RESUMEN
Chemoresistance is the main obstacle in the clinical treatment of osteosarcoma (OS). In this study, we investigated the role of EF-hand domain-containing protein 1 (EFHD1) in OS chemotherapy resistance. We found that the expression of EFHD1 was highly correlated with the clinical outcome after chemotherapy. We overexpressed EFHD1 in 143B cells and found that it increased their resistance to cell death after drug treatment. Conversely, knockdown of EFHD1 in 143BR cells (a cisplatin-less-sensitive OS cell line derived from 143B cells) increased their sensitivity to treatment. Mechanistically, EFHD1 bound to adenine nucleotide translocase-3 (ANT3) and inhibited its conformational change, thereby inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP). This effect could maintain mitochondrial function, thereby favoring OS cell survival. The ANT3 conformational inhibitor carboxyatractyloside (CATR), which can promote mPTP opening, enhanced the chemosensitivity of EFHD1-overexpressing cells when combined with cisplatin. The ANT3 conformational inhibitor bongkrekic acid (BKA), which can inhibit mPTP opening, restored the resistance of EFHD1 knockdown cells. In conclusion, our results suggest that EFHD1-ANT3-mPTP might be a promising target for OS therapy in the future.
Asunto(s)
Proliferación Celular , Cisplatino , Resistencia a Antineoplásicos , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Osteosarcoma , Humanos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Línea Celular Tumoral , Cisplatino/farmacología , Neoplasias Óseas/patología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Translocador 3 del Nucleótido Adenina/metabolismo , Translocador 3 del Nucleótido Adenina/genética , Antineoplásicos/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Animales , Ratones , Unión ProteicaRESUMEN
The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.
Asunto(s)
Síndrome de Klinefelter , Síndrome de QT Prolongado , Síndrome de Turner , Animales , Femenino , Humanos , Masculino , Adenosina Trifosfato , Electrocardiografía , Síndrome de QT Prolongado/genética , Cromosoma X , Pez Cebra/genética , Translocador 3 del Nucleótido AdeninaRESUMEN
Therapy resistance to a selective B-Raf inhibitor (BRAFi) poses a challenge in treating patients with BRAF-mutant melanomas. Here, we report that RNA interference of mortalin (HSPA9/GRP75), a mitochondrial molecular chaperone often upregulated and mislocalized in melanoma, can effectively induce death of vemurafenib-resistant progenies of human B-RafV600E melanoma cell lines, A375 and Colo-829. Mortalin depletion induced death of vemurafenib-resistant cells at similar efficacy as observed in vemurafenib-naïve parental cells. This lethality was correlated with perturbed mitochondrial permeability and was attenuated by knockdown of adenine nucleotide translocase (ANT) and cyclophilin D (CypD), the key regulators of mitochondrial permeability. Chemical inhibition of MEK1/2 and ERK1/2 also suppressed mortalin depletion-induced death and mitochondrial permeability in these cells. These data suggest that mortalin and MEK/ERK regulate an ANT/CypD-associated mitochondrial death mechanism(s) in B-RafV600E melanoma cells and that this regulation is conserved even after these cells develop BRAFi resistance. We also show that doxycycline-induced mortalin depletion can effectively suppress the xenografts of vemurafenib-resistant A375 progeny in athymic nude mice. These findings suggest that mortalin has potential as a candidate therapeutic target for BRAFi-resistant BRAF-mutant tumors.
