RESUMO
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.
Assuntos
Rutênio Vermelho/metabolismo , Canal de Ânion 1 Dependente de Voltagem/biossíntese , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Citocromos c/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Rutênio Vermelho/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Estaurosporina/farmacologia , Células U937 , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
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.
Assuntos
Cálcio/metabolismo , Modelos Moleculares , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Apoptose/efeitos dos fármacos , Azidas/farmacologia , Sítios de Ligação/genética , Linhagem Celular Transformada , Expressão Gênica , Humanos , Camundongos , Mutação de Sentido Incorreto , Compostos Organometálicos/farmacologia , Peptídeos/antagonistas & inibidores , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Raios Ultravioleta , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
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.
Assuntos
Apoptose/fisiologia , Fluoxetina/metabolismo , Mitocôndrias Hepáticas/metabolismo , Porinas/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Animais , Linhagem Celular , Citocromos c/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Ratos , Estaurosporina/metabolismo , Canais de Ânion Dependentes de VoltagemRESUMO
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.
Assuntos
Apoptose , Hexoquinase/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Citocromos c/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica , Hexoquinase/antagonistas & inibidores , Hexoquinase/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Alinhamento de Sequência , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+-independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with approximately 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel.