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In the last few years, many efforts have been devoted to the recovery and valorization of olive oil by-products because of their potentially high biological value. The olive mill wastewater (OMWW), a dark-green brown colored liquid that mainly consists of olive fruit vegetation water, is particularly exploited in this regard for its great content in phenolic compounds with strong antioxidant properties. In our previous work, we produced different OMWW fractions enriched in hydroxytyrosol- and hydroxytyrosol/oleuropein (i.e. C and OPE extracts, respectively) that exhibited considerable anti-microbial and radical-scavenging activities in vitro. Based on these findings, the present study aimed to assess the impact of C and OPE samples on mitochondrial function and oxidative stress response in mouse fibroblast-like cells (NCTC). Accordingly, OMWW phenolic extracts proved to enhance mitochondrial biogenesis and to reduce cellular sensitivity to hydrogen peroxide. Moreover, high-resolution respirometry experiments first time revealed the efficiency of OMWW phenols recovered by selective resin extraction in preventing mitochondrial respiration failure upon oxidative insult. Collected data definitely demonstrate the bioactivity of our phenolic-rich fractions, supporting the advantages of reusing the olive mill wastewater to generate, at low-cost, high added value molecules that could be useful for the improvement of health and nutrition products., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships. that could have appeared to influence the work reported in this article., (© 2024 The Authors. Published by Elsevier Ltd.)

One of the most robust approaches to the prediction of causal driver genes of complex diseases is to apply reverse engineering methods to infer a gene regulatory network (GRN) from gene expression profiles (GEPs). In this work, we analysed 794 GEPs of 1117 human whole-blood samples from Amyotrophic Lateral Sclerosis (ALS) patients and healthy subjects reported in the GSE112681 dataset. GRNs for ALS and healthy individuals were reconstructed by ARACNe-AP (Algorithm for the Reconstruction of Accurate Cellular Networks - Adaptive Partitioning). In order to examine phenotypic differences in the ALS population surveyed, several datasets were built by arranging GEPs according to sex, spinal or bulbar onset, and survival time. The designed reverse engineering methodology identified a significant number of potential ALS-promoting mechanisms and putative transcriptional biomarkers that were previously unknown. In particular, the characterization of ALS phenotypic networks by pathway enrichment analysis has identified a gender-specific disease signature, namely network activation related to the radiation damage response, reported in the networks of bulbar and female ALS patients. Also, focusing on a smaller interaction network, we selected some hub genes to investigate their inferred pathological and healthy subnetworks. The inferred GRNs revealed the interconnection of the four selected hub genes ( TP53, SOD1, ALS2, VDAC3 ) with p53-mediated pathways, suggesting the potential neurovascular response to ALS neuroinflammation. In addition to being well consistent with literature data, our results provide a novel integrated view of ALS transcriptional regulators, expanding information on the possible mechanisms underlying ALS and also offering important insights for diagnostic purposes and for developing possible therapies for a disease yet incurable., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Male infertility affects nearly 50% of infertile couples, with various underlying causes, including endocrine disorders, testicular defects, and environmental factors. Spermatozoa rely on mitochondrial oxidative metabolism for motility and fertilization, with mitochondria playing a crucial role in sperm energy production, calcium regulation, and redox balance. Voltage-dependent anion channels (VDACs), located on the outer mitochondrial membrane, regulate energy and metabolite exchange, which are essential for sperm function. This review offers an updated analysis of VDACs in the male reproductive system, summarizing recent advances in understanding their expression patterns, molecular functions, and regulatory mechanisms. Although VDACs have been widely studied in other tissues, their specific roles in male reproductive physiology still remain underexplored. Special attention is given to the involvement of VDAC2/3 isoforms, which may influence mitochondrial function in sperm cells and could be implicated in male fertility disorders. This update provides a comprehensive framework for future research in reproductive biology, underscoring the significance of VDACs as a molecular link between mitochondrial function and male fertility.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

The voltage-dependent anion-selective channel isoform 1 (VDAC1) is a pivotal component in cellular metabolism and apoptosis with a prominent role in many cancer types, offering a unique therapeutic intervention point. Through an in-silico -to- in-vitro approach we identified a set of VA molecules (VDAC Antagonists) that selectively bind to VDAC1 and display specificity toward cancer cells. Biochemical characterization showed that VA molecules can directly interact with VDAC1 with micromolar affinity by competing with the endogenous ligand NADH for a partially shared binding site. NADH displacement results in mitochondrial distress and reduced cell proliferation, especially when compared to non-cancerous cells. Experiments performed on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules with lower impact on healthy cells than conventional treatments like gemcitabine. VA molecules are chemical entities representing promising candidates for further optimization and development as cancer therapy strategies through precise metabolic interventions., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD + /NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis., (© 2024. The Author(s).)

COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the world. The viral positive-sense single-stranded RNA encodes 31 proteins among which the spike (S) is undoubtedly the best known. Recently, protein E has been reputed as a potential pharmacological target as well. It is essential for the assembly and release of the virions in the cell. Literature describes protein E as a voltage-dependent channel with preference towards monovalent cations whose intracellular expression, though, alters Ca 2+ homeostasis and promotes the activation of the proinflammatory cascades. Due to the extremely high sequence identity of SARS-CoV-2 protein E (E-2) with the previously characterized E-1 (i.e., protein E from SARS-CoV) many data obtained for E-1 were simply adapted to the other. Recent solid state NMR structure revealed that the transmembrane domain (TMD) of E-2 self-assembles into a homo-pentamer, albeit the oligomeric status has not been validated with the full-length protein. Prompted by the lack of a common agreement on the proper structural and functional features of E-2, we investigated the specific mechanism/s of pore-gating and the detailed molecular structure of the most cryptic protein of SARS-CoV-2 by means of MD simulations of the E-2 structure and by expressing, refolding and analyzing the electrophysiological activity of the transmembrane moiety of the protein E-2, in its full length. Our results show a clear agreement between experimental and predictive studies and foresee a mechanism of activity based on Ca 2+ affinity., Competing Interests: SR, VD were members by We.MitoBiotech S.R.L. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cubisino, Milenkovic, Conti-Nibali, Musso, Bonacci, De Pinto, Ceccarelli and Reina.)

Background: Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered., Results: We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML)., Conclusions: Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene., (© 2023. The Author(s).)

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.

Olive mill wastewater, a by-product of the olive oil industry, represents an important resource, rich in bioactive compounds with antioxidant activity. In this study, two strategies to concentrate the bioactive components were used: the tangential membrane filtration (ultrafiltration and reverse osmosis) and the selective resin extraction. The concentrates were evaluated for physico-chemical characteristics and antioxidant activity. Furthermore, the antimicrobial activity and the effect on the mitochondrial voltage-dependent anion selective channel 1 were evaluated. The chemical results highlighted that the highest concentration of hydroxytyrosol (as 7204 mg/L) was revealed in the sample obtained by inverse osmosis while the highest concentration of oleuropein (10005 mg/L) was detected in the sample obtained by resin extraction. The latter sample exhibited the highest antimicrobial effects against Listeria monocytogenes , Escherichia coli , Staphylococcus aureus and Pseudomonas aeruginosa . Both samples exhibited a high impact on the electrophysiological parameters of VDAC1 activity. These results showed that both valorization techniques, which can be reproduced at industrial scale, provided phenolic concentrates with antioxidant and antimicrobial activity useful for different future perspectives.

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.

α-synuclein (αSyn) is a small neuronal protein whose accumulation correlates with Parkinson's disease. αSyn A53T mutant impairs mitochondrial functions by affecting substrate import within the organelle, activity of complex I and the maximal respiratory capacity. However, the precise mechanism initiating the bioenergetic dysfunction is not clearly understood yet. By overexpressing αSyn A53T in SH-SY5Y cells, we investigated the specific changes in the mitochondrial respiratory profile using High-Resolution Respirometry. We found that αSyn A53T increases dissipative fluxes across the intermembrane mitochondrial space: this does not compromise the oxygen flows devoted to ATP production while it reduces the bioenergetic excess capacity of mitochondria, providing a possible explanation of the increased cell susceptibility observed in the presence of further stress stimuli.

