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Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity. Ceramides are lipids that act directly on mitochondria to trigger apoptosis, but the underlying mechanism remains largely unclear. Here authors use a photoactivatable ceramide probe combined with a computation approach and functional studies to identify the voltage-dependent anion channel VDAC2 as a direct effector of ceramide-mediated cell death.

Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type I interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.

Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Erastin, the ferroptosis activator, binds to voltage-dependent anion channels VDAC2 and VDCA3, but treatment with erastin can result in the degradation of the channels. Here, the authors show that Nedd4 is induced following erastin treatment, which leads to the ubiquitination and subsequent degradation of the channels. Depletion of Nedd4 limits the protein degradation of VDAC2/3, which increases the sensitivity of cancer cells to erastin. By understanding the molecular mechanism of erastin-induced cellular resistance, we can discover how cells adapt to new molecules to maintain homeostasis. Furthermore, erastin-induced resistance mediated by FOXM1-Nedd4-VDAC2/3 negative feedback loop provides an initial framework for creating avenues to overcome the drug resistance of ferroptosis activators. Erastin, the ferroptosis activator, binds to voltage gated ion channels CDAC2 and VDCA3 but treatment with erastin can result in the degradation of the channels. Here, the authors show that Nedd4 is induced following erastin treatment, which leads to the ubiquitination and subsequent degradation of the channels.

Mitochondria are critically involved in necrotic cell death induced by Ca 2+ overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore — a protein complex that spans both the outer and inner mitochondrial membranes — is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix 1 , 2 , 3 . Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1 -, Vdac3 -, and Vdac1 – Vdac3 -null mice exhibited a Ca 2+ - and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca 2+ - and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1–Vdac3 and Vdac1–Vdac2–Vdac3. Wild-type and Vdac -deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.

The current molecular docking study provided the Free Energy of Binding (FEB) for the interaction (nanotoxicity) between VDAC mitochondrial channels of three species (VDAC1- Mus musculus , VDAC1 -Homo sapiens , VDAC2 -Danio rerio ) with SWCNT-H, SWCNT-OH, SWCNT-COOH carbon nanotubes. The general results showed that the FEB values were statistically more negative (p < 0.05) in the following order : ( SWCNT-VDAC2- Danio rerio ) > (SWCNT-VDAC1- Mus musculus ) > (SWCNT-VDAC1- Homo sapiens ) > (ATP-VDAC). More negative FEB values for SWCNT-COOH and OH were found in VDAC2- Danio rerio when compared with VDAC1- Mus musculus and VDAC1 -Homo sapiens (p < 0.05). In addition, a significant correlation (0.66 > r 2  > 0.97) was observed between n -Hamada index and VDAC nanotoxicity (or FEB) for the zigzag topologies of SWCNT-COOH and SWCNT-OH. Predictive Nanoparticles-Quantitative-Structure Binding-Relationship models (nano-QSBR) for strong and weak SWCNT-VDAC docking interactions were performed using Perturbation Theory, regression and classification models. Thus, 405 SWCNT-VDAC interactions were predicted using a nano-PT-QSBR classifications model with high accuracy, specificity, and sensitivity (73–98%) in training and validation series, and a maximum AUROC value of 0.978. In addition, the best regression model was obtained with Random Forest (R 2 of 0.833, RMSE of 0.0844), suggesting an excellent potential to predict SWCNT-VDAC channel nanotoxicity. All study data are available at https://doi.org/10.6084/m9.figshare.4802320.v2 .

Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse. Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV. IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

The voltage-dependent anion-selective channel (VDAC) plays a crucial role in mitochondrial function, and VDAC paralogs are considered to ensure the differential integration of mitochondrial functions with cellular activities. Heterologous expression of VDAC paralogs in the yeast Saccharomyces cerevisiae por1Δ mutant cells is often employed in studies of functional differentiation of human VDAC paralogs (hVDAC1-hVDAC3) regardless of the presence of the yeast second VDAC paralog (yVDAC2) encoded by the POR2 gene. Here, we applied por1Δpor2Δ double mutants and relevant por1Δ and por2Δ single mutants, derived from two S. cerevisiae strains (M3 and BY4741) differing distinctly in auxotrophic markers but commonly used for heterologous expression of hVDAC paralogs, to study the effect of the presence of yVDAC2 and cell genotypes including MET15, the latter resulting in a low level of hydrogen sulfide (H2S), on the complementation potential of heterologous expression of hVDAC paralogs. The results indicated that yVDAC2 might contribute to the complementation potential. Moreover, the possibility to reverse the growth phenotype through heterologous expression of hVDAC paralogs in the presence of the applied yeast cell genotype backgrounds was particularly diverse for hVDAC3 and depended on the presence of the protein cysteine residues and expression of MET15. Thus, the difference in the set of auxotrophic markers in yeast cells, including MET15 contributing to the H2S level, may create a different background for the modification of cysteine residues in hVDAC3 and thus explain the different effects of the presence and deletion of cysteine residues in hVDAC3 in M3-Δpor1Δpor2 and BY4741-Δpor1Δpor2 cells. The different phenotypes displayed by BY4741-Δpor1Δpor2 and M3-Δpor1Δpor2 cells following heterologous expression of a particular hVDAC paralog make them valuable models for the study of human VDAC proteins, especially hVDAC3, as a representative of VDAC protein sensitive to the reduction–oxidation state.

Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of VDACs in plants is largely unknown. In order to determine the individual function of plant VDACs, molecular and genetic analysis was performed on four VDAC genes, VDAC1–VDAC4, found in Arabidopsis thaliana. VDAC1 and VDAC3 possess the eukaryotic mitochondrial porin signature (MPS) in their C-termini, while VDAC2 and VDAC4 do not. Localization analysis of VDAC–green fluorescent protein (GFP) fusions and their chimeric or mutated derivatives revealed that the MPS sequence is important for mitochondrial localization. Through the functional analysis of vdac knockout mutants due to T-DNA insertion, VDAC2 and VDAC4 which are expressed in the whole plant body are important for various physiological functions such as leaf development, the steady state of the mitochondrial membrane potential, and pollen development. Moreover, it was demonstrated that VDAC1 is not only necessary for normal growth but also important for disease resistance through regulation of hydrogen peroxide generation.

The voltage-dependent anion channel (VDAC) is the main gateway for metabolites across the mitochondrial outer membrane. VDAC oligomers are connected to apoptosis induced by various stimuli. However, the mechanistic and structural basis of apoptosis induction by VDAC remains poorly understood. Here, using cryo-EM and NMR we show that VDAC1 oligomerization or confinement in small lipid nanodiscs triggers the exposure of its N-terminal α-helix (VDAC1-N) which becomes available for partner protein binding. NMR and X-ray crystallography data show that VDAC1-N forms a complex with the BH3 binding groove of the anti-apoptotic Bcl2 protein BclxL. Biochemical assays demonstrate that VDAC1-N exhibits a pro-apoptotic function by promoting pore formation of the executor Bcl2 protein Bak via neutralization of BclxL. This mechanism is reminiscent of BH3-only sensitizer Bcl2 proteins that are efficient inducers of Bax/Bak-mediated mitochondrial outer membrane permeabilization and ultimately apoptosis. The VDAC pathway most likely responds to mitochondrial stress or damage. TGF-β is a latent complex (L-TGF-β). Latency is conferred by a homodimeric prodomain with a previously undefined domain architecture. Here we define the architecture of the prodomain as domain-swapped providing structural insights into the mechanism of activation of L-TGF-β.