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1.
Int J Mol Sci ; 23(17)2022 Sep 01.
Article in English | MEDLINE | ID: mdl-36077343

ABSTRACT

Impaired mitochondrial function has been proposed as a causative factor in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), caused by motor neuron degeneration. Mutations in superoxide dismutase (SOD1) cause ALS and SOD1 mutants were shown to interact with the voltage-dependent anion channel 1 (VDAC1), affecting its normal function. VDAC1 is a multi-functional channel located at the outer mitochondrial membrane that serves as a mitochondrial gatekeeper controlling metabolic and energetic crosstalk between mitochondria and the rest of the cell and it is a key player in mitochondria-mediated apoptosis. Previously, we showed that VDAC1 interacts with SOD1 and that the VDAC1-N-terminal-derived peptide prevented mutant SOD1 cytotoxic effects. In this study, using a peptide array, we identified the SOD1 sequence that interacts with VDAC1. Synthetic peptides generated from the identified VDAC1-binding sequences in SOD1 directly interacted with purified VDAC1. We also show that VDAC1 oligomerization increased in spinal cord mitochondria isolated from mutant SOD1G93A mice and rats. Thus, we used the novel VDAC1-specific small molecules, VBIT-4 and VBIT-12, inhibiting VDAC1 oligomerization and subsequently apoptosis and associated processes such as ROS production, and increased cytosolic Ca2+. VBIT-12 was able to rescue cell death induced by mutant SOD1 in neuronal cultures. Finally, although survival was not affected, VBIT-12 administration significantly improved muscle endurance in mutant SOD1G93A mice. Therefore, VBIT-12 may represent an attractive therapy for maintaining muscle function during the progression of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Disease Models, Animal , Mice , Mice, Transgenic , Mitochondrial Proteins/metabolism , Rats , Superoxide Dismutase/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 1/metabolism
2.
Front Cell Neurosci ; 13: 346, 2019.
Article in English | MEDLINE | ID: mdl-31474832

ABSTRACT

Mutations in superoxide dismutase (SOD1) are the second most common cause of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the death of motor neurons in the brain and spinal cord. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles, such as mitochondria and ER, disrupting their functions. Here, we demonstrate that mutant SOD1 binds specifically to the N-terminal domain of the voltage-dependent anion channel (VDAC1), an outer mitochondrial membrane protein controlling cell energy, metabolic and survival pathways. Mutant SOD1G93A and SOD1G85R, but not wild type SOD1, directly interact with VDAC1 and reduce its channel conductance. No such interaction with N-terminal-truncated VDAC1 occurs. Moreover, a VDAC1-derived N-terminal peptide inhibited mutant SOD1-induced toxicity. Incubation of motor neuron-like NSC-34 cells expressing mutant SOD1 or mouse embryonic stem cell-derived motor neurons with different VDAC1 N-terminal peptides resulted in enhanced cell survival. Taken together, our results establish a direct link between mutant SOD1 toxicity and the VDAC1 N-terminal domain and suggest that VDAC1 N-terminal peptides targeting mutant SOD1 provide potential new therapeutic strategies for ALS.

3.
Cell Death Dis ; 9(2): 107, 2018 01 25.
Article in English | MEDLINE | ID: mdl-29371591

ABSTRACT

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)-a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties-inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.


Subject(s)
Amyloid/chemistry , Amyotrophic Lateral Sclerosis/pathology , Macrophage Migration-Inhibitory Factors/pharmacology , Protein Aggregates/drug effects , Protein Folding , Superoxide Dismutase-1/chemistry , Superoxide Dismutase-1/toxicity , Active Transport, Cell Nucleus/drug effects , Amyotrophic Lateral Sclerosis/metabolism , Biocatalysis , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Humans , Models, Biological , Mutant Proteins/metabolism , Mutant Proteins/toxicity , Protein Binding/drug effects , Protein Folding/drug effects , Protein Multimerization/drug effects , Recombinant Proteins/pharmacology
4.
ACS Chem Neurosci ; 8(10): 2225-2234, 2017 10 18.
Article in English | MEDLINE | ID: mdl-28715630

