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Varda Shoshanbarmatz - One of the best experts on this subject based on the ideXlab platform.
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metabolic reprograming via silencing of mitochondrial VDAC1 expression encourages differentiation of cancer cells
Molecular therapy. Nucleic acids, 2019Co-Authors: Tasleem Arif, Zohar Amsalem, Varda ShoshanbarmatzAbstract:The mitochondrial gatekeeper voltage-dependent anion channel 1 (VDAC1) controls metabolic and energy cross-talk between mitochondria and the rest of the cell and is involved in mitochondria-mediated apoptosis. Here, we compared the effects of downregulated VDAC1 expression in the U-87MG glioblastoma, MDA-MB-231 triple-negative breast cancer (TNBC), and A549 lung cancer cell lines, using small interfering RNA (siRNA) specific to human VDAC1 (si-hVDAC1). The cells were subjected to si-hVDAC1 (50 nM) treatment for 5–20 days. Although VDAC1 silencing occurred within a day, the cells underwent reprograming with respect to rewiring metabolism, elimination of cancer stem cells (CSCs), and alteration of transcription factor (TF) expression and proteins associated with differentiation, with maximal changes being observed after 3 weeks of silencing VDAC1 expression. The differentiation into fewer tumorigenic cells may be associated with the elimination of CSCs. These alterations are interconnected, as protein up- or downregulation occurred simultaneously, starting 15–20 days after VDAC1 levels were first decreased. Moreover, the VDAC1 depletion-mediated effects on a network of key regulators of cell metabolism, CSCs, TFs, and other factors leading to differentiation are coordinated and are common to the glioblastoma multiforme (GBM) and lung and breast cancer cell lines, despite differing in origin and carried mutations. Thus, our study showed that VDAC1 depletion triggers reprograming of malignant cancer cells into terminally differentiated cells and that this may be a promising therapeutic approach for various cancers.
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VDAC1 and the tspo expression interactions and associated functions in health and disease states
International Journal of Molecular Sciences, 2019Co-Authors: Varda Shoshanbarmatz, Srinivas Pittala, Dario MizrachiAbstract:The translocator protein (TSPO), located at the outer mitochondrial membrane (OMM), serves multiple functions and contributes to numerous processes, including cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca2+ signaling, oxidative stress, and inflammation. TSPO forms a complex with the voltage-dependent anion channel (VDAC), a protein that mediates the flux of ions, including Ca2+, nucleotides, and metabolites across the OMM, controls metabolism and apoptosis and interacts with many proteins. This review focuses on the two OMM proteins TSPO and VDAC1, addressing their structural interaction and associated functions. TSPO appears to be involved in the generation of reactive oxygen species, proposed to represent the link between TSPO activation and VDAC, thus playing a role in apoptotic cell death. In addition, expression of the two proteins in healthy brains and diseased states is considered, as is the relationship between TSPO and VDAC1 expression. Both proteins are over-expressed in in brains from Alzheimer’s disease patients. Finally, TSPO expression levels were proposed as a biomarker of some neuropathological settings, while TSPO-interacting ligands have been considered as a potential basis for drug development.
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VDAC1 as a player in mitochondria mediated apoptosis and target for modulating apoptosis
Current Medicinal Chemistry, 2018Co-Authors: Varda Shoshanbarmatz, Yakov Krelin, Quan ChenAbstract:The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane protein, functions as a mitochondrial governor, controlling transport of metabolites in and out of the mitochondria and energy production, while also coordinating glycolysis and oxidative phosphorylation (OXPHOS). . VDAC1 plays a key role in mitochondria-mediated apoptosis by functioning in the release of apoptotic proteins located in the inter-membranal space (IMS) and due to its association with pro- and anti-apoptotic proteins. Thus, VDAC1 is considered as a promising target for controlling apoptosis. This review provides insight into the central role of VDAC1 in mammalian cell life and death and emphasizes VDAC1 function in apoptosis, focusing on VDAC1 oligomerization as an important step in the intrinsic apoptosis pathway involving mitochondria. Accumulated evidence suggests that VDAC1 oligomerization leads to the formation of a large pore that allows the passage of proteins, like cytochrome c (Cyto c). Here, we focus on the mechanism leading to VDAC1 oligomerization, presenting evidence for the relationship between VDAC1 expression levels and induction of apoptosis. Moreover, we present evidence showing that almost all apoptosis stimuli induce VDAC1 over-expression and suggest that these high levels of VDAC1 shift VDAC from a monomeric to an oligomeric assembly, corresponding to the Cyto c release channel. Several compounds or conditions inducing apoptosis also induce VDAC1 over-expression leading to VDAC1 oligomerization, such as cyathin-R, cisplatin and selenite, are presented. Likewise, VDAC1-interacting molecules, such as 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), diphenylamine-2-carboxylate (DPC), ruthenium red and VBIT, that inhibit VDAC1 oligomerization and apoptosis are also presented. VDAC1 oligomerization as a potential target for controlling apoptosis, specifically using drugs to induce apoptotic cell death in cancer and inhibit apoptosis in neurodegenerative diseases, as well as possible VDAC1-based therapeutic applications, are highlighted.
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VDAC1 functions in ca2 homeostasis and cell life and death in health and disease
Cell Calcium, 2018Co-Authors: Varda Shoshanbarmatz, Yakov Krelin, Anna ShteinferkuzmineAbstract:Abstract In the outer mitochondrial membrane (OMM), the voltage-dependent anion channel 1 (VDAC1) serves as a mitochondrial gatekeeper, controlling the metabolic and energy cross-talk between mitochondria and the rest of the cell. VDAC1 also functions in cellular Ca 2+ homeostasis by transporting Ca 2+ in and out of mitochondria. VDAC1 has also been recognized as a key protein in mitochondria-mediated apoptosis, contributing to the release of apoptotic proteins located in the inter-membranal space (IMS) and regulating apoptosis via association with pro- and anti-apoptotic members of the Bcl-2 family of proteins and hexokinase. VDAC1 is highly Ca 2+ -permeable, transporting Ca 2+ to the IMS and thus modulating Ca 2+ access to Ca 2+ transporters in the inner mitochondrial membrane. Intra-mitochondrial Ca 2+ controls energy metabolism via modulating critical enzymes in the tricarboxylic acid cycle and in fatty acid oxidation. Ca 2+ also determines cell sensitivity to apoptotic stimuli and promotes the release of pro-apoptotic proteins. However, the precise mechanism by which intracellular Ca 2+ mediates apoptosis is not known. Here, the roles of VDAC1 in mitochondrial Ca 2+ homeostasis are presented while emphasizing a new proposed mechanism for the mode of action of pro-apoptotic drugs. This view, proposing that Ca 2+ -dependent enhancement of VDAC1 expression levels is a major mechanism by which apoptotic stimuli induce apoptosis, position VDAC1 oligomerization at a molecular focal point in apoptosis regulation. The interactions of VDAC1 with many proteins involved in Ca 2+ homeostasis or regulated by Ca 2+ , as well as VDAC-mediated control of cell life and death and the association of VDAC with disease, are also presented.
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ca2 mediated regulation of VDAC1 expression levels is associated with cell death induction
Biochimica et Biophysica Acta, 2014Co-Authors: Shira Weisthal, Tasleem Arif, Nurit Keinan, Danya Benhail, Varda ShoshanbarmatzAbstract:Abstract 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 H 2 O 2 or As 2 O 3 , 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
Vito De Pinto - One of the best experts on this subject based on the ideXlab platform.
