer. However, how opposing Bcl-2 members meet and form a sophisticated regulative network there, is still poorly understood due to the lack of structural and functional information for these proteins acting at the the mitochondrial membraneous surface. To provide structural about this molecular regulation mechanism occurring at the mitochondrial outer membrane system, we apply solid-state/liquid NMR approaches together with neutron reflectometry on two representative Bcl-2 family members, namely the anti-apoptotic Bcl-2 protein itself which is an integral membrane protein, and its counterpart, the pro-apoptotic, membrane perforating Bax protein. i) Using MOM-like membranes doped with oxidized lipids to simulate apoptosis triggering oxidative stress, we investigate changes in the dynamic and structural organization of these membranes in the presence of Bax protein to identify the key structural elements which regulate Bax perforation activity in response to type and level of oxidative membrane damage. ii) The atomic structure of the Bcl-2 membrane protein is still undetermined, preventing a detailed mechanistic insight into its function, especially its loops as anti-apoptotic molecular switches. We are going to provide this information by combining liquid-state NMR on Bcl-2 in micelles with solid-state NMR on Bcl-2/lipid assemblies.

Insecticidal and antifungal activities of Rheum palmatum L. anthraquinones and structurally related compounds

As a well-known herb worldwide, rhubarb presents a wide range of pharmacological activities. In our studies, Rheum palmatum L. extracts and five isolated anthraquinones possessed insecticidal activity. The acetone extract (yield, 8.84%), which contained the highest amount of compounds (82.87 mg/g), showed stronger insecticidal activity than did the aqueous extract (yield, 37.17%) and the ethanol extract (yield, 36.92%). Notable amounts of compounds were also found in water extract residues (18.2 mg/g). Among the isolated quinones and structurally related compounds, emodin exhibited the highest insecticidal activity (LC50 = 84.30 μg/mL) against Nilarparvata lugens; and very moderate activity against Mythimna separata (LC50 = 548.74 μg/mL). The LC50 values of positive agent toosendanin against two insects were 89.34 and 418.24 μg/mL, respectively. Moreover, emodin exhibited significant AChE and GST inhibitory activities (with respective IC50 values of 11.36 and 4.18 μg/mL). Plumbagin exhibited stronger and broader antifungal activity against eight phytopathogenic fungi (with EC50 values ranging from 2.84 to 10.53 μg/mL) compared to a commercial fungicide, azoxystrobin (EC50 ranging from 20.40 to >100.00 μg/mL). Plumbagin significantly damaged the cell and mitochondrial membrane systems, changed the morphology, triggered the release of cellular contents and generate reactive oxygen species in Rhizoctonia solani mycelia. This study laid the foundation for the future development of emodin and plumbagin as alternative, environmentally friendly bioinsecticide and biofungicide, respectively. Rhubarb and its residues can be used as renewable sources of anthraquinone-based products.

Mitochondria in Obliquely Striated Muscles of the Horsehair Worm Gordionus alpestris (Nematomorpha, Gordioidea) with Structural Organization Typical of Cells with Energy-Intensive Processes.

The ultrastructure of mitochondria in the flattened circomyarian fibers of the horsehair worm Gordionus alpestris (Nemathelminthes) was examined. In contrast to the previously published data, we showed these mitochondria to be giant elongated organelles that densely fill the central cytoplasmic space of the ribbon-like muscle fibers. No fundamental differences were found in the ultrastructure of the muscle tissue mitochondria in actively moving free-living and parasitic G. alpestris worms. The functional significance of the observed ultrastructural organization of mitochondria is discussed in connection with the necessity for an extended mitochondrial membrane system for a uniform supply of active muscle tissue with energy.

Differential interaction of α-synuclein N-terminal segment with mitochondrial model membranes

Alpha-synuclein (α-syn) is an intrinsically-disordered protein that has been associated with Parkinson's disease through its deposition in an amyloid fibril form within Lewy Body. Several lines of evidence suggest that the physical association of α-syn with the mitochondrial membranes may cause membrane damage and mitochondrial dysfunction, playing an important role in disease progression. Although there is strong evidence that the N-terminus part of α-syn is essential for membrane affinity, cooperative formation of helical domains and regulation of mitochondria membrane permeability, the amino acids involve in this membrane binding is still controversial. Fluorescence spectroscopy, circular dichroism and Langmuir monolayer technique were used to elucidate this recognition process of mitochondrial membrane system by synthetic peptides derived from α-syn N-terminal segment. The results obtained in this work show that the first 15 amino acid of the α-syn N-terminal segment mainly participate in the anchoring, perturbing the membrane hydrophobic region, while the peptide corresponding to 16–30 residues interacts only with the phospholipid polar headgroup, confirming that the binding affinity of the N-terminus is nonuniform.

Mitochondrial cristae remodelling is associated with disrupted OPA1 oligomerisation in the Huntington's disease R6/2 fragment model

There is evidence of an imbalance of mitochondrial fission and fusion in patients with Huntington's disease (HD) and HD animal models. Fission and fusion are important for mitochondrial homeostasis including mitochondrial DNA (mtDNA) maintenance and may be relevant for the selective striatal mtDNA depletion that we observed in the R6/2 fragment HD mouse model. We aimed to investigate the fission/fusion balance and the integrity of the mitochondrial membrane system in cortex and striatum of end-stage R6/2 mice and wild-type animals. Mitochondrial morphology was determined using electron microscopy, and transcript and protein levels of factors that play a key role in fission and fusion, including DRP1, mitofusin 1 and 2, mitofilin and OPA1, and cytochrome c and caspase 3 were assessed by RT-qPCR and immunoblotting. OPA1 oligomerisation was evaluated using blue native gels. In striatum and cortex of R6/2 mice, mitochondrial cristae morphology was abnormal. Mitofilin and the overall levels of the fission and fusion factors were unaffected; however, OPA1 oligomerisation was abnormal in striatum and cortex of R6/2 mice. Mitochondrial and cytoplasmic cytochrome c levels were similar in R6/2 and wild-type mice with no significant increase of activated caspase 3. Our results indicate that the integrity of the mitochondrial cristae is compromised in striatum and cortex of the R6/2 mice and that this is most likely caused by impaired OPA1 oligomerisation.

452 - Sciatic Nerve Ligation Causes Impairment of Mitochondria Associated with Changes in Distribution, Respiration and Cardiolipin Composition in Related Spinal Cord Neurons in Rats

Sciatic nerve irritation is often associated with disturbed Ca 2+ homeostasis in related neurons of the spinal cord. Since mitochondria substantially contribute to Ca 2+ homeostasis and little information is available we studied effects of loose sciatic nerve ligation, a chronic constriction injury (CCI), on neuronal mitochonderia of the L3-L6 region. Three groups of rats (untreated, sham operated, and ligated) were explored. For the characterization of mitochondria, specimens of the L3-L6 spinal cord region were evaluated with respect to intracellular localization using pyruvate dehydrogenase immunohistochemistry and Mitotracker Red, and the ATP producing machinery using LC-MS/MS technique for the analysis of cardiolipin and high-resolution respirometry for the measurement of oxygen consumption. Thereby, the phospholipid cardiolipin supports electron transfer within the respiratory chain as part of mitochondrial respiration and is of high impact on the physical properties of the mitochondrial membrane system. Histological analysis of spinal cord motor neurons revealed clustering of mitochondria in ipsilateral samples from ligated animals 14 d after the insult. This phenomenon was similarly evident in the respective contralateral side. The intensity of MT-Red staining was enhanced exclusively at the ipsilateral side indicating increased mitochondrial activity. CCI of the sciatic nerve caused massive changes in the composition of cardiolipin reflecting mitochondrial impairment in the early phase followed by regeneration processes as late response. Sciatic nerve CCI caused decrease in the capacity of mitochondrial ATP production that recovered within 14 d after treatment. In conclusion we provide evidence that clustering of mitochondria, already verified for the spinal cord sensory neurons after CCI, also occurs in the respective motor neurons. Further we have demonstrated transient impairment of the capacity of mitochondrial ATP production in tissue samples. Stress-dependent changes in cardiolipin composition are sensitive markers and mediators of the response process including impairment and regeneration.

Sciatic nerve ligation causes impairment of mitochondria associated with changes in distribution, respiration, and cardiolipin composition in related spinal cord neurons in rats.