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, with a 10% genetic linkage, of which 20% of these cases may be attributed to mutations in superoxide dismutase (SOD1). Specific mutations in SOD1 have been associated with disease duration, which can be highly variable ranging from a life expectancy of 3 to beyond 10 years. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles disrupting their function or forms insoluble toxic aggregates. To understand whether these biophysical properties of the mutant protein may influence disease onset and duration, we generated 19 point mutations in the SOD1 gene, based on available clinical data of disease onset and progression from patients. By overexpressing these mutants in motor-neuron-like NSC-34 cells, we demonstrate a variability in misfolding capacity between the different mutants with a correlation between the degree of protein misfolding and mutation severity. We also show a clear variation of the different SOD1 mutants to associate with mitochondrial-enriched fractions with a correlation between mutation severity and this association. In summary, these findings reveal a correlation between the accumulation of misfolded SOD1 species and their mitochondrial association with disease duration but not with disease onset, and they have implications for the potential therapeutic role of suppressing the accumulation of misfolded SOD1.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Mitochondria/metabolism , Motor Neurons/metabolism , Protein Folding , Superoxide Dismutase-1/metabolism , Animals , Disease Models, Animal , Humans , Mice , Mutation/genetics , Nerve Degeneration/metabolism , Superoxide Dismutase-1/genetics
5.
Proc Natl Acad Sci U S A ; 113(36): 10198-203, 2016 09 06.
Article in English | MEDLINE | ID: mdl-27551074

ABSTRACT

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons in the brain and spinal cord. It has been suggested that the toxicity of mutant SOD1 results from its misfolding and accumulation on the cytoplasmic faces of intracellular organelles, including the mitochondria and endoplasmic reticulum (ER) of ALS-affected tissues. Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit the accumulation of misfolded SOD1 and its binding to intracellular membranes, but the role of endogenous MIF in modulating SOD1 misfolding in vivo remains unknown. To elucidate this role, we bred MIF-deficient mice with SOD1(G85R) mice, which express a dismutase-inactive mutant of SOD1 and are considered a model of familial ALS. We found that the accumulation of misfolded SOD1, its association with mitochondrial and ER membranes, and the levels of sedimentable insoluble SOD1 aggregates were significantly higher in the spinal cords of SOD1(G85R)-MIF(-/-) mice than in their SOD1(G85R)-MIF(+/+) littermates. Moreover, increasing MIF expression in neuronal cultures inhibited the accumulation of misfolded SOD1 and rescued from mutant SOD1-induced cell death. In contrast, the complete elimination of endogenous MIF accelerated disease onset and late disease progression and shortened the lifespan of the SOD1(G85R) mutant mice. These findings indicate that MIF plays a significant role in the folding and misfolding of SOD1 in vivo, and they have implications for the potential therapeutic role of up-regulating MIF within the nervous system to modulate the selective accumulation of misfolded SOD1.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Intramolecular Oxidoreductases/genetics , Macrophage Migration-Inhibitory Factors/genetics , Mutation , Protein Aggregates , Spinal Cord/metabolism , Superoxide Dismutase-1/chemistry , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Cell Death/genetics , Disease Models, Animal , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/pathology , Female , Gene Expression , Humans , Intramolecular Oxidoreductases/deficiency , Longevity/genetics , Macrophage Migration-Inhibitory Factors/deficiency , Male , Mice , Mice, Knockout , Mitochondria/chemistry , Mitochondria/pathology , Neurons/metabolism , Neurons/pathology , Primary Cell Culture , Protein Folding , Spinal Cord/pathology , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism
6.
Rare Dis ; 3(1): e1061164, 2015.
Article in English | MEDLINE | ID: mdl-26459694

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons. Mutations in superoxide dismutase (SOD1) cause about 20 percent of familial ALS which is accompanied by accumulation of misfolded SOD1 onto intracellular organelles. Recently, we identified the 12 kDa macrophage migration inhibitory factor (MIF) as a chaperone for mutant SOD1 which is abundant in non-neuronal tissues. Purified recombinant MIF was shown to directly inhibit mutant SOD1 misfolding and association with mitochondria and ER. Elevating MIF in neuronal cells inhibited the accumulation of misfolded SOD1 and its association with mitochondria and ER, and extended survival of mutant SOD1-expressing motor neurons. Our results revealed that the levels of MIF protein are very low in motor neurons, implicating low chaperone activity as a component of selective vulnerability of motor neurons to mutant SOD1 misfolding and toxicity.