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Live cell interactome of the human voltage dependent anion channel 3 (VDAC3) revealed in HeLa cells by affinity purification tag technique
Molecular BioSystems, 2020Co-Authors: Angela Messina, Andrea Magrì, Francesca Guarino, Simona Reina, Flora Tomasello, Rona R. Ramsay, Richard E Clark, Vito De PintoAbstract:In higher eukaryotes three different VDAC genes encode three homologous proteins which do not show the same activity. VDAC1 and VDAC2 isoforms have been characterized while VDAC3 isoform is still elusive. To explore VDAC3 protein interactions, we have established a stable cell line expressing a fluorescent and dual-tagged construct. This clone expresses a stable amount of VDAC3. Live cell imaging shows that fluorescent VDAC3 localizes in the mitochondria. Proteins interacting with VDAC3 have been separated by tandem-affinity purification and 2-D gel electrophoresis and identified by mass spectrometry. In the list of putative interacting proteins, there are cytosolic, mitochondrial, cytoskeletal and ER proteins. Coherent pathways like cell redox homeostasis, response to stress, formation/rearrangement of disulfide bonds, response to unfolded proteins or protein folding have been found to be related to clusters of proteins identified in this experiment. The list of associated proteins has been validated by immunoprecipitation experiments utilizing specific antibodies. Likely biological and pathological processes have been analyzed. Cytosolic proteins associated with VDAC3 include tubulins and cytoskeletal proteins, stress sensors, chaperones and proteasome components, redox-mediating enzymes such as protein disulphide isomerase. The overall picture points to a role for VDAC3 as mediator for the organization of protein complexes and regulator of the traffic of misfolded or non-folded proteins evoked from different stimuli.
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post translational modifications of VDAC1 and vdac2 cysteines from rat liver mitochondria
Biochimica et Biophysica Acta, 2018Co-Authors: Rosaria Saletti, Andrea Magrì, Simona Reina, Maria G G Pittala, Vincenzo Cunsolo, Salvatore Foti, Vito De PintoAbstract:Abstract 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.
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overexpression of human sod1 in VDAC1 less yeast restores mitochondrial functionality modulating beta barrel outer membrane protein genes
Biochimica et Biophysica Acta, 2016Co-Authors: Andrea Magrì, Maria Carmela Di Rosa, Francesca Guarino, Simona Reina, Angela Messina, Marianna Flora Tomasello, Vito De PintoAbstract: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.
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the voltage dependent anion selective channel 1 VDAC1 topography in the mitochondrial outer membrane as detected in intact cell
PLOS ONE, 2013Co-Authors: Marianna Flora Tomasello, Francesca Guarino, Simona Reina, Angela Messina, Vito De PintoAbstract: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.
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swapping of the n terminus of VDAC1 with vdac3 restores full activity of the channel and confers anti aging features to the cell
FEBS Letters, 2010Co-Authors: Simona Reina, Francesca Guarino, Angela Messina, Vanessa Palermo, Andrea Guarnera, Cristina Mazzoni, Vito De PintoAbstract: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.
Nir Arbel - One of the best experts on this subject based on the ideXlab platform.
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VDAC1 Cysteine Residues: Topology and Function in Channel Activity and Apoptosis
Biophysical Journal, 2020Co-Authors: Varda Shoshan-barmatz, Shay Geula, Lior Aram, Nir ArbelAbstract:VDAC is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of the VDAC1 cysteines in channel activity and to probe cysteine topology, with respect to facing the pore or the bilayer, we used thiol-modifying agents; membrane permeable N-ethyl-maleimide (NEM); bulky, charged 5-fluorescein-maleimide (5-FM) and cross-linking reagent bis[maleimido]ethane (BMOE). Bilayer-reconstituted VDAC conductance was decreased by 5-FM but not by NEM, while 5-FM had no effect on NEM-labeled VDAC conductance. BMOE formed dimeric VDAC1, suggesting that a VDAC1 cysteine residues is exposed and available for cross-linking. The results suggest that one of the VDAC1 cysteine residues faces the VDAC pore while the second is oriented toward the lipid bilayer. The positions of VDAC1 Cys127 and Cys232 with respect to the membrane and channel pore, were considered in light of proposed VDAC1 topology models. Mutated VDAC1 in which Cys127 and Cys232 were replaced by alanines showed channel activity as of native VDAC1 and when expressed in cells was localized to mitochondria. As with over-expression of native rVDAC1, cysteine-less rVDAC1 induced apoptotic cell death and underwent oligomerization upon apoptosis induction. The results suggest that the two cysteine residues are not required for VDAC1 oligomerization or apoptosis, as induced by H2O2, As2O3 or selenite, ROS producing agents.