Asunto(s)
Doxiciclina/administración & dosificación , Proteínas HSP70 de Choque Térmico/genética , Melanoma/tratamiento farmacológico , Proteínas Mitocondriales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Vemurafenib/administración & dosificación , Translocador 3 del Nucleótido Adenina/genética , Animales , Línea Celular Tumoral , Ciclofilinas/genética , Doxiciclina/farmacología , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Melanoma/genética , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación , Vemurafenib/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mortalin [also known as heat shock protein family A (HSP70) member 9 (HSPA9) or glucose-regulated protein 75 (GRP75)] is a mitochondrial molecular chaperone that is often up-regulated and mislocalized in tumors with abnormal activation of the kinases MEK and ERK. Here, we found that mortalin depletion was selectively lethal to tumor and immortalized normal cells expressing the mutant kinase B-RafV600E or the chimeric protein ΔRaf-1:ER and that MEK-ERK-sensitive regulation of the peptide-binding domain in mortalin was critical to cell survival or death. Proteomics screening identified adenine nucleotide translocase 3 (ANT3) as a previously unknown mortalin substrate and cell survival/death effector. Mechanistically, increased MEK-ERK signaling activity and mortalin function converged opposingly on the regulation of mitochondrial permeability. Specifically, whereas MEK-ERK activity increased mitochondrial permeability by promoting the interaction between ANT3 and the peptidyl-prolyl isomerase cyclophilin D (CypD), mortalin decreased mitochondrial permeability by inhibiting this interaction. Hence, mortalin depletion increased mitochondrial permeability in MEK-ERK-deregulated cells to an extent that triggered cell death. HSP70 inhibitor derivatives that effectively inhibited mortalin suppressed the proliferation of B-RafV600E tumor cells in culture and in vivo, including their B-Raf inhibitor-resistant progenies. These findings suggest that targeting mortalin has potential as a selective therapeutic strategy in B-Raf-mutant or MEK-ERK-driven tumors.
Asunto(s)
Translocador 3 del Nucleótido Adenina/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Mutación , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Translocador 3 del Nucleótido Adenina/genética , Línea Celular Tumoral , Proteínas HSP70 de Choque Térmico/genética , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Neoplasias/genética , Permeabilidad , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as "staurosporine" or the AAC highly selective inhibitor "carboxyatractyloside". Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.
Asunto(s)
Translocador 2 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/genética , Arilamina N-Acetiltransferasa/genética , Isoenzimas/genética , Neoplasias Renales/genética , Translocasas Mitocondriales de ADP y ATP/genética , Secuencia de Aminoácidos/genética , Apoptosis/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Estimación de Kaplan-Meier , Riñón/metabolismo , Riñón/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Translocasas Mitocondriales de ADP y ATP/metabolismo , Fosforilación OxidativaRESUMEN
Protein post-translational modification by ubiquitin-fold modifier 1, UFM1, regulates many biological processes such as response to endoplasmic reticulum stress and regulation of tumor progression. A recent study has indicated that the UFM1-binding and PCI domain-containing protein 1 (UFBP1) is required for the conjugation of UFM1 to a substrate. However, other biological functions of UFBP1 have not been explored. Here, we use immunoprecipitation and label-free quantitative proteomics to identify UFBP1-interacting proteins in a mammalian cell line. About 80 potential interacting proteins are obtained from MS analyses of three biological replicates. Bioinformatics analyses of these proteins suggest that UFBP1 may participate in the regulation of protein folding, stability, and trafficking. Biochemical experiments discover that UFBP1 expression downregulates the protein level and reduces the stability of several of its interacting proteins, while UFBP1 knockdown increases their protein levels. Protein synthesis inhibition and proteasomal inhibition experiments reveal that UFBP1 promotes their ubiquitination and degradation. Experiments using a model UFBP1-interacting protein ANT3 demonstrate that UFBP1 enhances the interaction between ANT3 and its E3 ligase and thus promotes its ubiquitination and degradation. Our work elucidates a novel molecular mechanism by which UFBP1 regulates protein ubiquitination and degradation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Procesamiento Proteico-Postraduccional , Proteolisis , Proteómica , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Translocador 3 del Nucleótido Adenina/metabolismo , Animales , Línea Celular , Regulación hacia Abajo , Células HEK293 , Humanos , Mamíferos , Unión Proteica , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by the viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed not only that the activity of caspase-3 and -9 is increased in PCV2-infected and ORF4-transfected cells but also that cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 protein colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, coimmunoprecipitation and pulldown analyses confirmed that the ORF4 protein interacts directly with mitochondrial ANT3 (mtANT3). Binding domain analysis further confirmed that N-terminal residues 1 to 30 of the ORF4 protein, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 protein and PCV2, indicating that ANT3 plays an important role in ORF4 protein-induced apoptosis during PCV2 infection. Taken together, these data indicate that the ORF4 protein is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE The porcine circovirus type 2 (PCV2) protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by the ORF4 protein. The ORF4 protein contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspase-3- and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We conclude that the ORF4 protein is a mitochondrial targeting protein and reveal a mechanism whereby circovirus recruits ANT3 to induce apoptosis.