7.
Neuron ; 86(1): 218-32, 2015 Apr 08.
Article in English | MEDLINE | ID: mdl-25801706

ABSTRACT

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and the endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in nonneuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and the ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity.


Subject(s)
Macrophage Migration-Inhibitory Factors/metabolism , Protein Folding , Superoxide Dismutase/metabolism , Acid Phosphatase/genetics , Animals , Cell Differentiation/genetics , Cells, Cultured , Choline O-Acetyltransferase/genetics , Choline O-Acetyltransferase/metabolism , Endoplasmic Reticulum/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Induced Pluripotent Stem Cells , Isoenzymes/genetics , Liver/metabolism , Liver/ultrastructure , Mice , Mice, Transgenic , Mitochondria/metabolism , Motor Neurons/physiology , Mutation/genetics , Nerve Tissue Proteins/metabolism , Protein Transport/genetics , Rats , Rats, Transgenic , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Spinal Cord/metabolism , Spinal Cord/ultrastructure , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Tartrate-Resistant Acid Phosphatase
8.
Biochem J ; 429(1): 147-55, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20420578

ABSTRACT

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.


Subject(s)
Apoptosis/genetics , Genes, Dominant , Mitochondria/genetics , Mutation , Voltage-Dependent Anion Channel 1/genetics , Voltage-Dependent Anion Channel 1/metabolism , Animals , Cell Line , Genes, Dominant/physiology , Humans , Protein Binding/genetics , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Voltage-Dependent Anion Channel 1/biosynthesis
9.
Biochim Biophys Acta ; 1797(6-7): 1281-91, 2010.
Article in English | MEDLINE | ID: mdl-20214874

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.


Subject(s)
Apoptosis/physiology , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/metabolism , Animals , Apoptosis Inducing Factor/metabolism , Apoptosis Regulatory Proteins , Base Sequence , Cell Line, Tumor , Cytochromes c/metabolism , DNA Primers/genetics , HEK293 Cells , HeLa Cells , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mitochondrial Proteins/metabolism , Models, Biological , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Multimerization , Protein Structure, Quaternary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Voltage-Dependent Anion Channel 1/antagonists & inhibitors , Voltage-Dependent Anion Channel 1/genetics
10.
J Cell Sci ; 122(Pt 11): 1906-16, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19461077

ABSTRACT

The release of mitochondrial-intermembrane-space pro-apoptotic proteins, such as cytochrome c, is a key step in initiating apoptosis. Our study addresses two major questions in apoptosis: how are mitochondrial pro-apoptotic proteins released and how is this process regulated? Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) plays a central role in mitochondria-mediated apoptosis. Here, we demonstrate that the N-terminal domain of VDAC1 controls the release of cytochrome c, apoptosis and the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to apoptosis, induced by various stimuli. Employing a variety of experimental approaches, we show that hexokinase and Bcl2 confer protection against apoptosis through interaction with the VDAC1 N-terminal region. We also demonstrate that apoptosis induction is associated with VDAC oligomerization. These results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the mechanism of cytochrome c release and how anti-apoptotic proteins regulate apoptosis and promote tumor cell survival.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/physiology , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/metabolism , Animals , Cell Line , Cytochromes c/metabolism , Hexokinase/genetics , Hexokinase/metabolism , Humans , Mice , Mitochondria/metabolism , Models, Molecular , Mutation , Peptides/genetics , Peptides/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Voltage-Dependent Anion Channel 1/genetics
11.
Biochim Biophys Acta ; 1787(5): 421-30, 2009 May.
Article in English | MEDLINE | ID: mdl-19094960

