Voltage-dependent anion channel (VDAC) as mitochondrial governator—Thinking outside the box

Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease.     

Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect

Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Role of voltage-dependent anion channels of the mitochondrial outer membrane in regulation of cell metabolism

The role of voltage-dependent anion channels (VDAC/porins) of the mitochondrial outer membrane in the regulation of cell metabolism is assessed using an experimental model of ethanol toxicity in cultured hepatocytes. It is demonstrated that ethanol inhibits the phosphorylating and the uncoupled mitochondrial respiration, decreases the accessibility of mitochondrial adenylate kinase in the intermembrane space, and suppresses ureagenic respiration in the cells. Treatment with digitonin at high concentrations (>80 μM)—which creates pores in the mitochondrial outer membrane, allowing bypass of closed VDAC—restores all the processes suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial function because of closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space. Our studies also reveal the role of VDAC in the regulation of liver-specific intracellular processes such as ureagenesis. The data obtained can be used in development of pharmaceuticals that would prevent VDAC closure in mitochondria of ethanol-oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.     

The role of the voltage-dependent anion channels in the outer membrane of mitochondria in the regulation of cellular metabolism

The role of the voltage-dependent anion channels (VDAC) harbored in the outer membrane of mitochondria in the regulation of cellular metabolism was investigated using an experimental model of ethanol toxicity in cultured hepatocytes. It was demonstrated that ethanol inhibits State 3 and uncoupled mitochondrial respirations, decreases the accessibility of mitochondrial adenylate kinase localized in the intermembrane space of mitochondria, and suppresses ureagenic respiration and synthesis of urea in cultured hepatocytes. Increasing the permeability of the outer mitochondrial membrane with closed VDAC with high concentrations of digitonin (> 80 microM), which creates pores in the membrane, allowing the alternative bypass of closed VDAC, and restores all reactions suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial functions due to the closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space of mitochondria. Our studies demonstrated that ethanol affects the main mitochondrial functions and revealed the role of VDAC channels in the outer mitochondrial membrane in the regulation of liver specific intracellular processes such as ureagenesis. The data obtained can be used for the development of pharmaceutical drugs that prevent the closure of VDAC in mitochondria of ethanol oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.     

Regulation of respiration in muscle cells in vivo by VDAC through interaction with the cytoskeleton and MtCK within Mitochondrial Interactosome

This review describes the recent experimental data on the importance of the VDAC-cytoskeleton interactions in determining the mechanisms of energy and metabolite transfer between mitochondria and cytoplasm in cardiac cells. In the intermembrane space mitochondrial creatine kinase connects VDAC with adenine nucleotide translocase and ATP synthase complex, on the cytoplasmic side VDAC is linked to cytoskeletal proteins. Applying immunofluorescent imaging and Western blot analysis we have shown that β2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αβ-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the β-tubulin subunit is bound on the cytoplasmic side and α-tubulin C-terminal tail is inserted into VDAC. Other cytoskeletal proteins, such as plectin and desmin may be involved in this process. The result of VDAC-cytoskeletal interactions is selective restriction of the channel permeability for adenine nucleotides but not for creatine or phosphocreatine that favors energy transfer via the phosphocreatine pathway. In some types of cancer cells these interactions are altered favoring the hexokinase binding and thus explaining the Warburg effect of increased glycolytic lactate production in these cells. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

The role of the mitochondrial outer membrane in energy metabolism of tumor cells

The outer mitochondrial membrane contains a pore structure which is composed of a 30,000 Da protein, porin. The pore has an internal diameter of 2 nm and exhibits a molecular-sieving exclusion limit between 3000 and 6000 Da. These pores, therefore, provide the exit/entrance port for metabolites moving between mitochondria and the cytosol. Hexokinase binds to porin on the outer surface of mitochondria. The location of hexokinase has evoked a number of theories in which bound hexokinase is given a central role in regulating glycolysis, and, perhaps, the metabolic communication between oxidative and glycolytic metabolism. This is of particular importance in rapidly growing tumor cells in which the aerobic production of lactate and hexokinase activity are highly induced. In the present paper, we summarize the suggested roles of the outer membrane and bound hexokinase in regulation glycolysis of tumor cells. Experiments attempting to elucidate the role of hexokinase binding in the regulation of tumor cell metabolism are presented.     

Regulation of hexokinase binding to VDAC

Hexokinase isoforms I and II bind to mitochondrial outer membranes in large part by interacting with the outer membrane voltage-dependent anion channel (VDAC). This interaction results in a shift in the susceptibility of mitochondria to pro-apoptotic signals that are mediated through Bcl2-family proteins. The upregulation of hexokinase II expression in tumor cells is thought to provide both a metabolic benefit and an apoptosis suppressive capacity that gives the cell a growth advantage and increases its resistance to chemotherapy. However, the mechanisms responsible for the anti-apoptotic effect of hexokinase binding and its regulation remain poorly understood. We hypothesize that hexokinase competes with Bcl2 family proteins for binding to VDAC to influence the balance of pro-and anti-apoptotic proteins that control outer membrane permeabilization. Hexokinase binding to VDAC is regulated by protein kinases, notably glycogen synthase kinase (GSK)-3β and protein kinase C (PKC)-ɛ. In addition, there is evidence that the cholesterol content of the mitochondrial membranes may contribute to the regulation of hexokinase binding. At the same time, VDAC associated proteins are critically involved in the regulation of cholesterol uptake. A better characterization of these regulatory processes is required to elucidate the role of hexokinases in normal tissue function and to apply these insights for optimizing cancer treatment.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris)

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S–13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.     

Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.

Proapoptotic proteins such as Bax, undergo translocation to the mitochondria during apoptosis, where they mediate the release of intermembrane space proteins including cytochrome c. Bax binds to the voltage-dependent anion channel (VDAC). VDAC is a beta-barrel protein located in the outer mitochondrial membrane. In planar lipid bilayers, Bax and VDAC form a channel through which cytochrome c can pass. Hexokinase II (HXK II) also binds to VDAC. HXK II catalyzes the first step of glycolysis and is highly expressed in transformed cells, where over 70% of it is bound to the mitochondria. The present study demonstrates that HXK II interferes with the ability of Bax to bind to mitochondria and release cytochrome c. Detachment of HXK II from the mitochondria-enriched fraction isolated from HeLa cells promoted the binding of recombinant Bax-Delta19 and subsequent cytochrome c release. Similarly, the addition of recombinant HXK II to the mitochondria-enriched fraction isolated from hepatocytes, cells that do not express HXK II endogenously, prevented the ability of recombinant Bax-Delta19 to bind to the mitochondria and promote cytochrome c release. Similar results were found in intact cells, in which the detachment of mitochondrial bound HXK II or its overexpression potentiated and inhibited, respectively, Bax-induced mitochondrial dysfunction and cell death.     

VDAC electronics: 5. Mechanism and computational model of hexokinase-dependent generation of the outer membrane potential in brain mitochondria

Glycolysis plays a key role in brain energy metabolism. The initial and rate-limiting step of brain glycolysis is catalyzed mainly by hexokinase I (HKI), the majority of which is bound to the mitochondrial outer membrane (MOM), mostly through the mitochondrial inter-membrane contact sites formed by the voltage-dependent anion channel (VDAC, outer membrane) and the adenine nucleotide translocator (ANT, inner membrane). Earlier, we proposed a mechanism for the generation of the mitochondrial outer membrane potential (OMP) as a result of partial application of the inner membrane potential (IMP) to MOM through the electrogenic ANT-VDAC-HK inter-membrane contact sites. According to this previous mechanism, the Gibbs free energy of the hexokinase reaction might modulate the generated OMP (Lemeshko, Biophys. J., 2002). In the present work, a new computational model was developed to perform thermodynamic estimations of the proposed mechanism of IMP-HKI-mediated generation of OMP. The calculated OMP was high enough to electrically regulate MOM permeability for negatively charged metabolites through free, unbound VDACs in MOM. On the other hand, the positive-inside polarity of OMP generated by the IMP-HKI-mediated mechanism is expected to protect mitochondria against elevated concentrations of cytosolic Ca²⁺. This computational analysis suggests that metabolically-dependent generation of OMP in the brain mitochondria, controlled by many factors that modulate VDAC1-HKI interaction, VDAC's voltage-gating properties and permeability, might represent one of the physiological mechanisms of regulation of the brain energy metabolism and of neuronal death resistance, and might also be involved in various neurodegenerative disorders, such as Alzheimer's disease.     