Asunto(s)
Translocador 3 del Nucleótido Adenina/metabolismo , Apoptosis/genética , Circovirus/genética , Circovirus/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Translocador 3 del Nucleótido Adenina/genética , Animales , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular , Infecciones por Circoviridae/inmunología , Infecciones por Circoviridae/virología , Citocromos c/metabolismo , Células HEK293 , Humanos , Mitocondrias/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , PorcinosRESUMEN
Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT was not mediating mitochondrial ATP/ADP exchange in cancer cells [corrected]. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.
Asunto(s)
Translocador 2 del Nucleótido Adenina/metabolismo , Translocador 3 del Nucleótido Adenina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Hepatocitos/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Glucólisis/efectos de los fármacos , Hepatocitos/patología , Masculino , Mitocondrias Hepáticas/patología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Teleosts living in seawater continually absorb water across the intestine to compensate for branchial water loss to the environment. The present study reveals that the Gulf toadfish (Opsanus beta) rectum plays a comparable role to the posterior intestine in ion and water absorption. However, the posterior intestine appears to rely more on SLC26a6 (a HCO3 (-)/Cl(-) antiporter) and the rectum appears to rely on NKCC2 (SLC12a1) for the purposes of solute-coupled water absorption. The present study also demonstrates that the rectum responds to renoguanylin (RGN), a member of the guanylin family of peptides that alters the normal osmoregulatory processes of the distal intestine, by inhibited water absorption. RGN decreases rectal water absorption more greatly than in the posterior intestine and leads to net Na(+) and Cl(-) secretion, and a reversal of the absorptive short-circuit current (ISC). It is hypothesized that maintaining a larger fluid volume within the distal segments of intestinal tract facilitates the removal of CaCO3 precipitates and other solids from the intestine. Indeed, the expression of the components of the Cl(-)-secretory response, apical CFTR, and basolateral NKCC1 (SLC12a2), are upregulated in the rectum of the Gulf toadfish after 96 h in 60 ppt, an exposure that increases CaCO3 precipitate formation relative to 35 ppt. Moreover, the downstream intracellular effects of RGN appear to directly inhibit ion absorption by NKCC2 and anion exchange by SLC26a6. Overall, the present findings elucidate key electrophysiological differences between the posterior intestine and rectum of Gulf toadfish and the potent regulatory role renoguanylin plays in osmoregulation.
Asunto(s)
Translocador 3 del Nucleótido Adenina/metabolismo , Batrachoidiformes/metabolismo , Hormonas Gastrointestinales/farmacología , Péptidos Natriuréticos/farmacología , Osmorregulación/efectos de los fármacos , Recto/efectos de los fármacos , Animales , Bicarbonatos/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Carbonato de Calcio/farmacología , Cloruros/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Agua/metabolismoRESUMEN
Human cytomegalovirus (HCMV) encodes at least 26 microRNAs (miRNA). These miRNAs are utilized by HCMV to regulate its own genes as well as the genes of the host cell during infection. It has been reported that a cellular gene, solute carrier family 25, member 6 (SLC25A6), which is also designated adenine nucleotide translocator 3 (ANT3), was identified as a candidate target of hcmv-miR-UL36-5p by hybrid PCR. In this study, ANT3 was further demonstrated to be a direct target of hcmv-miR-UL36-5p by luciferase reporter assays. The expression level of ANT3 protein was confirmed, by western blotting, to be directly downregulated by overexpression of hcmv-miR-UL36-5p in HEK293 cells, U373 cells and HELF cells. Moreover, HCMV-infected cells showed a decrease in the ANT3 protein level. Using ANT3-specific small interfering RNA (siRNA) and an inhibitor for hcmv-miR-UL36-5p, it was shown that inhibition of apoptosis by hcmv-miR-UL36-5p in these cells specifically occurred via inhibition of ANT3 expression. These results imply that hcmv-miR-UL36-5 may play the same role during actual HCMV infection in order to establish a balance between the host cell and the virus.