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mediation of the antiapoptotic activity of bcl xl protein upon interaction with VDAC1 protein
Journal of Biological Chemistry, 2012Co-Authors: Nir Arbel, Danya Benhail, Varda ShoshanbarmatzAbstract:The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity. Here, using a variety of approaches, we show the interaction of the antiapoptotic protein, Bcl-xL, with VDAC1 and reveal that this interaction mediates Bcl-xL protection against apoptosis. C-terminally truncated Bcl-xL(Δ21) interacts with purified VDAC1, as revealed by microscale thermophoresis and as reflected in the reduced channel conductivity of bilayer-reconstituted VDAC1. Overexpression of Bcl-xL prevented staurosporine-induced apoptosis in cells expressing native VDAC1 but not certain VDAC1 mutants. Having identified mutations in VDAC1 that interfere with the Bcl-xL interaction, certain peptides representing VDAC1 sequences, including the N-terminal domain, were designed and generated as recombinant and synthetic peptides. The VDAC1 N-terminal region and two internal sequences were found to bind specifically, and in a concentration- and time-dependent manner, to immobilized Bcl-xL(Δ21), as revealed by surface plasmon resonance. Moreover, expression of the recombinant peptides in cells overexpressing Bcl-xL prevented protection offered by the protein against staurosporine-induced apoptosis. These results point to Bcl-xL acting as antiapoptotic protein, promoting tumor cell survival via binding to VDAC1. These findings suggest that interfering with Bcl-xL binding to the mitochondria by VDAC1-based peptides may serve to induce apoptosis in cancer cells and to potentiate the efficacy of conventional chemotherapeutic agents.
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dominant negative VDAC1 mutants reveal oligomeric VDAC1 to be the active unit in mitochondria mediated apoptosis
Biochemical Journal, 2010Co-Authors: Asaf Mader, Salah Abuhamad, Nir Arbel, Manuel Gutierrasuilar, Varda ShoshanbarmatzAbstract:Mitochondria play a central role in the intrinsic pathway of apoptosis. Oligomerization of the mitochondrial protein VDAC1 (voltage-dependent anion channel 1) has been proposed to play a role in apoptosis in various studies. In the present study, we have generated dimeric fusion proteins consisting of tandem-linked wild-type and RuR (Ruthenium Red)-insensitive mutant VDAC1 monomers and studied the capacity of RuR to protect against apoptosis, as induced by various means. Fusion proteins composed of wild-type and/or E72Q-VDAC1 were successfully expressed in T-REx-293 cells. Bilayer-reconstituted dimeric rVDAC1 (rat VDAC1) functions as a channel-forming protein, showing typical voltage-dependence conductance, but with a unitary conductance higher than that of monomeric VDAC. As with wild-type VDAC1, overexpression of either the wild-type or mutated VDAC1 dimeric fusion protein induced apoptotic cell death. In addition, as shown previously, the anti-apoptotic effect of RuR was not observed in cells expressing E72Q-VDAC1, despite endogenous VDAC1 being present in these cells. Similar RuR insensitivity governed the VDAC1 fusion proteins comprising the E72Q mutation in either the first, second or both VDAC1 monomers of the same dimer. RuR-mediated protection against apoptosis in T-REx-293 cells, as induced by staurosporine, was observed in cells expressing VDAC1 or dimeric wild-type VDAC1. However, RuR offered no protection against staurosporine-induced apoptosis in cells expressing E72Q-VDAC1 or E72Q-containing dimeric VDAC1. These results suggest that E72Q-VDAC1 has a dominant-negative effect and implies that VDAC1 homo-oligomerization, involving intermolecular interactions, might be involved in the apoptotic process. Abbreviations: AIF, apoptosis-inducing factor; DMEM, Dulbecco's modified Eagle's medium; EGS, ethylene glycol bis(succinimidylsuccinate); GFP, green fluorescent protein; HEK-293T cells, HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40); HK, hexokinase; HRP, horseradish peroxidase; LDAO, N,N-dimethyldodecylamine-N-oxide; OMM, outer mitochondrial membrane; PLB, planar lipid bilayer; RuR, Ruthenium Red; siRNA, small interfering RNA; STS, staurosporine; TEV, tobacco etch virus; T-REx-293, HEK-293 cells that express the tetracycline repressor; VDAC, voltage-dependent anion channel; hVDAC1, human VDAC1; mVDAC1, murine VDAC1; rVDAC1, rat VDAC1