ABSTRACT

The voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, and thus controls cross-talk between mitochondria and the cytosol. VDAC also serves as a site for the docking of cytosolic proteins, such as hexokinase, and is recognized as a key protein in mitochondria-mediated apoptosis. The role of VDAC in apoptosis has emerged from various studies showing its involvement in cytochrome c release and apoptotic cell death as well as its interaction with proteins regulating apoptosis, including the mitochondria-bound isoforms of hexokinase (HK-I, HK-II). Recently, the functional HK-VDAC association has shifted from being considered in a predominantly metabolic light to the recognition of its major impact on the regulation of apoptotic responsiveness of the cell. Here, we demonstrate that the HK-VDAC1 interaction can be disrupted by mutating VDAC1 and by VDAC1-based peptides, consequently leading to diminished HK anti-apoptotic activity, suggesting that disruption of HK binding to VDAC1 can decrease tumor cell survival. Indeed, understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of differing functions, all important for cell life and death. By expressing VDAC1 mutants and VDAC1-based peptides, we have identified VDAC1 amino acid residues and domains important for interaction with HK and protection against apoptosis. These include negatively- and positively-charged residues, some of which are located within beta-strands of the protein. The N-terminal region of VDAC1 binds HK-I and prevents HK-mediated protection against apoptosis induced by STS, while expression of a VDAC N-terminal peptide detaches HK-I-GFP from mitochondria. These findings indicate that the interaction of HK with VDAC1 involves charged residues in several beta-strands and in the N-terminal domain. Displacing HK, serving as the 'guardian of the mitochondrion', from its binding site on VDAC1 may thus be exploited as an approach to cancer therapy.


Subject(s)
Hexokinase/metabolism , Voltage-Dependent Anion Channel 1/physiology , Apoptosis , Breast Neoplasms , Cell Line , Cell Line, Tumor , Female , HeLa Cells , Homeostasis , Humans , Ion Channels/physiology , Kidney , Kinetics , Transfection , U937 Cells , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/genetics
12.
J Biol Chem ; 283(19): 13482-90, 2008 May 09.
Article in English | MEDLINE | ID: mdl-18308720

ABSTRACT

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents.


Subject(s)
Apoptosis , Hexokinase/metabolism , Voltage-Dependent Anion Channel 1/metabolism , Amino Acid Sequence , Animals , Apoptosis/drug effects , Binding Sites , Cell Line , Conserved Sequence , Cytochromes c/metabolism , Cytosol/metabolism , Gene Expression Regulation , Hexokinase/antagonists & inhibitors , Hexokinase/genetics , Humans , Mice , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Kinase Inhibitors/pharmacology , Sequence Alignment , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/genetics
13.
Cell Calcium ; 43(2): 196-204, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17590433

ABSTRACT

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.


Subject(s)
Ruthenium Red/metabolism , Voltage-Dependent Anion Channel 1/biosynthesis , Amino Acid Sequence , Animals , Apoptosis/drug effects , Binding Sites , Cytochromes c/metabolism , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Ruthenium Red/pharmacology , Saccharomyces cerevisiae Proteins/metabolism , Staurosporine/pharmacology , U937 Cells , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/genetics
14.
J Cell Physiol ; 212(2): 551-61, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17503466