Asunto(s)
Translocador 3 del Nucleótido Adenina/genética , Apoptosis , Infecciones por Citomegalovirus/genética , Citomegalovirus/metabolismo , MicroARNs/metabolismo , ARN Viral/metabolismo , Proteínas Virales/metabolismo , Translocador 3 del Nucleótido Adenina/metabolismo , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/fisiopatología , Infecciones por Citomegalovirus/virología , Regulación hacia Abajo , Regulación Viral de la Expresión Génica , Humanos , MicroARNs/genética , ARN Viral/genética , Proteínas Virales/genéticaRESUMEN
In aerobic eukaryotic cells, the high energy metabolite ATP is generated mainly within the mitochondria following the process of oxidative phosphorylation. The mitochondrial ATP is exported to the cytoplasm using a specialized transport protein, the ADP/ATP carrier, to provide energy to the cell. Any deficiency or dysfunction of this membrane protein leads to serious consequences on cell metabolism and can cause various diseases such as muscular dystrophy. Described as a decisive player in the programmed cell death, it was recently shown to play a role in cancer. The objective of this review is to summarize the current knowledge of the involvement of the ADP/ATP carrier, encoded by the SLC25A4, SLC25A5, SLC25A6 and SLC25A31 genes, in human diseases and of the efforts made at designing different model systems to study this carrier and the associated pathologies through biochemical, genetic, and structural approaches.
Asunto(s)
Apoptosis/fisiología , Redes y Vías Metabólicas/fisiología , Translocasas Mitocondriales de ADP y ATP/genética , Translocasas Mitocondriales de ADP y ATP/fisiología , Modelos Moleculares , Familia de Multigenes/genética , Neoplasias/fisiopatología , Translocador 1 del Nucleótido Adenina/metabolismo , Translocador 2 del Nucleótido Adenina/metabolismo , Translocador 3 del Nucleótido Adenina/metabolismo , Secuencia de Aminoácidos , Apoptosis/genética , Humanos , Redes y Vías Metabólicas/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias/genética , Conformación Proteica , Alineación de SecuenciaRESUMEN
BACKGROUND: The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. RESULTS: From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases. CONCLUSIONS: Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.
Asunto(s)
Translocador 1 del Nucleótido Adenina/genética , Translocador 2 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/genética , Regulación de la Expresión Génica/genética , Translocasas Mitocondriales de ADP y ATP/genética , Transcripción Genética/genética , Biología Computacional , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genéticaRESUMEN
BACKGROUND: The ANT1 gene, encoding ADP/ATP translocase 1, was investigated in an adult patient with an autosomal recessive mitochondrial disorder characterised by congenital cataracts, hypertrophic cardiomyopathy, myopathy and lactic acidosis. METHODS AND RESULTS: ANT1 sequencing showed that the patient was homozygous for a new nucleotide variation, c.111+1GâA, abolishing the invariant GT splice donor site of intron 1. The ANT1 transcript was undetectable in both muscle and skin fibroblasts. A markedly abnormal metabolic profile was found, and skeletal muscle showed a dramatic proliferation of abnormal mitochondria, increased mitochondrial mass, and multiple mitochondrial DNA deletions. No compensating increase in the transcript level of the ANT3 gene, which encodes the human ubiquitous isoform of the ADP/ATP translocase, was observed. The patient's heterozygous mother had normal clinical, biochemical and pathological features. CONCLUSIONS: Complete loss of expression of the ANT1 gene causes a clinical syndrome mainly characterised by cardiomyopathy and myopathy. This report expands the clinical spectrum of ANT1-related human diseases, and emphasises the crucial role of the mitochondrial ADP/ATP carriers in muscle function and pathophysiology of human myopathies.