Sciatic nerve irritation is often associated with disturbed Ca(2+) homeostasis in related neurons of the spinal cord. Since mitochondria substantially contribute to Ca(2+) homeostasis and little information is available, we studied the effects of loose sciatic nerve ligation, a chronic constriction injury (CCI), on neuronal mitochondria of the L3-L6 regions. Three groups of rats (untreated, sham operated, and ligated) were explored. For the characterization of mitochondria, specimens of the L3-L6 spinal cord regions were evaluated with respect to intracellular localization using pyruvate dehydrogenase immunohistochemistry and Mitotracker Red, and the ATP producing machinery by LC-MS/MS technique for the analysis of cardiolipin and high-resolution respirometry for the measurement of oxygen consumption. Therefore, the phospholipid cardiolipin supports electron transfer within the respiratory chain as part of mitochondrial respiration and is of high impact on the physical properties of the mitochondrial membrane system. Histological analysis of spinal cord motor neurons revealed clustering of mitochondria in ipsilateral samples from ligated animals 14days after the insult. This phenomenon was similarly evident in the respective contralateral side. The intensity of MT-Red staining was enhanced exclusively at the ipsilateral side, indicating increased mitochondrial activity. CCI of the sciatic nerve caused massive changes in the composition of cardiolipin reflecting mitochondrial impairment in the early phase followed by regeneration processes as late response. Sciatic nerve CCI caused decrease in the capacity of mitochondrial ATP production that recovered within 14days after treatment. In conclusion, we provide evidence that clustering of mitochondria, already verified for the spinal cord sensory neurons after CCI, also occurs in the respective motor neurons. Further we have demonstrated transient impairment of the capacity of mitochondrial ATP production in tissue samples. Stress-dependent changes in cardiolipin composition are sensitive markers and mediators of the response process including impairment and regeneration.

The Role of Lipids in Regulation of Programmed Cell Death

In the mitochondrial apoptotic pathway; a major regulator in mammalian cells death, meet pro- and anti-apoptotic Bcl-2 proteins at the mitochondrial outer membrane (MOM) and tightly regulate the fate of a cell. In case of a death sentence the membrane becomes permeable via the action of the apoptotic Bax protein and apoptotic factors like cytochrome c get released. Recently it was found, that the mitochondrial membrane system seems to play an active and crucial role with its lipids being directly involved. Under oxidative stress conditions oxidized lipids can be generated with direct implication on mitochondrial apoptosis via direct modification of membrane properties and/or lipid-protein interactions with Bcl-2 family members. We therefore devised an oxidative stress emulating MOM model system. By embedding OxPls into membranes we were able to study their impact on the recruitment of pro-apoptotic Bax protein and its membrane action. By using a biophysical approach ranging from calorimetry and fluorescence leakage to solid state NMR and labelled proteins we investigated following three key steps: i) Bax mediated pore formation in membranes containing oxidized lipids (OxPls) without the requirement of further mediator proteins such as tBid. ii) specificity of the pore formation on the type of OxPls species used and iii) concentration dependence of the membrane pore formation on OxPls and Bax concentrations. Using fluorescence leakage studies of GUVs and LUVs we found that Bax induced leakage which was sensitive to the type and concentration of OxPL in the membrane. And the leakage rate was correlated to the Bax level. Additional solid state NMR studies confirmed that the incorporation of OxPls distorts the membrane organization, which is essential for the Bax to penetrate into the membrane without a high energy penalty. Further calorimetric experiments support these NMR results.

REPROGRAMMING OF MITOCHONDRIAL ENERGY METABOLISM IN MALIGNANT NEOPLASMS

The novel ideas of fundamental role of mitochondria in the maintenance of viability of malignant cells have been reviewed. The modern state of research is considered in detail, including: mitochondrial control of the cellular redox state, sites of reactive oxygen species (ROS) production in inner mitochondrial membrane and antioxidant protection systems. Specificities of the structural-functional mitochondrial remodelling in malignant tumors, the mechanisms of the energy metabolism reprogramming, enhancement of the ROS production and adaptation to the hypoxic conditions and metabolic stress are analyzed. The available data including our research on transplanted tumors indicate that cytotoxic action of sodium dichloroacetate (the inhibitor of pyruvate dehydrogenase kinase) depends on biological properties of tumors and intensity of structural-functional mitochondrial rearrangement. Dichloroacetate turned out to be effective for sarcoma 37, but not for Lewis lung carcinoma.

Role of mitochondrial network stabilisation by a human small heat shock protein in tumour malignancy.

Previously, we found that the unconventional small human heat-shock protein HSPB11 inhibits cell death by HSP90 mediated cholesterol-rich membrane microdomain dependent activation of phosphatidylinositol-3 kinase/protein kinase B pathway and by stabilising the mitochondrial membrane systems. Also, progressive cytoplasmic expression of HSPB11 correlated with brain tumor malignancy. In the present study we investigated how cytoplasmic abundance of HSPB11 augments tumor malignancy. We up- and downregulated the cytoplasmic level of HSPB11 before paclitaxel exposure in NIH3T3 and HeLa cells, which normally express low and high levels, respectively, of the HSPB11 protein. We examined the paclitaxel-mediated induction of cell death, mitochondrial fission, HSPB11 mitochondrial translocation and inhibitory phosphorylation of dynamin-like protein-1 (DLP1). We found that increasing cytoplasmic abundance of HSPB11 in NIH3T3 cells protected against paclitaxel-induced apoptosis, while suppressing HSPB11 in HeLa cells sensitised the cells toward paclitaxel. Also, paclitaxel enhanced mitochondrial translocation of HSPB11 in wild type HeLa but not in NIH3T3 cells. More importantly, increased cytoplasmic level of HSPB11 in NIH3T3 cells enhanced the inhibitory phosphorylation of DLP1 and attenuated paclitaxel-induced mitochondrial fission. All these results suggest that increased cytoplasmic abundance of HSPB11 augments inhibitory phosphorylation of DLP1 thereby reduces mitochondrial fission that eventually leads to decreased apoptosis. This novel mechanism may explain the resistance to apoptosis and increased malignancy of HSPB11-overexpressing tumours. The clinical significance of this mechanism has already been highlighted by the finding that the kinase inhibitor tyrphostin A9 induces cancer cell death by DLP1-mediated mitochondrial fragmentation.

Role of β-hydroxybutyrate, its polymer poly-β-hydroxybutyrate and inorganic polyphosphate in mammalian health and disease.

We provide a comprehensive review of the role of β-hydroxybutyrate (β-OHB), its linear polymer poly-β-hydroxybutyrate (PHB), and inorganic polyphosphate (polyP) in mammalian health and disease. β-OHB is a metabolic intermediate that constitutes 70% of ketone bodies produced during ketosis. Although ketosis has been generally considered as an unfavorable pathological state (e.g., diabetic ketoacidosis in type-1 diabetes mellitus), it has been suggested that induction of mild hyperketonemia may have certain therapeutic benefits. β-OHB is synthesized in the liver from acetyl-CoA by β-OHB dehydrogenase and can be used as alternative energy source. Elevated levels of PHB are associated with pathological states. In humans, short-chain, complexed PHB (cPHB) is found in a wide variety of tissues and in atherosclerotic plaques. Plasma cPHB concentrations correlate strongly with atherogenic lipid profiles, and PHB tissue levels are elevated in type-1 diabetic animals. However, little is known about mechanisms of PHB action especially in the heart. In contrast to β-OHB, PHB is a water-insoluble, amphiphilic polymer that has high intrinsic viscosity and salt-solvating properties. cPHB can form non-specific ion channels in planar lipid bilayers and liposomes. PHB can form complexes with polyP and Ca2+ which increases membrane permeability. The biological roles played by polyP, a ubiquitous phosphate polymer with ATP-like bonds, have been most extensively studied in prokaryotes, however polyP has recently been linked to a variety of functions in mammalian cells, including blood coagulation, regulation of enzyme activity in cancer cells, cell proliferation, apoptosis and mitochondrial ion transport and energy metabolism. Recent evidence suggests that polyP is a potent activator of the mitochondrial permeability transition pore in cardiomyocytes and may represent a hitherto unrecognized key structural and functional component of the mitochondrial membrane system.

Ultrastructural Examination of Renal Tubular Epithelial Cells and Hepatocytes in the Course of Chronic Cyclosporin A Treatment—A Possible Link to Oxidative Stress

Cyclosporin A is an immunosuppressant drug used to prevent graft rejection in organ transplantation. Side effects include toxicity to the kidney and liver. Recently, many experimental and clinical data have demonstrated that CsA-induced toxicity is associated with increased production of reactive oxygen species (ROS) and lipid peroxidation in the kidney and liver. The aim of the study was ultrastructural examination of renal and liver epithelial cells in the course of chronic experimental CsA treatment. Rats were treated with CsA in a dose of 25 mg/kg/day. Animals developed failure of the kidney and the liver functions manifested by an increase in serum levels of creatinine, urea, uric acid, bilirubin, AST, and ALT and a decrease in total proteins. Ultrastructural examination of tubular epithelial cells and hepatocytes revealed dilatation of endoplasmic reticulum and injury to mitochondria, formation of autolysosomes, and presence of single apoptotic cells. On the basis of a review of the literature the authors suggest that mitochondrial and cell membrane system changes are, at least in part, a result of oxidative damage to the liver and renal tubular epithelial cells.

