RESUMO
BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.
Assuntos
Retrovirus Endógenos/genética , Epigenômica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Redutases do Citocromo/genética , Retrovirus Endógenos/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas/genética , Pirofosfatases/genética , RNA Longo não Codificante , Taxa de Sobrevida , Sequências Repetidas Terminais/genética , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Liver regeneration is a well-orchestrated process that is triggered by tissue loss due to trauma or surgical resection and by hepatocellular death induced by toxins or viral infections. Due to the central role of the liver for body homeostasis, intensive research was conducted to identify factors that might contribute to hepatic growth and regeneration. Using a model of partial hepatectomy several factors including cytokines and growth factors that regulate this process were discovered. Among them, a protein was identified to specifically support liver regeneration and therefore was named ALR (Augmenter of Liver Regeneration). ALR protein is encoded by GFER (growth factor erv1-like) gene and can be regulated by various stimuli. ALR is expressed in different tissues in three isoforms which are associated with multiple functions: The long forms of ALR were found in the inner-mitochondrial space (IMS) and the cytosol. Mitochondrial ALR (23 kDa) was shown to cooperate with Mia40 to insure adequate protein folding during import into IMS. On the other hand short form ALR, located mainly in the cytosol, was attributed with anti-apoptotic and anti-oxidative properties as well as its inflammation and metabolism modulating effects. Although a considerable amount of work has been devoted to summarizing the knowledge on ALR, an investigation of ALR expression in different organs (location, subcellular localization) as well as delineation between the isoforms and function of ALR is still missing. This review provides a comprehensive evaluation of ALR structure and expression of different ALR isoforms. Furthermore, we highlight the functional role of endogenously expressed and exogenously applied ALR, as well as an analysis of the clinical importance of ALR, with emphasis on liver disease and in vivo models, as well as the consequences of mutations in the GFER gene.
Assuntos
Redutases do Citocromo/genética , Regeneração Hepática/genética , Fígado/fisiologia , Animais , Apoptose , Regulação da Expressão Gênica , Humanos , Hepatopatias , Camundongos , Mitocôndrias , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Dobramento de ProteínaRESUMO
Mitochondria are the center of energy metabolism in the cell and the preferential target of various toxicants and ischemic injury. Renal ischemia-reperfusion (I/R) injury triggers proximal tubule injury and the mitochondria are believed to be the primary subcellular target of I/R injury. The promotion of mitochondrial biogenesis (MB) is critical for the prevention I/R injury. The results of our previous study showed that augmenter of liver regeneration (ALR) has anti-apoptotic and anti-oxidant functions. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. To gain further insight into the role of ALR in MB, human kidney (HK)-2 cells were treated with lentiviruses carrying ALR short interfering RNA (siRNA) and a model of hypoxia reoxygenation (H/R) injury in vitro was created. We observed that knockdown of ALR promoted apoptosis of renal tubular cells and aggravated mitochondrial injury, as evidenced by the decrease in the mitochondrial respiratory proteins adenosine triphosphate (ATP) synthase subunit ß, cytochrome c oxidase subunit 1, and nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) beta subcomplex 8. Meanwhile, the production of reactive oxygen species was increased and ATP levels were decreased significantly in HK-2 cells, as compared with the siRNA/control group (p < 0.05). In addition, the mitochondrial DNA copy number and membrane potential were markedly decreased. Furthermore, critical transcriptional regulators of MB (i.e., peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, mitochondrial transcription factor A, sirtuin-1, and nuclear respiratory factor-1) were depleted in the siRNA/ALR group. Taken together, these findings unveil essential roles of ALR in the inhibition of renal tubular cell apoptosis and attenuation of mitochondrial dysfunction by promoting MB in AKI.