Asunto(s)
Translocador 1 del Nucleótido Adenina/genética , Cardiomiopatía Hipertrófica/genética , Miopatías Mitocondriales/genética , Translocador 3 del Nucleótido Adenina/genética , Adulto , Secuencia de Bases , Cardiomiopatía Hipertrófica/diagnóstico , Células Cultivadas , ADN Polimerasa gamma , ADN Polimerasa Dirigida por ADN/genética , Exones , Femenino , Expresión Génica , Humanos , Imagen por Resonancia Magnética , Miopatías Mitocondriales/diagnóstico , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Mutación , Neuroimagen , Linaje , Adulto JovenRESUMEN
Most vertebrates have three paralogous genes with identical intron-exon structures and a high degree of sequence identity that encode mitochondrial adenine nucleotide translocase (Ant) proteins, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant3 (Slc25a6). Recently, we and others identified a fourth mammalian Ant paralog, Ant4 (Slc25a31), with a distinct intron-exon structure and a lower degree of sequence identity. Ant4 was expressed selectively in testis and sperm in adult mammals and was indeed essential for mouse spermatogenesis, but it was absent in birds, fish and frogs. Since Ant2 is X-linked in mammalian genomes, we hypothesized that the autosomal Ant4 gene may compensate for the loss of Ant2 gene expression during male meiosis in mammals. Here we report that the Ant4 ortholog is conserved in green anole lizard (Anolis carolinensis) and demonstrate that it is expressed in the anole testis. Further, a degenerate DNA fragment of putative Ant4 gene was identified in syntenic regions of avian genomes, indicating that Ant4 was present in the common amniote ancestor. Phylogenetic analyses suggest an even more ancient origin of the Ant4 gene. Although anole lizards are presumed male (XY) heterogametic, like mammals, copy numbers of the Ant2 as well as its neighboring gene were similar between male and female anole genomes, indicating that the anole Ant2 gene is either autosomal or located in the pseudoautosomal region of the sex chromosomes, in contrast to the case to mammals. These results imply the conservation of Ant4 is not likely simply driven by the sex chromosomal localization of the Ant2 gene and its subsequent inactivation during male meiosis. Taken together with the fact that Ant4 protein has a uniquely conserved structure when compared to other somatic Ant1, 2 and 3, there may be a specific advantage for mammals and lizards to express Ant4 in their male germ cells.
Asunto(s)
Genómica/métodos , Lagartos/genética , Mamíferos/genética , Translocasas Mitocondriales de ADP y ATP/genética , Proteínas de Reptiles/genética , Espermatozoides/metabolismo , Translocador 1 del Nucleótido Adenina/genética , Translocador 1 del Nucleótido Adenina/metabolismo , Translocador 2 del Nucleótido Adenina/genética , Translocador 2 del Nucleótido Adenina/metabolismo , Translocador 3 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos , Mapeo Cromosómico , Evolución Molecular , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Lagartos/metabolismo , Masculino , Mamíferos/metabolismo , Ratones , Ratones Endogámicos C57BL , Translocasas Mitocondriales de ADP y ATP/clasificación , Translocasas Mitocondriales de ADP y ATP/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas de Reptiles/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Espermatozoides/enzimología , Sintenía , Testículo/citología , Testículo/enzimología , Testículo/metabolismoRESUMEN
The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.