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Dominant-negative VDAC1 mutants reveal oligomeric VDAC1 to be the active unit in mitochondria-mediated apoptosis
Biochemical Journal, 2010Co-Authors: Asaf Mader, Salah Abu-hamad, Nir Arbel, Manuel Gutierr-asuilar, Varda Shoshan-barmatzAbstract:Mitochondria play a central role in the intrinsic pathway of apoptosis. Oligomerization of the mitochondrial protein VDAC1 (voltage-dependent anion channel 1) has been proposed to play a role in apoptosis in various studies. In the present study, we have generated dimeric fusion proteins consisting of tandem-linked wild-type and RuR (Ruthenium Red)-insensitive mutant VDAC1 monomers and studied the capacity of RuR to protect against apoptosis, as induced by various means. Fusion proteins composed of wild-type and/or E72Q-VDAC1 were successfully expressed in T-REx-293 cells. Bilayer-reconstituted dimeric rVDAC1 (rat VDAC1) functions as a channel-forming protein, showing typical voltage-dependence conductance, but with a unitary conductance higher than that of monomeric VDAC. As with wild-type VDAC1, overexpression of either the wild-type or mutated VDAC1 dimeric fusion protein induced apoptotic cell death. In addition, as shown previously, the anti-apoptotic effect of RuR was not observed in cells expressing E72Q-VDAC1, despite endogenous VDAC1 being present in these cells. Similar RuR insensitivity governed the VDAC1 fusion proteins comprising the E72Q mutation in either the first, second or both VDAC1 monomers of the same dimer. RuR-mediated protection against apoptosis in T-REx-293 cells, as induced by staurosporine, was observed in cells expressing VDAC1 or dimeric wild-type VDAC1. However, RuR offered no protection against staurosporine-induced apoptosis in cells expressing E72Q-VDAC1 or E72Q-containing dimeric VDAC1. These results suggest that E72Q-VDAC1 has a dominant-negative effect and implies that VDAC1 homo-oligomerization, involving intermolecular interactions, might be involved in the apoptotic process.
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VDAC1 cysteine residues topology and function in channel activity and apoptosis
Biochemical Journal, 2010Co-Authors: Lior Aram, Shay Geula, Nir Arbel, Varda ShoshanbarmatzAbstract:The VDAC (voltage-dependent anion channel) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of VDAC1 cysteine residues in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane-permeable NEM ( N -ethylmaleimide), bulky charged 5-FM (fluorescein-5-maleimide) and the cross-linking reagent BMOE [bis(maleimido)ethane]. Bilayer-reconstituted VDAC conductance was decreased by 5-FM, but not by NEM, whereas 5-FM had no effect on NEM-labelled VDAC conductance. BMOE caused the formation of dimeric VDAC1, suggesting that one of the two VDAC1 cysteine residues is exposed and available for cross-linking. The results thus suggest that one of the VDAC1 cysteine residues faces the VDAC pore, whereas the second is oriented towards the lipid bilayer. Mutated rat VDAC1 in which the two cysteine residues, Cys 127 and Cys 232 , were replaced by alanine residues showed channel activity like native VDAC1 and, when expressed in cells, was localized to mitochondria. Human VDAC1-shRNA (small hairpin RNA)- or -siRNA (small interfering RNA)-treated cells, expressing low levels of endogenous human VDAC1 together with native or cysteine-less rat VDAC1, undergo apoptosis as induced by overexpression of these VDAC1 or upon treatment with reactive oxygen species-producing agents, H 2 O 2 , As 2 O 3 or selenite, suggesting that the two cysteine residues are not required for apoptosis or VDAC1 oligomerization.