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

Voltage-dependent anion-selective channel (VDAC) is one of the main proteins of the outer mitochondrial membrane of all eukaryotes, where it forms aqueous, voltage-sensitive, and ion-selective channels. Its electrophysiological properties have been thoroughly analyzed with the planar lipid bilayer technique. To date, however, available results are based on isolations of VDACs from tissue or from recombinant VDACs produced in bacterial systems. It is well known that the cytosolic overexpression of highly hydrophobic membrane proteins often results in the formation of inclusion bodies containing insoluble aggregates. Purification of properly folded proteins and restoration of their full biological activity requires several procedures that considerably lengthen experimental times. To overcome these restraints, we propose a one-step reaction that combines in vitro cell-free protein expression with nanodisc technology to obtain human VDAC isoforms directly integrated in a native-like lipid bilayer. Reconstitution assays into artificial membranes confirm the reliability of this new methodological approach and provide results comparable to those of VDACs prepared with traditional protein isolation and reconstitution protocols. The use of membrane-mimicking nanodisc systems represents a breakthrough in VDAC electrophysiology and may be adopted to further structural studies., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

VDACs are pore-forming proteins, coating the mitochondrial outer membrane, and playing the role of main regulators for metabolites exchange between cytosol and mitochondria. In mammals, three isoforms have evolutionary originated, VDAC1, VDAC2, and VDAC3. Despite similarity in sequence and structure, evidence suggests different biological roles in normal and pathological conditions for each isoform. We compared Homo sapiens and Mus musculus VDAC genes and their regulatory elements. RNA-seq transcriptome analysis shows that VDAC isoforms are expressed in human and mouse tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3, with the exception of reproductive system. Numerous transcript variants for each isoform suggest specific context-dependent regulatory mechanisms. Analysis of VDAC core promoters has highlighted that, both in a human and a mouse, VDAC genes show features of TATA-less ones. The level of CG methylation of the human VDAC genes revealed that VDAC1 promoter is less methylated than other two isoforms. We found that expression of VDAC genes is mainly regulated by transcription factors involved in controlling cell growth, proliferation and differentiation, apoptosis, and bioenergetic metabolism. A non-canonical initiation site termed "the TCT/TOP motif," the target for translation regulation by the mTOR pathway, was identified in human VDAC2 and VDAC3 and in every murine VDACs promoter. In addition, specific TFBSs have been identified in each VDAC promoter, supporting the hypothesis that there is a partial functional divergence. These data corroborate our experimental results and reinforce the idea that gene regulation could be the key to understanding the evolutionary specialization of VDAC isoforms., Competing Interests: AM, VDP, and FG have an intellectual collaboration with the company, we.MitoBiotech.srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zinghirino, Pappalardo, Messina, Nicosia, De Pinto and Guarino.)

Mitochondrial porins, also known as voltage-dependent anion selective channels (VDACs), are pore-forming molecules of the outer mitochondrial membranes, involved in the regulation of metabolic flux between cytosol and mitochondria. Playing such an essential role, VDAC proteins are evolutionary conserved and isoforms are present in numerous species. The quest for specific function(s) related to the raise of multiple isoforms is an intriguing theme. The yeast Saccharomyces cerevisiae genome is endowed with two different VDAC genes encoding for two distinct porin isoforms, definitely less characterized in comparison to mammalian counterpart. While yVDAC1 has been extensively studied, the second isoform, yVDAC2, is much less expressed, and has a still misunderstood function. This review will recapitulate the known and poorly known information in the literature, in the light of the growing interest about the features of VDAC isoforms in the cell., Competing Interests: FG, AM, and VDP are affiliated with we. MitoBiotech S.R.L, a spin-off company to the University of Catania. All the authors declare that the research was conducted in the absence of any commercial of financial relationships that could be construed a potential conflict of interest., (Copyright © 2021 Di Rosa, Guarino, Conti Nibali, Magrì and De Pinto.)