Asunto(s)
Apoptosis , Mitocondrias/fisiología , Translocasas Mitocondriales de ADP y ATP/fisiología , Translocador 3 del Nucleótido Adenina/biosíntesis , Translocador 3 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/fisiología , Antineoplásicos/farmacología , Caspasa 9/metabolismo , Proliferación Celular , Forma de la Célula , Citoprotección , Células HeLa , Humanos , Peróxido de Hidrógeno/análisis , Indazoles/farmacología , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/fisiología , Translocasas Mitocondriales de ADP y ATP/biosíntesis , Translocasas Mitocondriales de ADP y ATP/sangre , Translocasas Mitocondriales de ADP y ATP/genética , Fosforilación Oxidativa , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Estaurosporina/farmacología , Superóxidos/análisisRESUMEN
The mossy fiber (MF) system targets the apical dendrites of CA3 pyramidal cells in the stratum lucidum (SL). In mice overexpressing the growth-associated protein GAP-43 there is an apparent ectopic growth of these MFs into the stratum oriens (SO) targeting the basal dendrites of these same pyramidal cells (Aigner et al. (1995) Cell 83:269-278). This is the first evidence to our knowledge that links increased GAP-43 expression with growth of central axons. Here we studied the Aigner et al. transgenic mice but were unable to confirm such growth into SO. However, using quantitative methods we did observe enhanced growth within the regions normally targeted by MFs, for example, the SL in the CA3a region. These contrasting results led us to study MFs with double-immunostaining using an immunohistochemical marker for MFs, the zinc transporter, ZnT3, to visualize the colocalization of transgenic GAP-43 within MFs. Unexpectedly, using both fluorescence and confocal microscopy, we were unable to detect colocalization of GAP-43-positive axons with ZnT3-positive MF axons within the MF pathways, either in the region of the MF axons or in the SL, where MF terminals are abundant. In contrast, the plasma membrane-associated presynaptic marker SNAP-25 did colocalize with transgenic GAP-43-positive terminals in the SL. Synaptophysin, the vesicle-associated presynaptic terminal marker, colocalized with ZnT3 but did not appear to colocalize with GAP-43. The present findings raise important questions about the properties of granule cells and the MF mechanisms that differentially regulate axonal remodeling in the adult hippocampus: (1) Because there appears to be at least two populations of granule cells defined by their differential protein expression, this points to the existence of an intrinsic heterogeneity of granule cell expression beyond that contributed by adult neurogenesis; (2) Giventhe present evidence that growth is induced in mice overexpressing GAP-43 in adjacent non-GAP-43 containing MFs, the potential exists for a heretofore unexplored interaxonal communication mechanism.
Asunto(s)
Axones/fisiología , Proteína GAP-43/metabolismo , Fibras Musgosas del Hipocampo/fisiología , Translocador 3 del Nucleótido Adenina/metabolismo , Animales , Astrocitos/metabolismo , Región CA3 Hipocampal/fisiología , Aumento de la Célula , Pollos , Proteína GAP-43/genética , Hipocampo/fisiología , Ratones , Ratones Transgénicos , Neuronas/fisiología , ARN Mensajero/metabolismo , Sinaptofisina/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
The complete coding sequences of three of sheep genes SLC25A4, SLC25A5 and SLC25A6 were firstly amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Sheep SLC25A4, SLC25A5 and SLC25A6 genes encode three corresponding proteins of 298 amino acids which contain the identically conserved putative mitochondrial carrier protein domain. Sheep SLC25A4 protein has high homology with the SLC25A4 proteins of six species-cattle (99%), human (95%), rat (95%), mouse (94%), dog (94%) and chicken (89%). Sheep SLC25A5 protein has high identity with the SLC25A5 proteins of five species-cattle (100%), dog (99%), mouse (98%), rat (98%) and human (98%). Sheep SLC25A6 protein also has high homology with the SLC25A6 proteins of four species-cattle (99%), human (97%), pig (97%) and chicken (93%). The phylogenetic tree analysis demonstrated that sheep SLC25A4, SLC25A5 and SLC25A6 proteins share a common ancestor. Moreover, SLC25A4, SLC25A5 and SLC25A6 proteins present stronger interaction each other. The tissue expression analysis indicated that sheep SLC25A4, SLC25A5 and SLC25A6 genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.