Lior Aram - One of the best experts on this subject based on the ideXlab platform.
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VDAC1 Cysteine Residues: Topology and Function in Channel Activity and Apoptosis
Biophysical Journal, 2020Co-Authors: Varda Shoshan-barmatz, Shay Geula, Lior Aram, Nir ArbelAbstract:VDAC is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of the VDAC1 cysteines in channel activity and to probe cysteine topology, with respect to facing the pore or the bilayer, we used thiol-modifying agents; membrane permeable N-ethyl-maleimide (NEM); bulky, charged 5-fluorescein-maleimide (5-FM) and cross-linking reagent bis[maleimido]ethane (BMOE). Bilayer-reconstituted VDAC conductance was decreased by 5-FM but not by NEM, while 5-FM had no effect on NEM-labeled VDAC conductance. BMOE formed dimeric VDAC1, suggesting that a VDAC1 cysteine residues is exposed and available for cross-linking. The results suggest that one of the VDAC1 cysteine residues faces the VDAC pore while the second is oriented toward the lipid bilayer. The positions of VDAC1 Cys127 and Cys232 with respect to the membrane and channel pore, were considered in light of proposed VDAC1 topology models. Mutated VDAC1 in which Cys127 and Cys232 were replaced by alanines showed channel activity as of native VDAC1 and when expressed in cells was localized to mitochondria. As with over-expression of native rVDAC1, cysteine-less rVDAC1 induced apoptotic cell death and underwent oligomerization upon apoptosis induction. The results suggest that the two cysteine residues are not required for VDAC1 oligomerization or apoptosis, as induced by H2O2, As2O3 or selenite, ROS producing agents.
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Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c, AIF and Smac/Diablo
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2020Co-Authors: Varda Shoshan-barmatz, Salah Abu-hamad, Nurit Keinan, Dalia Tyomkin, Lior AramAbstract:AbstractMitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2 or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2
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apoptosis is regulated by the VDAC1 n terminal region and by vdac oligomerization release of cytochrome c aif and smac diablo
Biochimica et Biophysica Acta, 2010Co-Authors: Varda Shoshanbarmatz, Salah Abuhamad, Nurit Keinan, Dalia Tyomkin, Lior AramAbstract:Abstract Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2 or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.
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VDAC1 cysteine residues topology and function in channel activity and apoptosis
Biochemical Journal, 2010Co-Authors: Lior Aram, Shay Geula, Nir Arbel, Varda ShoshanbarmatzAbstract:The VDAC (voltage-dependent anion channel) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of VDAC1 cysteine residues in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane-permeable NEM ( N -ethylmaleimide), bulky charged 5-FM (fluorescein-5-maleimide) and the cross-linking reagent BMOE [bis(maleimido)ethane]. Bilayer-reconstituted VDAC conductance was decreased by 5-FM, but not by NEM, whereas 5-FM had no effect on NEM-labelled VDAC conductance. BMOE caused the formation of dimeric VDAC1, suggesting that one of the two VDAC1 cysteine residues is exposed and available for cross-linking. The results thus suggest that one of the VDAC1 cysteine residues faces the VDAC pore, whereas the second is oriented towards the lipid bilayer. Mutated rat VDAC1 in which the two cysteine residues, Cys 127 and Cys 232 , were replaced by alanine residues showed channel activity like native VDAC1 and, when expressed in cells, was localized to mitochondria. Human VDAC1-shRNA (small hairpin RNA)- or -siRNA (small interfering RNA)-treated cells, expressing low levels of endogenous human VDAC1 together with native or cysteine-less rat VDAC1, undergo apoptosis as induced by overexpression of these VDAC1 or upon treatment with reactive oxygen species-producing agents, H 2 O 2 , As 2 O 3 or selenite, suggesting that the two cysteine residues are not required for apoptosis or VDAC1 oligomerization.