The oxidized phospholipid PazePC modulates interactions between Bax and mitochondrial membranes

Activation of the pro-apoptotic protein Bax under intracellular oxidative stress is closely related to its association with the mitochondrial outer membrane (MOM) system, ultimately resulting in cell death. The precise mechanism by which this activation and the subsequent structural changes in the protein occur is currently unknown. In addition to triggering the onset of apoptosis, oxidative stress generates oxidized lipids whose impact on mitochondrial membrane integrity and the activity of membrane-associated Bax is unclear. We therefore devised a model system that mimics oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into mitochondria-like liposomes, and studied the OxPls' impact on Bax-membrane interactions. Differential scanning calorimetry (DSC) was used to study membrane organization and protein stability, while conformational changes in the protein upon contact with lipid vesicles were monitored using far-UV circular dichroism (CD) spectroscopy. The thermograms for liposomes containing the OxPl 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) differed dramatically from those for unmodified liposomes. Moreover, Bax exhibited enhanced thermal stability in the presence of the modified liposomes, indicating that it interacted strongly with PazePC-containing membranes. The presence of PazePC also increased the α-helical character of Bax compared to the protein alone or with PazePC-free vesicles, at 10°C, 20°C, and 37°C. Presumably, the presence of PazePC-like OxPls a) increases the population of membrane-associated Bax and b) facilitates the protein's insertion into the membrane by distorting the bilayer's organization, as seen by solid-state high-resolution 1H and 31P magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy.

Induction of mitochondrial destabilization and necrotic cell death by apolar mitochondria-directed SOD mimetics

In this paper, we present evidence, for the first time, that increasing the lipophilicity of mitochondria targeting SOD mimetics reverses their cytoprotective properties, destabilizing the mitochondrial membrane system and promoting cell death. A new mitochondria-directed apolar SOD mimetic, HO-3814, was found to provoke mitochondrial swelling and loss of mitochondrial membrane potential, and these effects were not inhibited by cyclosporine A. HO-3814-induced cell death was predominantly necrotic, caspase-independent, and not affected by mitochondrial permeability transition inhibitors or cyclophilin D-suppression, inhibitors of mitogen-activated protein kinases or Akt, or various antioxidants. In contrast, Bcl-2 overexpression diminished the effects of HO-3814.

TIP47 confers resistance to taxol-induced cell death by preventing the nuclear translocation of AIF and Endonuclease G

Tail-interacting protein (TIP47, also named PP17) has been implicated in lipid droplet metabolism and in the development of late endosomes, to date however, no data about its possible role in regulating cell death processes has been available. Here, we provide evidence for the role of TIP47 in the regulation of mitochondrial membrane stability and cell death. Overexpression of TIP47 protected NIH3T3 cells from taxol-induced cell death, while suppression of TIP47 by siRNA facilitated cell death. TIP47, but not its truncated form, t-TIP47, decreased taxol-induced cell death as determined by propidium iodide and fluorescent Annexin V staining. Recombinant TIP47, but not t-TIP47, partially prevented taxol-induced depolarization of mitochondria in vitro. Overexpression of TIP47, but not its truncated form, prevented the taxol-induced nuclear and cytoplasmic translocation of AIF and Endonuclease G, as well as the taxol-induced depolarization of mitochondria in NIH3T3 cells. Furthermore, overexpression of TIP47 facilitated Bcl-2 expression and suppressed Bax expression in taxol-treated cells. These data show that besides its previously known functions, TIP47 is involved in the regulation of mitochondria-related cell death by directly stabilizing the mitochondrial membrane system and by favorably affecting the expression of Bcl-2 homologues. Since TIP47 is overexpressed in certain tumors, it is possible that TIP47 contributes to the development of cytostatic resistance.

Role of Carboxylic Groups in the Control of Nonspecific Permeability of Mitochondrial Membranes

Ionized COOH groups are present in molecular structures involved in the process of formation of mitochondrial permeability transition pores (MPTPs), in particular, in the ADP/ATP antiporter and/or voltage-dependent anion channels. In experiments on preparations of isolated mitochondria obtained from rat hepatocytes, we found that, in the case of induction of nonspecific permeability through mitochondrial membranes under the action of Cа2+ in a relatively low concentration (15 μM), modulation of the activity of COOH groups with the use of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1 mM) led to unidirectional effects, namely to acceleration of the processes of formation of MPTPs and transport of incubation solution and Са2+ through these megachannels, prolongation of the open state of the latter, as well as to increases in the final volume (swelling) of the mitochondria and to a rise in the amount of Са2+ released from these organelles. In contrast, when calcium was used in a high concentration (100 μM), the directions of the above processes were dissimilar. Slowing down of the flow of incubation solution through MPTPs and the process of their formation was observed; at the same time, Са2+ release from the mitochondria was accelerated. However, the final volume of the mitochondria and the amount of Са2+ released from these cellular structures increased. Differences between the effects of the used modulator of the activity of COOH groups on the nonspecific permeability of the mitochondria induced by calcium applied in low and high concentrations are perhaps determined by the following. The process of swelling of the mitochondria is saturable, while Са2+ release from these organelles shows an unlimited pattern. The latter process (Са2+ release) probably undergoes calcium-initiated inactivation. The mechanisms of induction of nonspecific permeability of the mitochondrial membranes under the action of low and high calcium concentrations differ from each other. The calcium uniporter in the mitochondria is sensitive to the modulator of the activity of COOH groups. Diffusion of water through the inner mitochondrial membrane and/or other systems provides some contribution to the studied processes; this can lead to changes in the transport of liquids in these organelles.

Application of atomic-force microscopy technology to a structural analysis of the mitochondrial inner membrane

For the first time, the surface of mitoplasts (mitochondria devoid of an outer membrane) was investigated by atomic-force microscopy (AFM). AFM has revealed folds on the surface of mitoplasts with thicknesses of 30–40 nm, which coincides with that of “dry cristae” in mitochondria measured with small-angle neutron scattering and electron microscopy. These results indicate the existence of a specific system maintaining the configuration of a mitoplast membrane similar to that of the inner membrane of intact mitochondria under their swelling in hypotonic conditions. The prospects of using AFM to study the configuration of the surface of the mitochondrial inner membrane and other biological membrane systems were demonstrated.

Enzymes in Energy Metabolism, Introduction

Several years ago, the bioenergetics subgroup decided to broaden the more narrow definition of bioenergetics to include all pathways involved in energy production. The importance of studying them in combination can be illustrated by the very different metabolic behavior of cancer cells that, in contrast to healthy cells, derive most of their energy from glycolysis and at the same time show a lack of apoptosis. While each presentation in this workshop will be focused on specific topics, glycolysis, the mitochondrial membrane system, the pyruvate dehydrogenase as part of the TCA cycle and the fumarate reductase as one of the enzymes in the mitochondrial respiratory chain, emphasis will be given to the interconnection between the different systems.

The Anti-apoptotic Mitochondrial Membrane Protein Bcl-2; An Achilles Heel Of Cancer Cells?

Escape from programmed cell death, apoptosis, is one of the main hallmarks of cancer.. The anti-apoptotic protein Bcl-2 belongs to the Bcl-2 protein family, which function as a major gatekeeper in the mitochondrial pathway. Bcl-2 is found to a great extent in many breast cancers and is highly involved in the inherent resistance to anti-cancer drugs. This protein is mitochondrial membrane-associated and we will use NMR spectroscopy to provide structural information of the membrane-mediated mechanism underlying the action of Bcl-2 as a potent inhibitor of cell death.For this purpose we express currently the full length protein and carry out various preliminary studies of the membrane dependent behaviour of synthesized segments of the proteins by a range of biophysical methods ranging from CD (Circular Dichroism), ATR (Attenuated Total Reflection), Calorimetry to solid state NMR spectroscopy.At present, we study the impact of the unique BH4 domain of the pro-survival Bcl-2 protein on the mitochondrial membranes, since this interaction seems to be essential to block any apoptotic activation with its connected pore-formation and cytochrome c release. Our first results reveal that the BH4 domain requires cardiolipin to be able to convert into an α-helix conformation. In contact with neutral membranes, the peptide seems to aggregate on the surface. Bcl-2's counterplayer is Bax protein which upon activation tranlocates to the mitochondrial membrane. In this process the first helix localized at the N-terminus of Bax (Bax-α1) can act as an addressing sequence, which upon activation directs the protein from the cytosol towards the mitochondrial surface. Our biophysical approach provided insight into the molecular mechanism behind the recognition of the mitochondrial membrane system by the Bax-α1 sequence.