ABSTRACT

In a previous study, we presented evidence for the existence of a nucleotide-binding site (NBS) in the N-terminal region of the voltage-dependent anion channel (VDAC1). In this study, further localization and possible roles of the proposed VDAC1-NBS were investigated using site-directed mutagenesis. The predicated NBS of murine VDAC1 (mVDAC1) was mutated by replacing two glycine residues with alanines or a conserved lysine residue with a serine. Expression of the G21A,G23A- and K20S-mVDAC1s in human T-REx-293 cells in which endogenous VDAC1 expression had been silenced affected cell growth and cytosolic ATP levels. While G21A,G23A-mVDAC1-expressing cells displayed growth rates similar to native-mVDAC1-expressing cells, the K20S-mVDAC1-expressing cells displayed significantly retarded growth and increased resistance to cell death. Cells expressing either mVDAC1 mutant also displayed significantly reduced cellular ATP levels. When K20S-mutant mVDAC1 was expressed in porin1-less yeast, the transformed cells grew slower on non-fermentable carbon sources, while isolated mitochondria expressing either mVDAC1 mutant showed significant reduction in ATP synthesis. Purified K20S-mVDAC1 displayed a significant decrease in [alpha-(32)P]BzATP-binding and altered channel properties, that is, reduced ion selectivity, while the G21A,G23A-mutant protein displayed only a mild reduction in channel selectivity. These results suggest that mutations in the proposed VDAC1-NBS, particularly the K20S, altered channel activity, thereby interfering with VDAC function as the major pathway for the transport of metabolites and adenine nucleotides across the outer mitochondrial membrane. Finally, involvement of the VDAC1-NBS in the control of mitochondrial ATP synthesis, cell growth and viability is discussed.


Subject(s)
Adenosine Triphosphate/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae/metabolism , Voltage-Dependent Anion Channel 1/metabolism , Adenosine Triphosphate/analogs & derivatives , Alanine/chemistry , Apoptosis , Binding Sites , Cell Line , Cell Proliferation , Cytosol/metabolism , Glycine/chemistry , Humans , Lysine/chemistry , Mitochondria/pathology , Mutation , Permeability , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Serine/chemistry , Time Factors , Transfection , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/genetics
15.
Cell Calcium ; 41(3): 235-44, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16930689

ABSTRACT

Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca(2+)-binding proteins and to strongly inhibit their Ca(2+)-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca(2+)-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca(2+), in the presence of 1M NaCl, but not Mg(2+), prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [(103)Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca(2+)-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca(2+)-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca(2+)-binding proteins and localizing their Ca(2+)-binding sites.


Subject(s)
Calcium/metabolism , Models, Molecular , Voltage-Dependent Anion Channel 1/metabolism , Animals , Apoptosis/drug effects , Azides/pharmacology , Binding Sites/genetics , Cell Line, Transformed , Gene Expression , Humans , Mice , Mutation, Missense , Organometallic Compounds/pharmacology , Peptides/antagonists & inhibitors , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Binding , Ultraviolet Rays , Voltage-Dependent Anion Channel 1/antagonists & inhibitors , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/genetics
16.
Proc Natl Acad Sci U S A ; 103(15): 5787-92, 2006 Apr 11.
Article in English | MEDLINE | ID: mdl-16585511

ABSTRACT

Mitochondria not only generate cellular energy, but also act as the point for cellular decisions leading to apoptosis. The voltage-dependent anion channel (VDAC), as a major mitochondrial outer-membrane transporter, has an important role in energy production by controlling metabolite traffic and is also recognized as a key protein in mitochondria-mediated apoptosis. In this study, the role of VDAC1 in regulating cell survival and death was investigated by silencing endogenous human (h)VDAC1 expression by using a short hairpin RNA (shRNA)-expressing vector. The shRNA effectively down-regulated the expression in human T-REx-293 cells of hVDAC1 but not murine (m)VDAC1. Cells in which hVDAC1 expression was decreased by approximately 90% proliferated extremely slowly. Normal growth was, however, restored upon expression of mVDAC1 in a tetracycline-regulated manner. Although low tetracycline concentrations promoted cell growth, high concentrations induced mVDAC1 overexpression, leading to cell death. Cells with low levels of VDAC1 showed 4-fold-lower ATP-synthesis capacity and contained low ATP and ADP levels, with a strong correlation between ATP levels and cell growth, suggesting limited metabolite exchange between mitochondria and cytosol. The possibility of suppressing endogenous hVDAC1 expression and introducing native and mutated mVDAC1 is used to further explore the involvement of VDAC1 in apoptosis. Cells suppressed for hVDAC1 but expressing either native mVDAC1 or an E72Q mutant underwent apoptosis induced by various stimuli that can be inhibited by ruthenium red in the native cells but not in the mutated cells, suggesting that VDAC1 regulates apoptosis independent of the apoptosis-inducing pathway.


Subject(s)
Cell Death/physiology , Cell Survival/physiology , Voltage-Dependent Anion Channel 1/genetics , Adenosine Triphosphate/metabolism , Apoptosis/physiology , Base Sequence , Cell Physiological Phenomena , Gene Expression Regulation , Humans , RNA Interference , RNA, Catalytic/genetics
17.
Chem Biol ; 12(11): 1169-78, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16298296

ABSTRACT

Ca2+ as a signaling molecule carries information pivotal to cell life and death via its reversible interaction with a specific site in a protein. Although numerous Ca2+-dependent activities are known, the proteins responsible for some of these activities remain unidentified. We synthesized and characterized a photoreactive reagent, azido ruthenium (AzRu), which interacts specifically with Ca2+ binding proteins and strongly inhibits their Ca2+-dependent activities, regardless of their catalytic mechanisms or functional state as purified proteins, embedded in the membrane or in intact cells. As expected from a Ca2+ binding protein-specific reagent, AzRu had no effect on Ca2+-independent and Mg2+-dependent activities. Az103Ru covalently bound, and specifically labeled, known Ca2+ binding proteins. AzRu is a photoreactive reagent that provides an approach for identification of Ca2+ binding proteins, characterization of their binding sites, and exploration of new Ca2+-dependent processes.


Subject(s)
Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/chemistry , Molecular Probes/analysis , Molecular Probes/chemistry , Apoptosis/drug effects , Azides/chemical synthesis , Azides/chemistry , Azides/pharmacology , Calcium/chemistry , Calcium/metabolism , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium-Transporting ATPases/metabolism , Cations, Divalent , Cell Line , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Humans , Mitochondria/chemistry , Mitochondria/metabolism , Mutation/genetics , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Photosensitizing Agents/chemistry , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum/radiation effects , Staurosporine/pharmacology , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolism
18.
FEBS Lett ; 579(22): 5105-10, 2005 Sep 12.
Article in English | MEDLINE | ID: mdl-16139271

ABSTRACT

Fluoxetine (Prozac) is a potent antidepressant compound inhibiting serotonin reuptake, but also Na+, K+ and Ca2+ channels and reported to both trigger and prevent apoptosis. Recently, fluoxetine was found to increase the voltage sensitivity of the mitochondrial voltage-dependent anion channel (VDAC). VDAC which functions in transporting metabolites across the mitochondria also plays a crucial role in apoptosis. Here, we demonstrate that fluoxetine interacted with VDAC and decreased its conductance. Fluoxetine inhibited the opening of the mitochondrial permeability transition pore, the release of cytochrome c, and protected against staurosporine-induced apoptotic cell death. These findings may explain some of the reported fluoxetine side effects.


Subject(s)
Apoptosis/physiology , Fluoxetine/metabolism , Mitochondria, Liver/metabolism , Porins/metabolism , Selective Serotonin Reuptake Inhibitors/metabolism , Animals , Cell Line , Cytochromes c/metabolism , Enzyme Inhibitors/metabolism , Humans , Rats , Staurosporine/metabolism , Voltage-Dependent Anion Channels
19.
Biochem J ; 377(Pt 2): 347-55, 2004 Jan 15.
Article in English | MEDLINE | ID: mdl-14561215

ABSTRACT

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.


Subject(s)
Apoptosis , Hexokinase/physiology , Mitochondria/metabolism , Porins/metabolism , Animals , Cell Line , Cytochromes c/metabolism , Glucose-6-Phosphate/pharmacology , Hexokinase/metabolism , Hexokinase/pharmacology , Humans , Ion Channels/metabolism , Mitochondria/drug effects , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Patch-Clamp Techniques , Rats , Staurosporine/pharmacology , U937 Cells , Voltage-Dependent Anion Channels
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