RESUMEN

Chlorinated gymnastatin and dankastatin alkaloids derived from the fungal strain Gymnascella dankaliensis have been reported to possess significant anticancer activity but their mode of action is unknown. These members possess electrophilic functional groups that can might undergo covalent bond formation with specific proteins to exert their biological activity. To better understand the mechanism of action of this class of natural products, we mapped the proteome-wide cysteine reactivity of the most potent of these alkaloids, dankastatin B, by using activity-based protein profiling chemoproteomic approaches. We identified a primary target of dankastatin B in breast cancer cells as cysteine C65 of the voltage-dependent anion-selective channel on the outer mitochondrial membrane VDAC3. We demonstrated direct and covalent interaction of dankastatin B with VDAC3. VDAC3 knockdown conferred hypersensitivity to dankastatin B-mediated antiproliferative effects in breast cancer cells, thus indicating that VDAC3 was at least partially involved in the anticancer effects of this natural product. Our study reveals a potential mode of action of dankastatin B through covalent targeting of VDAC3 and highlights the utility of chemoproteomic approaches in gaining mechanistic understanding of electrophilic natural products.

Asunto(s)

Productos Biológicos , Neoplasias de la Mama , Humanos , Femenino , Cisteína/química , Productos Biológicos/química , Mitocondrias/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

Enterovirus (EV) 71 caused episodes of outbreaks in China and Southeast Asia during the last few decades. We have previously reported that EV71 induces reactive oxygen species (ROS). However, the underlying mechanism remains elusive. Co-immunoprecipitation-proteomic analysis revealed that enteroviral 2B protein interacted with mitochondrial voltage-dependent anion channel 3 (VDAC3). Knockdown (KD) of VDAC3 expression specifically inhibited enteroviral replication. Single-round viral replication was also inhibited in KD cells, suggesting that VDAC3 plays an essential role in replication. Consistent with this, VDAC3 gene KD significantly reduced the EV71-induced mitochondrial ROS generation. Exogenous 2B expression could induce the mitochondrial ROS generation that was significantly reduced in VDAC3-KD cells or in the Mito-TEMPO-treated cells. Moreover, VDAC3 appears to be necessary for regulation of antioxidant metabolism. VDAC3 gene KD led to the enhancement of such pathways as hypotaurine/taurine synthesis in the infected cells. Taken together, these findings suggest that 2B and VDAC3 interact to enhance mitochondrial ROS generation, which promotes viral replication.

Asunto(s)

Enterovirus Humano A , Picornaviridae , Enterovirus Humano A/metabolismo , Mitocondrias/metabolismo , Picornaviridae/metabolismo , Proteómica , Especies Reactivas de Oxígeno/metabolismo , Replicación Viral , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

Ferroptosis is a novel form of cell death that is closely associated with the formation of many tumors. Our study focused on the mechanism by which long noncoding RNAs (lncRNAs) regulate ferroptosis in gastric cancer (GC) peritoneal metastasis (PM). We utilized lncRNA sequencing and protein profiling analysis to identify ferroptosis-associated lncRNAs and proteins. qRT-PCR was used to analyze the expression of BDNF-AS and FBXW7 in GC tissues and adjacent normal tissues. Chromatin isolation by RNA purification (ChIRP), RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) assays were performed to investigate the interaction between BDNF-AS and its downstream targets. Finally, the function of BDNF-AS was validated in vivo . We demonstrated that BDNF-AS was highly expressed in GC and PM tissues. High BDNF-AS expression was positively related to GC progression and poor prognosis. Functionally, BDNF-AS overexpression protected GC cells from ferroptosis and promoted the progression of GC and PM. Mechanistically, BDNF-AS could regulate FBXW7 expression by recruiting WDR5, thus affecting FBXW7 transcription, and FBXW7 regulated the protein expression of VDAC3 through ubiquitination. Conclusively, our research demonstrated that the BDNF-AS/WDR5/FBXW7 axis regulates ferroptosis in GC by affecting VDAC3 ubiquitination. BDNF-AS might be a biomarker for the evaluation of GC prognosis and the treatment of GC.

