Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC (PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one alpha-helix, 13 transmembrane beta-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis.     

A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells

The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak.     

The Voltage-Dependent Anion Channel (VDAC) of Pacific Oysters <Emphasis Type="Italic">Crassostrea gigas</Emphasis> Is Upaccumulated During Infection by the Ostreid Herpesvirus-1 (OsHV-1): an Indicator of the Warburg Effect

Voltage-dependent anion channel (VDAC) is a key mitochondrial protein. VDAC drives cellular energy metabolism by controlling the influx and efflux of metabolites and ions through the mitochondrial membrane, playing a role in its permeabilization. This protein exerts a pivotal role during the white spot syndrome virus (WSSV) infection in shrimp, through its involvement in a particular metabolism that plays in favor of the virus, the Warburg effect. The Warburg effect corresponds to an atypical metabolic shift toward an aerobic glycolysis that provides energy for rapid cell division and resistance to apoptosis. In the Pacific oyster Crassostrea gigas, the Warburg effect occurs during infection by Ostreid herpesvirus (OsHV-1). At present, the role of VDAC in the Warburg effect, OsHV-1 infection and apoptosis is unknown. Here, we developed a specific antibody directed against C. gigas VDAC. This tool allowed us to quantify the tissue-specific expression of VDAC, to detect VDAC oligomers, and to follow the amount of VDAC in oysters deployed in the field. We showed that oysters sensitive to a mortality event in the field presented an accumulation of VDAC. Finally, we propose to use VDAC quantification as a tool to measure the oyster susceptibility to OsHV-1 depending on its environment.     

Candidate tumour suppressor Fau regulates apoptosis in human cells: An essential role for Bcl-G

FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.     

Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells

In the p53-deficient human B lymphoma Namalwa cell line that quickly undergoes apoptosis after DNA topoisomerase I inhibitor (camptothecin, CPT) treatment, we observed rapid and slight induction of the pro-apoptotic BH3-only Bik, Bim-EL, Bim-L and Bim-S proteins. In contrast, the expression levels of Bad and multidomain Bax-alpha and Bak remained mostly unchanged after CPT treatment. However, multiple pro-apoptotic proteins, including Bax-alpha, Bak, Bik, Bim-EL and Bim-L, translocated rapidly to the mitochondria after CPT treatment. Gel filtration chromatography experiments demonstrated that somes of the pro-apoptotic proteins assemble themselves into high molecular weight protein complexes. The protein composition of these oligomers was further analyzed by co-immunoprecipitation experiments performed on highly purified mitochondrial fractions, which revealed the formation of Bax/Bak, Bax/VDAC1, Bak/VDAC1, Bim/VDAC1 and Bim/Bcl-2 complexes after DNA damage induction. Thus, it appeared that induction, mitochondrial translocation and assembly in multimeric protein complexes of several pro-apoptotic members of the Bcl-2 family correlated with the rapid activation of apoptosis in a p53-independent pathway after CPT-mediated DNA strand breaks.     

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.     

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.     

Ca-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca^2 + across the OMM and because Ca^2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca^2 + levels ([Ca^2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca^2 + and induce VDAC1 over-expression. Direct elevation of [Ca^2 +]i by the Ca^2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca^2 +]i using the cell-permeable Ca^2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca^2 +]i levels and apoptosis induction, as induced by H₂O₂ or As₂O₃, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca^2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca^2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca^2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau     

Voltage-dependent Anion Channel 1-based Peptides Interact with Bcl-2 to Prevent Antiapoptotic Activity

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.     

Metaxins 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery,are essential for Bak activation during TNF alpha triggered apoptosis

The proteins Bax and Bak are central in the execution phase of apoptosis; however, little is known about the partners involved in the control of this complex process. Here, we show that mitochondrial Bak is incorporated into a VDAC2/Mtx1/Mtx2 multi-protein complex in both resting and dying cells. VDAC2 is a porin that has previously been described as a partner of Bak while Mtx1 and Mtx2 are two proteins of the mitochondrial sorting and assembly machinery (SAM) that have been implicated in TNF-induced apoptosis. We show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to interact with Mtx1.     

A comparative study of the porin genes encoding VDAC, a voltage-dependent anion channel protein, in Anopheles gambiae and Drosophila melanogaster

The protein called voltage-dependent anion-selective channel (VDAC), or mitochondrial porin, forms channels that provide the major pathway for small metabolites across the mitochondrial outer membrane. We have identified and sequenced agporin, a gene of the malaria vector mosquito Anopheles gambiae that conceptually encodes a protein with 73% identity to the VDAC protein encoded by the porin gene in Drosophila melanogaster. By in situ hybridization, we have localized agporin at region 35D on the right arm of A. gambiae chromosome 3, which is homologous to the 2L chromosomal arm of D. melanogaster where the porin gene resides. The comparison of agporin with its putative Drosophila counterpart revealed that both the nucleotide sequence and the structural organization of the two genes are strikingly conserved even though the ancestral lines of A. gambiae and D. melanogaster are thought to have diverged about 250 million years ago. Our results suggest that, while in yeast, plants, and mammals, VDAC isoforms are encoded by small multigene families and are able to compensate for each other at least partially, in A. gambiae a single gene encodes the VDAC protein.     

Lysine methyltransferase Smyd2 suppresses p53-dependent cardiomyocyte apoptosis

Apoptosis, or programmed cell death, is an essential physiological process for proper embryogenesis as well as for homeostasis during aging. In addition, apoptosis is one of the major mechanisms causing cell loss in pathophysiological conditions such as heart failure. Thus, inhibition of apoptosis is an important approach for preventive and therapeutic strategies. Here we show that the histone 3 lysine 4- and lysine 36-specific methyltransferase Smyd2 acts as an endogenous antagonistic player of p53-dependent cardiomyocyte apoptosis. Smyd2 protein levels were significantly decreased in cardiomyocytes upon cobalt chloride-induced apoptosis or myocardial infarction, while p53 expression was enhanced. siRNA-mediated knockdown of Smyd2 in cultured cardiomyocytes further enhanced cobalt chloride-induced cardiomyocyte apoptosis. In contrast, Smyd2 overexpression resulted in marked methylation of p53 and prevented its accumulation as well as apoptotic cell death in an Hsp90-independent manner. Moreover, overexpression, of Smyd2, but not Smyd2Y240F lacking a methyl transferase activity, significantly rescued CoCl₂-induced apoptosis in H9c2 cardioblasts. Finally, Smyd2 cardiomyocyte-specific deletion in vivo promoted apoptotic cell death upon myocardial infarction, which correlated with enhanced expression of p53 and pro-apoptotic Bax. Collectively, our data indicate Smyd2 as a cardioprotective protein by methylating p53.     

Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.     

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9–13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3′ untranslated sequence (3′-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5′-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5′-UTR sequence, demonstrated to be transcribed by 5′-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2–1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.