Simona Reina

Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis

Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-XL, indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

VDAC isoforms in mammals

VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Characterization of human VDAC isoforms: A peculiar function for VDAC3?

VDACs are a family of pore-forming proteins mainly located in the mitochondrial outer membrane. In mammals three isoforms exist. In this work we review the information available about them with the addition of new results. We have compared the human VDACs transformed in a yeast strain lacking the endogenous porin. VDAC1 and 2 are able to complement the lack of porin in mitochondrial respiration and modulation of ROS. VDAC3 has a limited ability to support the mitochondrial respiration and has no influence in the control of ROS production. The over-expression of VDAC isoforms in wild type yeast strain led to a dramatic sensitivity to oxidative stress, especially for VDAC3, and a shorter lifespan in respiratory conditions. Real-time PCR comparison of the isoforms indicated that in HeLa cells VDAC1 is 10 times more abundant than VDAC2 and 100 times than VDAC3. The over-expression of any single isoform caused a 10 times increase of the transcripts of VDAC2 and VDAC3, while VDAC1 is not changed by the over-expression of the other isoforms. Models of VDAC2 and VDAC3 isoform structure showed that they could be made of a 19-strand beta-barrel and an N-terminal sequence with variable features. In this work we show for the first time a functional characterization of VDAC3 in a cellular context.

The Voltage-Dependent Anion Selective Channel 1 (VDAC1) Topography in the Mitochondrial Outer Membrane as Detected in Intact Cell

Voltage-Dependent Anion selective Channel maintains the permeability of the outer mitochondrial membrane and is relevant in bioenergetic metabolism and apoptosis. The structure of the protein was shown to be a β-barrel formed by 19 strands. The topology or sideness of the pore has been predicted with various approaches but a general consensus was never reached. This is an important issue since VDAC is considered receptor of Hexokinase and Bcl-2. We fused at VDAC1 C-terminus two tags separated by a caspase cleavage site. Activation in cellulo of caspases was used to eventually separate the two reporters. This experiment did not require the isolation of mitochondria and limited the possibility of outer membrane rupture due to similar procedures. Our results show that the C-terminus end of VDAC faces the mitochondrial inter-membrane space.

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage‐dependent anion‐selective channels (VDACs) are pore‐forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin‐1. We swapped the VDAC3 N‐terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N‐terminus with the VDAC1 N‐terminus caused the chimaera to become more active than VDAC1. The VDAC2 N‐terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N‐terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Recombinant Human Voltage Dependent Anion Selective Channel Isoform 3 (hVDAC3) Forms Pores with a Very Small Conductance

Background/Aims: Voltage-dependent anion channels (VDAC), also known as eukaryotic porins, are located in the outer mitochondrial membrane and allow the flux of ions and small metabolites. While the pore-forming ability of recombinant VDAC1 and VDAC2 has been extensively studied during the last decades, a clear-cut ion conducting channel activity has not been assigned to the VDAC3 isoform. Methods: Electrophysiological characterization of the recombinant protein purified and refolded was obtained after incorporation into planar lipid bilayers. Results: Here we report for the first time that recombinant hVDAC3, upon expression in E.coli and purification-refolding, shows a channel activity with a very small conductance (90 pS in 1 M KCl) with respect to the conductance of hVDAC1 (>3500 pS in 1 M KCl). Purified hVDAC3 allowed the passage of both chloride and gluconate anions and did not distinguish between potassium, sodium and calcium used as cations. In contrast to VDAC1, the channel was active also at transmembrane voltages higher than +/-40 mV and displayed a relatively high open probability even at +/-80 mV. hVDAC3 was only slightly voltage-dependent, displaying a tendency to adopt lower-conductance states at positive voltages applied to the cis chamber. In accordance with the small conductance of the pore, expression of hVDAC3 in a porin-less yeast strain allowed only partial recovery of the growth under non-permissive conditions. Conclusion: The observed electrophysiological properties of hVDAC3 are surprisingly different from the other isoforms and are discussed in relation to the proposed physiological role of the protein in mammalian cells.

Geographically widespread swordfish barcode stock identification: a case study of its application.

Background The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species. Methodology We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5′ hyper-variable fragment of the control region (5′dloop), were also used to validate the efficacy of COI as a stock-specific marker. Case Report This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products. Conclusions The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5′dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.

Deletion of β-strands 9 and 10 converts VDAC1 voltage dependence in an asymmetrical process

Voltage-dependent anion selective channel isoform1 maintains the permeability of the outer mitochondrial membrane. Its voltage-gating properties are relevant in bioenergetic metabolism and apoptosis. The N-terminal domain is suspected to be involved in voltage-gating, due to its peculiar localization. However this issue is still controversial. In this work we exchanged or deleted the β-strands that take contact with the N-terminal domain. The exchange of the whole hVDAC1 β-barrel with the homologous hVDAC3 β-barrel produces a chimeric protein that, in reconstituted systems, loses completely voltage-dependence. hVDAC3 β-barrel has most residues in common with hVDAC1, including V143 and L150 considered anchor points for the N-terminus. hVDAC1 mutants completely lacking either the β-strand 9 or both β-strands 9 and 10 were expressed, refolded and reconstituted in artificial bilayers. The mutants formed smaller pores. Molecular dynamics simulations of the mutant structure supported its ability to form smaller pores. The mutant lacking both β-strands 9 and 10 showed a new voltage-dependence feature resulting in a fully asymmetric behavior. These data indicate that a network of β-strands in the pore-walls, and not single residues, are required for voltage-gating in addition to the N-terminus.

Hexokinase I N-terminal based peptide prevents the VDAC1-SOD1 G93A interaction and re-establishes ALS cell viability.

Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies.