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1.
Biol Res ; 56(1): 33, 2023 Jun 22.
Article in English | MEDLINE | ID: mdl-37344914

ABSTRACT

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.


Subject(s)
Leukemia, Myeloid, Acute , Pseudogenes , Voltage-Dependent Anion Channels , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mitochondria , Pseudogenes/genetics , Transcriptome , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolism
2.
Int J Mol Sci ; 24(7)2023 Apr 02.
Article in English | MEDLINE | ID: mdl-37047622

ABSTRACT

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane [...].


Subject(s)
Mitochondrial Membranes , Voltage-Dependent Anion Channels , Voltage-Dependent Anion Channels/metabolism , Mitochondrial Membranes/metabolism , Mitochondria/metabolism , Ions/metabolism
3.
Int J Mol Sci ; 24(4)2023 Feb 12.
Article in English | MEDLINE | ID: mdl-36835102

ABSTRACT

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.


Subject(s)
Electron Transport Complex I , Mitochondria , Oxygen Consumption , Voltage-Dependent Anion Channel 1 , Humans , Electron Transport Complex I/metabolism , Electron Transport Complex I/physiology , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Oxygen Consumption/genetics , Porins/metabolism , Protein Isoforms/metabolism , Saccharomyces cerevisiae/metabolism , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 1/metabolism
4.
Int J Mol Sci ; 23(24)2022 Dec 13.
Article in English | MEDLINE | ID: mdl-36555496

ABSTRACT

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.


Subject(s)
Amyotrophic Lateral Sclerosis , Tandem Mass Spectrometry , Humans , Superoxide Dismutase-1/metabolism , Reactive Oxygen Species/metabolism , Voltage-Dependent Anion Channels/metabolism , Protein Processing, Post-Translational , Mitochondrial Membrane Transport Proteins/metabolism
5.
Cell Mol Life Sci ; 77(16): 3195-3213, 2020 Aug.
Article in English | MEDLINE | ID: mdl-31655859

ABSTRACT

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 ρ0 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.


Subject(s)
Mitochondria/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Voltage-Dependent Anion Channel 1/metabolism , Energy Metabolism/genetics , Energy Metabolism/physiology , Genes, Mitochondrial/genetics , Mitochondria/genetics , Mitochondrial Membranes/metabolism , Oxidation-Reduction , Porins/genetics , Porins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Voltage-Dependent Anion Channel 1/genetics
6.
Int J Mol Sci ; 22(23)2021 Nov 27.
Article in English | MEDLINE | ID: mdl-34884639

ABSTRACT

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.


Subject(s)
Mass Spectrometry/methods , Porins/metabolism , Voltage-Dependent Anion Channels/metabolism , Animals , Humans , Hydrophobic and Hydrophilic Interactions , Mass Spectrometry/instrumentation , Protein Processing, Post-Translational
7.
Int J Mol Sci ; 21(19)2020 Oct 07.
Article in English | MEDLINE | ID: mdl-33036380

ABSTRACT

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 (VDAC1-3), 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.


Subject(s)
Gene Expression Regulation , Voltage-Dependent Anion Channels/genetics , Animals , Base Sequence , Binding Sites , Cell Line, Tumor , Computational Biology/methods , Databases, Genetic , Gene Expression Profiling , HeLa Cells , Humans , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Multigene Family , Nucleotide Motifs , Promoter Regions, Genetic , Protein Isoforms , Transcription Factors/metabolism , Transcriptional Activation , Voltage-Dependent Anion Channels/metabolism
8.
Int J Mol Sci ; 21(4)2020 Feb 21.
Article in English | MEDLINE | ID: mdl-32098132

ABSTRACT

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.