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Live cell interactome of the human voltage dependent anion channel 3 (VDAC3) revealed in HeLa cells by affinity purification tag technique
Molecular BioSystems, 2020Co-Authors: Angela Messina, Andrea Magrì, Francesca Guarino, Simona Reina, Flora Tomasello, Rona R. Ramsay, Richard E Clark, Vito De PintoAbstract:In higher eukaryotes three different VDAC genes encode three homologous proteins which do not show the same activity. VDAC1 and VDAC2 isoforms have been characterized while VDAC3 isoform is still elusive. To explore VDAC3 protein interactions, we have established a stable cell line expressing a fluorescent and dual-tagged construct. This clone expresses a stable amount of VDAC3. Live cell imaging shows that fluorescent VDAC3 localizes in the mitochondria. Proteins interacting with VDAC3 have been separated by tandem-affinity purification and 2-D gel electrophoresis and identified by mass spectrometry. In the list of putative interacting proteins, there are cytosolic, mitochondrial, cytoskeletal and ER proteins. Coherent pathways like cell redox homeostasis, response to stress, formation/rearrangement of disulfide bonds, response to unfolded proteins or protein folding have been found to be related to clusters of proteins identified in this experiment. The list of associated proteins has been validated by immunoprecipitation experiments utilizing specific antibodies. Likely biological and pathological processes have been analyzed. Cytosolic proteins associated with VDAC3 include tubulins and cytoskeletal proteins, stress sensors, chaperones and proteasome components, redox-mediating enzymes such as protein disulphide isomerase. The overall picture points to a role for VDAC3 as mediator for the organization of protein complexes and regulator of the traffic of misfolded or non-folded proteins evoked from different stimuli.
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Deletion of Voltage-Dependent Anion Channel 1 knocks mitochondria down triggering metabolic rewiring in yeast
Cellular and Molecular Life Sciences, 2019Co-Authors: Andrea Magrì, Maria Carmela Di Rosa, Ivan Orlandi, Francesca Guarino, Simona Reina, Maria Guarnaccia, Giovanna Morello, Antonio Spampinato, Sebastiano Cavallaro, Angela MessinaAbstract: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 (Δ por1 ) 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, Δ por1 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, Δ por1 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.
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post translational modifications of VDAC1 and vdac2 cysteines from rat liver mitochondria
Biochimica et Biophysica Acta, 2018Co-Authors: Rosaria Saletti, Andrea Magrì, Simona Reina, Maria G G Pittala, Vincenzo Cunsolo, Salvatore Foti, Vito De PintoAbstract:Abstract 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.
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overexpression of human sod1 in VDAC1 less yeast restores mitochondrial functionality modulating beta barrel outer membrane protein genes
Biochimica et Biophysica Acta, 2016Co-Authors: Andrea Magrì, Maria Carmela Di Rosa, Francesca Guarino, Simona Reina, Angela Messina, Marianna Flora Tomasello, Vito De PintoAbstract: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.
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the voltage dependent anion selective channel 1 VDAC1 topography in the mitochondrial outer membrane as detected in intact cell
PLOS ONE, 2013Co-Authors: Marianna Flora Tomasello, Francesca Guarino, Simona Reina, Angela Messina, Vito De PintoAbstract: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.