It has become impossible to review all the existing literature on Voltage-Dependent Anion selective Channel (VDAC) in a single article. A real Renaissance of studies brings this protein to the center of decisive knowledge both for cell physiology and therapeutic application. This review, after highlighting the similarities between the cellular context and the study methods of the solute carriers present in the inner membrane and VDAC in the outer membrane of the mitochondria, will focus on the isoforms of VDAC and their biochemical characteristics. In particular, the possible reasons for their evolutionary onset will be discussed. The variations in their post-translational modifications and the differences between the regulatory regions of their genes, probably the key to understanding the current presence of these genes, will be described. Finally, the situation in the higher eukaryotes will be compared to that of yeast, a unicellular eukaryote, where there is only one active isoform and the role of VDAC in energy metabolism is better understood.

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs' presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon-intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes ( VDAC 1-3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

The Voltage-Dependent Anion-selective Channel (VDAC) is the pore-forming protein of mitochondrial outer membrane, allowing metabolites and ions exchanges. In Saccharomyces cerevisiae, inactivation of POR1, encoding VDAC1, produces defective growth in the presence of non-fermentable carbon source. Here, we characterized the whole-genome expression pattern of a VDAC1-null strain (Δpor1) by microarray analysis, discovering that the expression of mitochondrial genes was completely abolished, as consequence of the dramatic reduction of mtDNA. To overcome organelle dysfunction, Δpor1 cells do not activate the rescue signaling retrograde response, as ρ 0 cells, and rather carry out complete metabolic rewiring. The TCA cycle works in a "branched" fashion, shunting intermediates towards mitochondrial pyruvate generation via malic enzyme, and the glycolysis-derived pyruvate is pushed towards cytosolic utilization by PDH bypass rather than the canonical mitochondrial uptake. Overall, Δpor1 cells enhance phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, overproduce and excrete inositol. Such unexpected re-arrangement of whole metabolism suggests a regulatory role of VDAC1 in cell bioenergetics.

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed., (Copyright © 2020 Reina, Pittalà, Guarino, Messina, De Pinto, Foti and Saletti.)

The voltage-dependent anion-selective channels (VDACs), which are also known as eukaryotic porins, are pore-forming proteins, which allow for the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions that regulate organelle and cellular metabolism. We have recently reported the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing, for the first time, the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed for us to extend the investigation to the human VDACs. The over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as also certained in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs that are found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell. Data are available via ProteomeXchange with identifier PXD017482.

Voltage-dependent anion channel (VDAC) is the major pathway for the transport of ions and metabolites across the mitochondrial outer membrane. Among the three known mammalian VDAC isoforms, VDAC3 is the least characterized, but unique functional roles have been proposed in cellular and animal models. Yet, a high-sequence similarity between VDAC1 and VDAC3 is indicative of a similar pore-forming structure. Here, we conclusively show that VDAC3 forms stable, highly conductive voltage-gated channels that, much like VDAC1, are weakly anion selective and facilitate metabolite exchange, but exhibit unique properties when interacting with the cytosolic proteins α-synuclein and tubulin. These two proteins are known to be potent regulators of VDAC1 and induce similar characteristic blockages (on the millisecond time scale) of VDAC3, but with 10- to 100-fold reduced on-rates and altered α-synuclein blocking times, indicative of an isoform-specific function. Through cysteine scanning mutagenesis, we found that VDAC3's cysteine residues regulate its interaction with α-synuclein, demonstrating VDAC3-unique functional properties and further highlighting a general molecular mechanism for VDAC isoform-specific regulation of mitochondrial bioenergetics., (© 2019 Queralt-Martín et al.)

Voltage-dependent anion channel isoform 2 of the yeast Saccharomyces cerevisiae (yVDAC2) was believed for many years to be devoid of channel activity. Recently, we isolated yVDAC2 and showed that it exhibits channel-forming activity in the planar lipid bilayer system when in its so-called native form. Here, we describe an alternative strategy for yVDAC2 isolation, through heterologous expression in bacteria and refolding in vitro. Recombinant yVDAC2, like its native form, is able to form voltage-dependent channels. However, some differences between native and recombinant yVDAC2 emerged in terms of voltage dependence and ion selectivity, suggesting that, in this specific case, the recombinant protein might be depleted of post-translational modification(s) that occur in eukaryotic cells., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Voltage-dependent anion-selective channels (VDACs) are primarily located in the mitochondrial outer membrane (MOM). They are essential for the regulation of ion and metabolite exchanges. In particular, their role in energy-related nucleotide exchange has many implications in apoptosis, cancer, and neurodegenerative diseases. It has been proposed that VDACs' functions are regulated by mobility of the N-terminal helical domain, which is bound to the inner wall of the main β-barrel domain but exists in equilibrium between the bound-folded and the unbound-unfolded state. When the N-terminal domain detaches from the channel's wall and eventually leaves the lumen, it can either stay exposed to the cytosolic environment or interact with the outer leaflet of the MOM; then, it may also interact with other protein partners. In humans, three different VDAC isoforms are expressed at different tissue-specific levels with evidence of distinct roles. Although the N-terminal domains share high sequence similarity, important differences do exist, with the functionality of the entire protein mostly attributed to them. In this work, the three-dimensional structure and membrane affinity of the three isolated hVDAC N-terminal peptides have been compared through Fourier-transform infrared and NMR spectroscopy in combination with molecular dynamics simulations, and measurement of the surface pressure of lipid monolayers. Although peptides were studied as isolated from the β-barrel domain, the observed differences are relevant for those whole protein's functions in which a protein-protein interaction is mediated by the N-terminal domain., Competing Interests: The authors declare no competing financial interest.

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell., (Copyright © 2018. Published by Elsevier B.V.)

Cancer and neurodegeneration are different classes of diseases that share the involvement of mitochondria in their pathogenesis. Whereas the high glycolytic rate (the so-called Warburg metabolism) and the suppression of apoptosis are key elements for the establishment and maintenance of cancer cells, mitochondrial dysfunction and increased cell death mark neurodegeneration. As a main actor in the regulation of cell metabolism and apoptosis, VDAC may represent the common point between these two broad families of pathologies. Located in the outer mitochondrial membrane, VDAC forms channels that control the flux of ions and metabolites across the mitochondrion thus mediating the organelle's cross-talk with the rest of the cell. Furthermore, the interaction with both pro-apoptotic and anti-apoptotic factors makes VDAC a gatekeeper for mitochondria-mediated cell death and survival signaling pathways. Unfortunately, the lack of an evident druggability of this protein, since it has no defined binding or active sites, makes the quest for VDAC interacting molecules a difficult tale. Pharmacologically active molecules of different classes have been proposed to hit cancer and neurodegeneration. In this work, we provide an exhaustive and detailed survey of all the molecules, peptides, and microRNAs that exploit VDAC in the treatment of the two examined classes of pathologies. The mechanism of action and the potential or effectiveness of each compound are discussed.

The yeast Saccharomyces cerevisiae genome is endowed with two distinct isoforms of Voltage-Dependent Anion Channel (VDAC). The isoform yVDAC2 is currently understudied with respect to the best known yVDAC1. Yet, since the discovery, the function of yVDAC2 was unclear, leading to the hypothesis that it might be devoid of a channel function. In this work we have elucidated, by bioinformatics modeling and electrophysiological analysis, the functional activity of yVDAC2. The conformation of yVDAC2 and, for comparison, of yVDAC1 were modeled using a multiple template approach involving mouse, human and zebrafish structures and both showed to arrange the sequences as the typical 19-stranded VDAC β-barrel. Molecular dynamics simulations showed that yVDAC2, in comparison with yVDAC1, has a different number of permeation paths of potassium and chloride ions. yVDAC2 protein was over-expressed in the S. cerevisiae cells depleted of functional yVDAC1 (Δpor1 mutant) and, after purification, it was reconstituted in artificial membranes (planar lipid bilayer (PLB) system). The protein displayed channel-forming activity and the calculated conductance, voltage-dependence and ion selectivity values were similar to those of yVDAC1 and other members of VDAC family. This is the first time that yVDAC2 channel features are detected and characterized., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane (OMM) protein, serves as a mitochondrial gatekeeper, mediating the transport of nucleotides, Ca 2+ and other metabolites across the OMM. VDAC1 also plays a central role in mitochondria-mediated apoptosis by facilitating the release of apoptotic proteins and by association with both pro- and anti-apoptotic proteins. Tumor cells, which are constantly exposed to hypoxic conditions, affect the cell via the transcription factor hypoxia-inducible factor (HIF) that induces transcriptional activity. In cultured cells and in lung cancer patients, hypoxia induces VDAC1 truncation at the C-terminus (VDAC1-ΔC). However, the molecular mechanisms involved in VDAC1-ΔC formation are unknown. Here, we show that hypoxia-induced VDAC1-ΔC formation is inhibited by the Ca 2+ chelator BAPTA-AM, by calpain inhibitor-1, by inhibitor of the asparagine endopeptidase (AEP) and by si-RNA targeting HIF1-α or Ca 2+ -activated protease calpain-1 expression but not that of calpain-2. Finally, VDAC1-ΔC expressed in bacteria and reconstituted into a planar lipid bilayer exhibited decreased channel conductance relative to the full-length protein, yet retained voltage-dependent conductance. These findings suggest that hypoxia, acting via HIF-1α expression, leads to VDAC1 cleavage involving the activation of calpain 1 and AEP., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.

Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys 229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys 122 could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: VDAC (Voltage-Dependent Anion selective Channel) is a small family of abundant pore-forming proteins located in the outer mitochondrial membrane. Their role range from the most intuitive, the formation of a hydrophilic conduit through the membrane thanks to its beta-barrel structure, to less understood functions that make them essential actors in the cross-talk between the bioenergetics metabolism and the cytosol components. Due to this localization, VDAC1, in particular, has been reported to be involved in apoptosis, Hexokinase and tubulin binding, and in the Warburg effect. For these reasons, an involvement of VDAC in cancer is considered consequential and a number of compounds have been proposed and used in experimental trials to demonstrate the efficacy of molecules affecting the functions of VDAC., Objectives: In this work, we thus survey the literature describing drug compounds acting on the cancerous proliferation through VDAC. Three main categories have been assigned: molecules acting on the VDAC-Hexokinase binding, molecules directly inhibiting the VDAC conductance, molecules affecting the expression levels of the VDAC gene. The application of biological peptides for this purpose is also considered., Conclusion: Since the knowledges about the functional properties of VDAC protein are still insufficient, VDAC as a pharmacological target in the fight against cancer is still a very open, but very promising, field., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

VDAC3 is the least known isoform of the mammalian voltage-dependent anion selective channels of the outer mitochondrial membrane. It has been recently shown that cysteine residues of VDAC3 are found over-oxidized. The VDAC3 cysteine over-oxidation was associated with the oxidizing environment and the abundance of reactive oxygen species (ROS) in the intermembrane space. In this work, we have examined the role of VDAC3 in general pathogenic mechanisms at the basis of mitochondrial dysfunction and involving the mitochondrial quality control. Many of the diseases reported here, including cancer and viral infections, are often associated with significant changes in the intracellular redox state. In this sense, VDAC3 bearing oxidative modifications could become marker of the oxidative load in the mitochondria and part of the ROS signaling pathway.

Apoptosis is thought to play a critical role in several pathological processes, such as neurodegenerative diseases (i.e. Parkinson's and Alzheimer's diseases) and various cardiovascular diseases. Despite the fact that apoptotic mechanisms are well defined, there is still no substantial therapeutic strategy to stop or even slow this process. Thus, there is an unmet need for therapeutic agents that are able to block or slow apoptosis in neurodegenerative and cardiovascular diseases. The outer mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for a variety of cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondrion-mediated apoptosis. Thus, VDAC1 oligomerization represents a prime target for agents designed to modulate apoptosis. Here, high-throughput compound screening and medicinal chemistry were employed to develop compounds that directly interact with VDAC1 and prevent VDAC1 oligomerization, concomitant with an inhibition of apoptosis as induced by various means and in various cell lines. The compounds protected against apoptosis-associated mitochondrial dysfunction, restoring dissipated mitochondrial membrane potential, and thus cell energy and metabolism, decreasing reactive oxidative species production, and preventing detachment of hexokinase bound to mitochondria and disruption of intracellular Ca 2+ levels. Thus, this study describes novel drug candidates with a defined mechanism of action that involves inhibition of VDAC1 oligomerization, apoptosis, and mitochondrial dysfunction. The compounds VBIT-3 and VBIT-4 offer a therapeutic strategy for treating different diseases associated with enhanced apoptosis and point to VDAC1 as a promising target for therapeutic intervention., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