Subject(s)
Disulfides/metabolism , Mass Spectrometry , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Voltage-Dependent Anion Channel 2/metabolism , Voltage-Dependent Anion Channels/metabolism , Animals , Cell Line , Humans , Oxidation-Reduction , Protein Isoforms/metabolism , Rats
9.
Biochim Biophys Acta Bioenerg ; 1859(9): 806-816, 2018 09.
Article in English | MEDLINE | ID: mdl-29890122

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.


Subject(s)
Cysteine/chemistry , Mitochondria, Liver/metabolism , Protein Processing, Post-Translational , Voltage-Dependent Anion Channel 1/metabolism , Voltage-Dependent Anion Channel 2/metabolism , Amino Acid Sequence , Animals , Cysteine/metabolism , Male , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 2/chemistry , Voltage-Dependent Anion Channel 2/genetics
10.
Biochim Biophys Acta Bioenerg ; 1859(4): 270-279, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29408701

ABSTRACT

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 ß-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.


Subject(s)
Mitochondria/chemistry , Mitochondrial Membranes/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 2/chemistry , Animals , Binding Sites , Chlorides/chemistry , Chlorides/metabolism , Computational Biology , Gene Expression , Humans , Ion Transport , Kinetics , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Mice , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Molecular Dynamics Simulation , Potassium/chemistry , Potassium/metabolism , Protein Binding , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Structural Homology, Protein , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 1/metabolism , Voltage-Dependent Anion Channel 2/genetics , Voltage-Dependent Anion Channel 2/metabolism , Zebrafish
11.
J Biol Chem ; 291(48): 24986-25003, 2016 Nov 25.
Article in English | MEDLINE | ID: mdl-27738100

ABSTRACT

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 Ca2+ 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.


Subject(s)
Apoptosis/drug effects , Mitochondria/metabolism , Voltage-Dependent Anion Channel 1/antagonists & inhibitors , Animals , Apoptosis/genetics , Calcium Signaling/drug effects , Calcium Signaling/genetics , Energy Metabolism/drug effects , Energy Metabolism/genetics , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Knockout , Mitochondria/genetics , Protein Multimerization/drug effects , Protein Multimerization/genetics , Rats , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 1/metabolism
12.
Biochim Biophys Acta ; 1857(8): 1219-1227, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26947058

ABSTRACT

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.


Subject(s)
Cysteine/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membranes/metabolism , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 2/chemistry , Voltage-Dependent Anion Channels/chemistry , Animals , Conserved Sequence , Evolution, Molecular , Gene Expression , Humans , Ion Transport , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Mutation , Protein Multimerization , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 1/metabolism , Voltage-Dependent Anion Channel 2/genetics , Voltage-Dependent Anion Channel 2/metabolism , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolism
13.
Biochim Biophys Acta ; 1858(4): 813-23, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26806159

ABSTRACT

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.


Subject(s)
Cysteine/chemistry , Disulfides/chemistry , Mitochondrial Membrane Transport Proteins/chemistry , Protein Conformation , Voltage-Dependent Anion Channels/chemistry , Humans , Ion Transport , Molecular Dynamics Simulation , Protein Isoforms/chemistry
14.
Biochim Biophys Acta ; 1857(6): 789-98, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26947057

ABSTRACT

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 ß-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.


Subject(s)
Mitochondria/genetics , Mutation , Saccharomyces cerevisiae Proteins/genetics , Superoxide Dismutase/genetics , Voltage-Dependent Anion Channel 1/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal/drug effects , Herbicides/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Immunoblotting , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Oxidants/pharmacology , Paraquat/pharmacology , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Transformation, Genetic , Voltage-Dependent Anion Channel 1/metabolism
15.
Biochim Biophys Acta Biomembr ; 1859(3): 301-311, 2017 03.
Article in English | MEDLINE | ID: mdl-27989743

ABSTRACT

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. Cys229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122 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.