Asunto(s)

Ferroptosis , Neoplasias Peritoneales , ARN Largo no Codificante , Neoplasias Gástricas , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Ferroptosis/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Transporte de Membrana Mitocondrial/genética , Neoplasias Peritoneales/genética , ARN Largo no Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Ubiquitinación/genética , Canales Aniónicos Dependientes del Voltaje/genética

RESUMEN

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

Asunto(s)

Cisteína , Canales Aniónicos Dependientes del Voltaje , Cisteína/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Isoformas de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Aniónicos Dependientes del Voltaje/química , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

Voltage-dependent anion-selective channels (VDAC) are pore-forming proteins located in the outer mitochondrial membrane. Three isoforms are encoded by separate genes in mammals (VDAC1-3). These proteins play a crucial role in the cell, forming the primary interface between mitochondrial and cellular metabolisms. Research on the role of VDACs in the cell is a rapidly growing field, but the function of VDAC3 remains elusive. The high-sequence similarity between isoforms suggests a similar pore-forming structure. Electrophysiological analyzes revealed that VDAC3 works as a channel; however, its gating and regulation remain debated. A comparison between VDAC3 and VDAC1-2 underlines the presence of a higher number of cysteines in both isoforms 2 and 3. Recent mass spectrometry data demonstrated that the redox state of VDAC3 cysteines is evolutionarily conserved. Accordingly, these residues were always detected as totally reduced or partially oxidized, thus susceptible to disulfide exchange. The deletion of selected cysteines significantly influences the function of the channel. Some cysteine mutants of VDAC3 exhibited distinct kinetic behavior, conductance values and voltage dependence, suggesting that channel activity can be modulated by cysteine reduction/oxidation. These properties point to VDAC3 as a possible marker of redox signaling in the mitochondrial intermembrane space. Here, we summarize our current knowledge about VDAC3 predicted structure, physiological role and regulation, and possible future directions in this research field.

RESUMEN

Background: Hypothermia attenuates microglial activation and exerts a potential neuroprotective effect against cerebral ischemic-reperfusion (I/R) injury. However, the underlying mechanism remains to be elucidated. In this in vitro study, a model of oxygen-glucose deprivation, followed by recovery (OGD/R), was used to investigate whether hypothermia exerts anti-inflammatory and anti-apoptosis properties via enhanced ubiquitination and down-regulation of voltage-dependent anion channel 3 (VDAC3) expression. Methods: BV2 microglia were cultured under OGD for 4 h following reperfusion with or without hypothermia for 2, 4, or 8 h. M1 and M2 microglia markers [inducible nitric oxide synthase (iNOS) and arginase (Arg)1] were detected using immunofluorescence. The levels of pro-inflammatory cytokines [tumor necrosis factor (TNF) α, interleukin (IL)-1ß], and anti-inflammatory factor (IL-10) were determined using enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was assayed by JC-1 staining using a flow cytometer. Expression of caspase-3, cleaved caspase-3, and VDAC3 were assessed using western blot analysis. The cellular locations and interactions of ubiquitin and VDAC3 were identified using double immunofluorescence staining and immunoprecipitation (IP) assay. Also, the level of the VDAC3 mRNA was determined using a quantitative polymerase chain reaction (qPCR). Results: Hypothermia inhibited the OGD/R-induced microglia activation and differentiation into the M1 type with pro-inflammatory effect, whereas it promoted differentiation to the M2 type with anti-inflammatory effect. Hypothermia attenuated OGD/R-induced loss of Δψm, as well as the expression of apoptosis-associated proteins. Compared to normothermia, hypothermia increased the level of ubiquitinated VDAC3 in the BV2 microglia at both 2 and 8 h of reperfusion. Furthermore, hypothermia did not attenuate VDAC3 mRNA expression in OGD/R-induced microglia. Conclusions: Hypothermia treatment during reperfusion, attenuated OGD/R-induced inflammation, and apoptosis in BV2 microglia. This might be due to the promotion of VDAC3 ubiquitination, identifying VDAC3 as a new target of hypothermia.