In this mini-review, we analyze the influence of cysteines in the structure and activity of mitochondrial outer membrane mammalian VDAC isoforms. The three VDAC isoforms show conserved sequences, similar structures and the same gene organization. The meaning of three proteins encoded in different chromosomes must thus be searched for subtle differences at the amino acid level. Among others, cysteine content is noticeable. In humans, VDAC1 has 2, VDAC2 has 9 and VDAC3 has 6 cysteines. Recent works have shown that, at variance from VDAC1, VDAC2 and VDAC3 exhibit cysteines predicted to protrude towards the intermembrane space, making them a preferred target for oxidation by ROS. Mass spectrometry in VDAC3 revealed that a disulfide bridge can be formed and other cysteine oxidations are also detectable. Both VDAC2 and VDAC3 cysteines were mutagenized to highlight their role in vitro and in complementation assays in Δporin1 yeast. Chemico-physical techniques revealed an important function of cysteines in the structural stabilization of the pore. In conclusion, the works available on VDAC cysteines support the notion that the three proteins are paralogs with a similar pore-function and slightly different, but important, ancillary biological functions. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cu/Zn Superoxide Dismutase (SOD1), the most important antioxidant defense against ROS in eukaryotic cells, localizes in cytosol and intermembrane space of mitochondria (IMS). Several evidences show a SOD1 intersection with both fermentative and respiratory metabolism. The Voltage Dependent Anion Channel (VDAC) is the main pore-forming protein in the mitochondrial outer membrane (MOM), and is considered the gatekeeper of mitochondrial metabolism. Saccharomyces cerevisiae lacking VDAC1 (Δpor1) is a very convenient model system, since it shows an impaired growth rate on non-fermentable carbon source. Transformation of Δpor1 yeast with human SOD1 completely restores the cell growth deficit in non-fermentative conditions and re-establishes the physiological levels of ROS, as well as the mitochondrial membrane potential. No similar result was found upon yeast SOD1 overexpression. A previous report highlighted the action of SOD1 as a transcription factor. Quantitative Real-Time PCR showed that β-barrel outer-membrane encoding-genes por2, tom40, sam50 are induced by hSOD1, but the same effect was not obtained in Δpor1Δpor2 yeast, indicating a crucial function for yVDAC2. Since the lack of VDAC1 in yeast can be considered a stress factor for the cell, hSOD1 could relieve it stimulating the expression of genes bringing to the recovery of the MOM function. Our results suggest a direct influence of SOD1 on VDAC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The human VDAC channel exists in three isoforms characterized by high sequence homology and structural similarity. Yet the function and mode of action of hVDAC3 are still elusive. The presence of six surface cysteines exposed to the oxidizing environment of the mitochondrial inter-membrane space suggests the possible establishment of intramolecular disulfide bonds. Two natural candidates for disulfide bridge formation are Cys2 and Cys8 that, located on the flexible N-terminal domain, can easily come in contact. A third potentially important residue is Cys122 that is close to Cys2 in the homology model of VDAC3. Here we analyzed the impact of SS bonds through molecular dynamics simulations of derivatives of hVDAC3 (dubbed SS-2-8, SS-2-122, SS-8-122) including a single disulfide bond. Simulations showed that in SS-8-122, the fragment 1-7 crosses the top part of the barrel partially occluding the pore and causing a 20% drop of conductance. In order to identify other potential channel-occluding disulfide bonds, we used a set of neural networks and structural bioinformatics algorithms, after filtering with the steric constraints imposed by the 3D-structure. We identified other three species, namely SS-8-65, SS-2-36 and SS-8-36. While the conductance of SS-8-65 and SS-2-36 is about 30% lower than that of the species without disulfide bonds, the conductance of SS-8-36 was 40-50% lower. The results show how VDAC3 is able to modulate its pore size and current by exploiting the mobility of the N-terminal and forming, upon external stimuli, disulfide bridges with cysteine residues located on the barrel and exposed to the inter-membrane space., (Copyright © 2016 Elsevier B.V. All rights reserved)

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.

The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.