Asunto(s)
Translocador 1 del Nucleótido Adenina/genética , Translocador 2 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/genética , Perfilación de la Expresión Génica , Ovinos/genética , Translocador 1 del Nucleótido Adenina/química , Translocador 1 del Nucleótido Adenina/metabolismo , Translocador 2 del Nucleótido Adenina/química , Translocador 2 del Nucleótido Adenina/metabolismo , Translocador 3 del Nucleótido Adenina/química , Translocador 3 del Nucleótido Adenina/metabolismo , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
PB1-F2 is a recently described influenza A viral protein that induces apoptosis by binding with two mitochondrial membrane proteins, i.e. VDAC1 (outer membrane) and ANT3 (inner membrane). Knowledge of this binding mechanism could provide insights that would aid in the design of novel inhibitors against this protein. Therefore, to better understand these interactions, we have undertaken this study to model the PB1-F2 protein of the highly pathogenic influenza A virus subtype H5N1. Moreover, a model of human ANT3 was also established. The dynamics of the molecular interactions between the C-terminal region of PB1-F2 protein and VDAC1 and ANT3 were expounded by employing an in silico approach. Our results suggest the involvement of 12 amino acids of PB1-F2 protein, which form hydrophobic contacts with 22 amino acids of VDAC1. Of these, Leu64, Arg75 and Val76 were found to be crucial for mitochondrial targetting. In the case of the PB1-F2-ANT3 complex, 14 amino acids of ANT3 were found to make hydrophobic contacts with 9 amino acids of PB1-F2. Furthermore, two hydrogen bonds were predicted in both complexes PB1-F2/VDAC1 and PB1-F2/ANT3. This study reveals the molecular interactions required for PB1-F2-induced apoptosis and suggests a hypothetical model for future study.
Asunto(s)
Translocador 3 del Nucleótido Adenina/química , Translocador 3 del Nucleótido Adenina/metabolismo , Biología Computacional , Membranas Mitocondriales/química , Modelos Moleculares , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Virus de la Influenza A/clasificación , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Canal Aniónico 1 Dependiente del Voltaje/química , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Genome sequencing of the pig is being accelerated because of its importance as an evolutionary and biomedical model animal as well as a major livestock animal. However, information on expressed porcine genes is insufficient to allow annotation and use of the genomic information. A series of expressed sequence tags of 5' ends of five full-length enriched cDNA libraries (SUSFLECKs) were functionally characterized. SUSFLECKs were constructed from porcine abdominal fat, induced fat cells, loin muscle, liver, and pituitary gland, and were composed of non-normalized and normalized libraries. A total of 55,658 ESTs that were sequenced once from the 5' ends of clones were produced and assembled into 17,684 unique sequences with 7,736 contigs and 9,948 singletons. In Gene Ontology analysis, two significant biological process leaf nodes were found: gluconeogenesis and translation elongation. In functional domain analysis based on the Pfam database, the beta transducin repeat domain of WD40 protein was the most frequently occurring domain. Twelve genes, including SLC25A6, EEF1G, EEF1A1, COX1, ACTA1, SLA, and ANXA2, were significantly more abundant in fat tissues than in loin muscle, liver, and pituitary gland in the SUSFLECKs. These characteristics of SUSFLECKs determined by EST analysis can provide important insight to discover the functional pathways in gene networks and to expand our understanding of energy metabolism in the pig.
Asunto(s)
Tejido Adiposo/metabolismo , Biblioteca de Genes , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Porcinos/genética , Actinas/genética , Actinas/metabolismo , Translocador 3 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/metabolismo , Animales , Células Cultivadas , Citoesqueleto/genética , Metabolismo Energético/genética , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Mitocondrias Hepáticas/genética , Biosíntesis de Proteínas/genética , Alineación de SecuenciaRESUMEN
Adenine nucleotide translocase (ANT) is known as a core component of the mitochondrial permeability transition pore (MPTP) and a key player in cell death. However, its role in camptothecin (CPT)-induced apoptosis has not been examined. We showed that CPT-induced apoptosis in QGY7703 cells and down-regulated the expression of ANT3. Using ANT3 knock-out and overexpression experiments, we provide further evidence that ANT3 plays a contributive role in CPT-induced apoptosis through induction of MPTP. We speculate that the down-regulation of ANT3 upon stimulation with CPT may be part of the molecular basis underlying the mechanism of acquired resistance to CPT.