RESUMEN

Voltage-dependent anion channel isoform 3 (VDAC3), a channel in the mitochondrial outer membrane, has been suggested to play a role in the regulation of ATP transport and Ca2+ homeostasis. These processes are regarded as important for spermatozoa motility. Accordingly, in previous years, mutations in the VDAC3-encoding gene were detected in spermatozoa with low motility from infertile patients. Therefore, it can be assumed that these mutations would cause alteration of the structure and/or charge of the VDAC3 channel. The review is focused on current knowledge about contribution of VDAC3 activity to human spermatozoa motility and morphology. We also discuss the possibility of designing new molecules that could specifically block the VDAC3 channel and consequently act as male contraceptives.

Asunto(s)

Anticonceptivos Masculinos/farmacología , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Espermatozoides/efectos de los fármacos , Canales Aniónicos Dependientes del Voltaje/antagonistas & inhibidores , Canales Aniónicos Dependientes del Voltaje/metabolismo , Animales , Anticonceptivos Masculinos/química , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Humanos , Masculino , Motilidad Espermática/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/metabolismo

RESUMEN

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with high heritability, although their underlying genetic factors are still largely unknown. Here we present a comprehensive genetic characterization of two ASD siblings from Sardinia by genome-wide copy number variation analysis and whole exome sequencing (WES), to identify novel genetic alterations associated with this disorder. Single nucleotide polymorphism (SNP) array data revealed a rare microdeletion involving CAPG, ELMOD3, and SH2D6 genes, in both siblings. CAPG encodes for a postsynaptic density (PSD) protein known to regulate spine morphogenesis and synaptic formation. The reduced CAPG mRNA and protein expression levels in ASD patients, in the presence of hemizygosity or a particular genetic and/or epigenetic background, highlighted the functional relevance of CAPG as a candidate gene for ASD. WES analysis led to the identification in both affected siblings of a rare frameshift mutation in VDAC3, a gene intolerant to loss of function mutation, encoding for a voltage-dependent anion channel localized on PSD. Moreover, four missense damaging variants were identified in genes intolerant to loss of function variation encoding for PSD proteins: PLXNA2, KCTD16, ARHGAP21, and SLC4A1. This study identifies CAPG and VDAC3 as candidate genes and provides additional support for genes encoding PSD proteins in ASD susceptibility.

RESUMEN

BACKGROUND: Hepatitis B virus (HBV) plays a critical role in the tumorigenic behavior of human hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) have been reported to participate in HCC development via the regulation of their target genes. However, HBV-modulated miRNAs involved in tumorigenesis remain to be identified. Here, we found that a novel highly expressed miRNA, TLRC-m0008_3p (miR-3928v), may be an important factor that promotes the malignancy of HBV-related HCC. METHODS: Solexa sequencing was applied to profile miRNAs, and RT-qPCR was used to identify and quantitate miRNAs. We studied miR-3928v function in HCC cell lines by MTT, colony formation, migration/invasion, and vascular mimicry (VM) assays in vitro and by a xenograft tumor model in vivo. Finally, we predicted and verified the target gene of miR-3928v by a reporter assay, studied the function of this target gene, and cloned the promoter of miR-3928v and the transcription factor for use in dual-luciferase reporter assays and EMSAs. RESULTS: A variant of miR-3928 (miR-3928v) was identified and found to be highly expressed in HBV (+) HCC tissues. Voltage-dependent anion channel 3 (VDAC3) was validated as a target of miR-3928v and found to mediate the effects of miR-3928v in promoting HCC growth and migration/invasion. Furthermore, HBx protein increased early growth response 1 (EGR1) expression and facilitated its translocation into the nucleus to enhance miR-3928v promoter activity in an NF-κB signaling-dependent manner. CONCLUSIONS: miR-3928v is induced by HBx through the NF-κB/EGR1 signaling pathway and down-regulates the tumor suppressor gene VDAC3 to accelerate the progression of HCC.