The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis

The outer mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexokinase and Bax. Cytochrome c in the creatine kinase complex will be differently organised, not accessible to Bax and the ANT is run as anti-porter by the active creatine kinase octamer. However, when, for example, free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax-dependent cytochrome c release and risk of permeability transition pore opening.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.     

Structure–function relationships in feedback regulation of energy fluxes in vivo in health and disease: Mitochondrial Interactosome

The aim of this review is to analyze the results of experimental research of mechanisms of regulation of mitochondrial respiration in cardiac and skeletal muscle cells in vivo obtained by using the permeabilized cell technique. Such an analysis in the framework of Molecular Systems Bioenergetics shows that the mechanisms of regulation of energy fluxes depend on the structural organization of the cells and interaction of mitochondria with cytoskeletal elements. Two types of cells of cardiac phenotype with very different structures were analyzed: adult cardiomyocytes and continuously dividing cancerous HL-1 cells. In cardiomyocytes mitochondria are arranged very regularly, and show rapid configuration changes of inner membrane but no fusion or fission, diffusion of ADP and ATP is restricted mostly at the level of mitochondrial outer membrane due to an interaction of heterodimeric tubulin with voltage dependent anion channel, VDAC. VDAC with associated tubulin forms a supercomplex, Mitochondrial Interactosome, with mitochondrial creatine kinase, MtCK, which is structurally and functionally coupled to ATP synthasome. Due to selectively limited permeability of VDAC for adenine nucleotides, mitochondrial respiration rate depends almost linearly upon the changes of cytoplasmic ADP concentration in their physiological range. Functional coupling of MtCK with ATP synthasome amplifies this signal by recycling adenine nucleotides in mitochondria coupled to effective phosphocreatine synthesis. In cancerous HL-1 cells this complex is significantly modified: tubulin is replaced by hexokinase and MtCK is lacking, resulting in direct utilization of mitochondrial ATP for glycolytic lactate production and in this way contributing in the mechanism of the Warburg effect. Systemic analysis of changes in the integrated system of energy metabolism is also helpful for better understanding of pathogenesis of many other diseases.     

Negative regulation of mitochondrial VDAC channels by C-Raf kinase

Background

Growth of cancer cells results from the disturbance of positive and negative growth control mechanisms and the prolonged survival of these genetically altered cells due to the failure of cellular suicide programs. Genetic and biochemical approaches have identified Raf family serine/threonine kinases B-Raf and C-Raf as major mediators of cell survival. C-Raf cooperates with Bcl-2/Bcl-X_L in suppression of apoptosis by a mechanism that involves targeting of C-Raf to the outer mitochondrial membrane and inactivation of the pro-apoptotic protein Bad. However, apoptosis suppression by C-Raf also occurs in cells lacking expression of Bad or Bcl-2.

Results

Here we show that even in the absence of Bcl-2/Bcl-X_L, mitochondria-targeted C-Raf inhibits cytochrome c release and caspase activation induced by growth factor withdrawal. To clarify the mechanism of Bcl-2 independent survival control by C-Raf at the mitochondria a search for novel mitochondrial targets was undertaken that identified voltage-dependent anion channel (VDAC), a mitochondrial protein (porin) involved in exchange of metabolites for oxidative phosphorylation. C-Raf forms a complex with VDAC in vivo and blocks reconstitution of VDAC channels in planar bilayer membranes in vitro.

Conclusion

We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release from mitochondria in growth factor starved cells. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells.     

Targeting mitochondria as a therapeutic target in cancer

Knowledge of re-programming in cancer cells with metabolic differences from their normal counterparts has resulted in new examination of therapeutic approaches. Several studies of the role of tumor mitochondria in cancer have led to the development of non-genotoxic therapies which target mitochondrial proteins, function. The now well-established functions of mitochondria in apoptosis provide novel targets for tumor cell suicide. Mitochondria serve as a central hub for responses to cellular stress as well as injury. The alterations in cancer cells which result in protection from apoptosis can be targeted to inhibit proliferation. Because of the reprogramming of cancer cell metabolism involving increased glycolysis, it appears that blocking InsP(3)R Ca(2+) release or adaptive pathways in response to hypoxia by targeting HIF-1 or metabolic enzymes encoded by the HIF-1 gene represents a feasible therapeutic approach to cancer. A very early in vitro event found in tumor cells following resveratrol addition is an increase in intracellular Ca(2+), measurable within seconds. Ca(2+) release is also observed with non-toxic flavonoids and a goal to identify the sentinel targets of resveratrol as a model compound involved in calcium activation seems worthwhile. New findings of the relationship between autophagy and apoptosis are discussed. The contribution of reactive oxygen species (ROS) generated by mitochondria is also considered. New data as to how cyclophilins and VDAC are involved in mitochondrial hexokinase protection of factors that induce apoptosis are reviewed. In addition, chemotherapeutic approaches based on Akt-activated mTORC1 are described, and their relationship to the role of aerobic glycolysis in this protection.     

Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin

Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria.     

重组荞麦胰蛋白酶抑制剂通过下调己糖激酶Ⅱ抑制HepG2细胞生长

糖酵解过度活跃是肿瘤细胞能量代谢的显著特征。抑制过度糖酵解已经成为一种新的癌症疗法。重组荞麦胰蛋白酶抑制剂 (recombinant buckwheat trypsin inhibitor, rBTI)可以通过上调磷酸酶及张力蛋白同源基因 (PTEN) 进而抑制HepG2细胞增殖。有关rBTI对肿瘤细胞能量代谢的影响仍未见报道。本研究中的MTT和ATP检测分析表明,rBTI以剂量依赖性方式抑制细胞活力及胞内ATP含量。qRT-PCR和Western印迹分析表明,rBTI处理HepG2细胞后,己糖激酶Ⅱ转录显著下调,但是糖酵解过程中的其他酶及葡萄糖转运蛋白基因在转录水平未发生显著变化,同时己糖激酶Ⅱ蛋白水平的表达也显著下调。酶活性分析也表明,rBTI能显著降低己糖激酶的活性。进一步分析表明, rBTI使细胞内PTEN转录及表达水平明显上调,己糖激酶Ⅱ转录和p-AKT,p-mTOR、己糖激酶Ⅱ的表达下调。当PTEN抑制剂phen存在时,可阻断rBTI诱导的己糖激酶 Ⅱ表达下降,表明rBTI能通过上调PTEN进而影响己糖激酶Ⅱ的表达。免疫荧光及Western印迹分析显示,rBTI作用后减弱了己糖激酶 Ⅱ在线粒体的定位,导致己糖激酶Ⅱ与线粒体电压依赖性阴离子通道蛋白 (voltage-dependent anion channel, VDAC) 分离,促使己糖激酶Ⅱ从线粒体转位到细胞质,降低糖酵解的效率。上述结果证明,rBTI对肿瘤细胞能量代谢的调控作用主要通过抑制PI3K/AKT信号通路,下调己糖激酶Ⅱ的表达并影响空间定位,进而抑制肿瘤细胞糖酵解过程,导致癌细胞生长受到抑制。     

VDAC and peripheral channelling complexes in health and disease

VDAC changes its structure either voltage dependent in artificial membranes or physiologically by interaction with the c conformation of the adenine nucleotide translocator (ANT). This interaction creates contact sites and leads to a specific organisation of cytochrome c in the VDAC ANT complexes. The VDAC structure specific for contact sites thus generates a signal at the surface for several proteins in the cytosol to bind with high affinity such as hexokinase, glycerolkinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC ANT complex has two critical qualities: firstly, external Bax gets access to the cytochrome c and secondly the ANT stays in the c conformation that easily changes the structure to an unspecific uni-porter causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as specific anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly hinders direct interaction between VDAC and ANT and secondly changes porin structure into low affinity for hexokinase and external Bax. Cytochrome c in the creatine kinase complex will be differently organised not accessible to Bax and the ANT is run as anti-porter by the active octamer. However, when free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax dependent cytochrome c release and risk of permeability transition pore opening.