Subject(s)
Cysteine/chemistry , Methionine/chemistry , Mitochondrial Membrane Transport Proteins/metabolism , Tandem Mass Spectrometry , Voltage-Dependent Anion Channels/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Oxidation-Reduction , Peptides/analysis , Rats , Trypsin/metabolism , Voltage-Dependent Anion Channels/chemistry
16.
J Neurosci ; 34(23): 7988-98, 2014 Jun 04.
Article in English | MEDLINE | ID: mdl-24899720

ABSTRACT

Elevated mammalian target of rapamycin (mTOR) signaling has been found in Alzheimer's disease (AD) patients and is linked to diabetes and aging, two known risk factors for AD. However, whether hyperactive mTOR plays a role in the cognitive deficits associated with AD remains elusive. Here, we genetically reduced mTOR signaling in the brains of Tg2576 mice, a widely used animal model of AD. We found that suppression of mTOR signaling reduced amyloid-ß deposits and rescued memory deficits. Mechanistically, the reduction in mTOR signaling led to an increase in autophagy induction and restored the hippocampal gene expression signature of the Tg2576 mice to wild-type levels. Our results implicate hyperactive mTOR signaling as a previous unidentified signaling pathway underlying gene-expression dysregulation and cognitive deficits in AD. Furthermore, hyperactive mTOR signaling may represent a molecular pathway by which aging contributes to the development of AD.


Subject(s)
Alzheimer Disease/complications , Cognition Disorders , Gene Expression Regulation/genetics , Gene Expression/genetics , Hippocampus/physiopathology , TOR Serine-Threonine Kinases/metabolism , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cognition Disorders/etiology , Cognition Disorders/pathology , Cognition Disorders/therapy , Disease Models, Animal , Gene Expression/drug effects , Gene Expression Profiling , Hippocampus/drug effects , Humans , Immunosuppressive Agents/pharmacology , Maze Learning/drug effects , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Oligonucleotide Array Sequence Analysis , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , TOR Serine-Threonine Kinases/genetics
17.
Biochemistry ; 54(36): 5646-56, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26303511

ABSTRACT

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 ß-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.


Subject(s)
Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Proteins/chemistry , Peptides/chemistry , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 2/chemistry , Voltage-Dependent Anion Channels/chemistry , Circular Dichroism , Computational Biology , Humans , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Isoforms/chemistry , Protein Structure, Secondary , Spectrophotometry, Infrared
18.
Biochim Biophys Acta ; 1827(6): 793-805, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23541892

ABSTRACT

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


Subject(s)
Voltage-Dependent Anion Channel 1/chemistry , Amino Acid Sequence , Membrane Potentials , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Sequence Alignment , Voltage-Dependent Anion Channel 1/physiology
19.
Cell Physiol Biochem ; 34(3): 842-53, 2014.
Article in English | MEDLINE | ID: mdl-25171321

ABSTRACT

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


Subject(s)
Mitochondrial Membrane Transport Proteins/metabolism , Voltage-Dependent Anion Channels/metabolism , Crystallization , Humans , Membrane Potentials , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/physiology , Nuclear Magnetic Resonance, Biomolecular , Porins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Voltage-Dependent Anion Channels/chemistry , Voltage-Dependent Anion Channels/physiology
20.
J Am Soc Mass Spectrom ; 35(7): 1422-1433, 2024 Jul 03.
Article in English | MEDLINE | ID: mdl-38832804

ABSTRACT

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.


Subject(s)
Amino Acid Sequence , Disulfides , Tandem Mass Spectrometry , Voltage-Dependent Anion Channel 2 , Animals , Voltage-Dependent Anion Channel 2/chemistry , Voltage-Dependent Anion Channel 2/metabolism , Voltage-Dependent Anion Channel 2/analysis , Rats , Disulfides/chemistry , Disulfides/analysis , Disulfides/metabolism , Tandem Mass Spectrometry/methods , Oxidation-Reduction , Cysteine/chemistry , Cysteine/analysis , Molecular Sequence Data , Chromatography, High Pressure Liquid/methods
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