Asunto(s)

Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , MicroARNs/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Transactivadores/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Regiones no Traducidas 3' , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Ratones , MicroARNs/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , FN-kappa B/metabolismo , Conformación de Ácido Nucleico , Oncogenes , Interferencia de ARN , Transducción de Señal , Proteínas Reguladoras y Accesorias Virales , Canales Aniónicos Dependientes del Voltaje/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

It has been suggested that voltage-dependent anion channels (VDACs) control the release of superoxide from mitochondria. We have previously shown that reactive oxygen species (ROS) such as superoxide (O2̇̄) and hydrogen peroxide (H2O2) stimulate epithelial sodium channels (ENaCs) in sodium-transporting epithelial tissue, including cortical collecting duct (CCD) principal cells. Therefore, we hypothesized that VDACs could regulate ENaC by modulating cytosolic ROS levels. Herein, we find that VDAC3-knockout(KO) mice can maintain normal salt and water balance on low-salt and high-salt diets. However, on a high-salt diet for 2 weeks, VDAC3-KO mice had significantly higher systolic blood pressure than wildtype mice. Consistent with this observation, after a high-salt diet for 2 weeks, ENaC activity in VDAC3-KO mice was significantly higher than wildtype mice. EM analysis disclosed a significant morphological change of mitochondria in the CCD cells of VDAC3-KO mice compared with wildtype mice, which may have been caused by mitochondrial superoxide overload. Of note, compared with wildtype animals, ROS levels in VDAC3-KO animals fed a normal or high-salt diet were consistently and significantly increased in renal tubules. Both the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL) and the mitochondrial ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mito-TEMPO) could reverse the effect of high-salt on ENaC activity and systolic blood pressure in the VDAC3-KO mice. Mito-TEMPO partially correct the morphological changes in VDAC3-KO mice. Our results suggest that knocking out mitochondrial VDAC3 increases ROS, alters renal sodium transport, and leads to hypertension.

Asunto(s)

Canales Epiteliales de Sodio/metabolismo , Peróxido de Hidrógeno/metabolismo , Riñón/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Sodio/metabolismo , Superóxidos/metabolismo , Canales Aniónicos Dependientes del Voltaje/deficiencia , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Óxidos N-Cíclicos/farmacología , Canales Epiteliales de Sodio/genética , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/patología , Transporte Iónico/efectos de los fármacos , Transporte Iónico/genética , Riñón/patología , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/patología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Compuestos Organofosforados/farmacología , Piperidinas/farmacología , Marcadores de Spin , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

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.

Asunto(s)

Cisteína/química , Metionina/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Espectrometría de Masas en Tándem , Canales Aniónicos Dependientes del Voltaje/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Oxidación-Reducción , Péptidos/análisis , Ratas , Tripsina/metabolismo , Canales Aniónicos Dependientes del Voltaje/química

RESUMEN

It has been previously demonstrated that cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H2O2 requires the presence of functional VDAC (YVDAC1). Thus, we decided to transform YVDAC1-depleted yeast cells (Δpor1 cells) with plasmids expressing human VDAC isoforms (HVDAC1, HVDAC2, HVDAC3) to estimate their involvement in the minocycline cytoprotective effect. We observed that only expression of HVDAC3 in Δpor1 cells provided minocycline-mediated cytoprotection against H2O2 although all human isoforms are functional in Δpor1 cells. The observation appears to be important for on-going discussion concerning VDAC isoform roles in mitochondria and cell functioning.

Asunto(s)

Antioxidantes/metabolismo , Antioxidantes/farmacología , Citoprotección , Minociclina/metabolismo , Minociclina/farmacología , Isoformas de Proteínas/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Unión Proteica , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Canales Aniónicos Dependientes del Voltaje/genética

RESUMEN

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