Assuntos
Redutases do Citocromo/metabolismo , Rim/patologia , Mitocôndrias/patologia , Biogênese de Organelas , Traumatismo por Reperfusão/patologia , Trifosfato de Adenosina/metabolismo , Apoptose , Linhagem Celular Transformada , Redutases do Citocromo/antagonistas & inibidores , Redutases do Citocromo/genética , DNA Mitocondrial/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
BACKGROUND: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein-coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. METHODS: Lipidomic plasma analysis were performed after EPA supplementation in Apoe-/- mice. Erv1/Chemr23-/- xApoe-/- mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. RESULTS: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe-/- mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1-mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. CONCLUSIONS: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Ácido Eicosapentaenoico/farmacologia , Lipoproteínas LDL/metabolismo , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Placa Aterosclerótica , Receptores Acoplados a Proteínas G/agonistas , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Dieta Ocidental , Modelos Animais de Doenças , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/sangue , Ácido Eicosapentaenoico/metabolismo , Predisposição Genética para Doença , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Necrose , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Fenótipo , Receptores de Quimiocinas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Augmenter of liver regeneration (ALR) plays crucial roles in cell survival and growth. Previous studies have demonstrated that ALR exerts a protective effect on toxic agentinduced cell death in acute T lymphoblastic leukemia cells and ALR knockdown can sensitize cancer cells to radiation. However, the biological functions of ALR against drug resistance in T-cell acute lymphoblastic leukemia are mostly unknown. In the present study, we investigated the effect of small interfering RNA (siRNA)-induced ALR silencing on cell proliferation and sensitivity to vincristine (VCR) of Jurkat cells. We found that ALR siRNA effectively decreased the ALR expression, then inhibited cell growth and increased sensitivity to VCR in Jurkat cells. Flow cytometry assay revealed that the downregulation of ALR expression promoted cell apoptosis and regulated cell cycle distribution. Following incubation with VCR, apoptosis-related proteins, such as pro-PARP, pro-caspase 8, pro-caspase 3 and Bcl-2 were downregulated in the siRNA/ALR group. Pretreatment with siRNA/ALR in combination with VCR resulted in prolonged G2/M arrest, accompanied by downregulation of cdc25c and cdc2 expression and dissociation of cyclin B1. In conclusion, the results of this study demonstrated that targeted inhibition of the ALR expression in Jurkat cells triggered cell growth inhibition and sensitized cells to VCR via promoting apoptosis and regulating the cell cycle.
Assuntos
Apoptose/efeitos dos fármacos , Redutases do Citocromo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Vincristina/administração & dosagem , Proteína 5 Relacionada à Autofagia/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Redutases do Citocromo/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , RNA Interferente Pequeno/genética , Vincristina/efeitos adversosRESUMO
BACKGROUND: The Associating Liver Partition and Portal Ligation for Staged Hepatectomy (ALPPS) depends on a significant inter-stages kinetic growth rate (KGR). Liver regeneration is highly energy-dependent. The metabolic adaptations in ALPPS are unknown. AIMS: i) Assess bioenergetics in both stages of ALPPS (T1 and T2) and compare them with control patients undergoing minor (miHp) and major hepatectomy (MaHp), respectively; ii) Correlate findings in ALPPS with volumetric data; iii) Investigate expression of genes involved in liver regeneration and energy metabolism. METHODS: Five patients undergoing ALPPS, five controls undergoing miHp and five undergoing MaHp. Assessment of remnant liver bioenergetics in T1, T2 and controls. Analysis of gene expression and protein content in ALPPS. RESULTS: Mitochondrial function was worsened in T1 versus miHp; and in T2 versus MaHp (p < 0.05); but improved from T1 to T2 (p < 0.05). Liver bioenergetics in T1 strongly correlated with KGR (p < 0.01). An increased expression of genes associated with liver regeneration (STAT3, ALR) and energy metabolism (PGC-1α, COX, Nampt) was found in T2 (p < 0.05). CONCLUSION: Metabolic capacity in ALPPS is worse than in controls, improves between stages and correlates with volumetric growth. Bioenergetic adaptations in ALPPS could serve as surrogate markers of liver reserve and as target for energetic conditioning.
Assuntos
Metabolismo Energético , Hepatectomia/métodos , Regeneração Hepática , Fígado/cirurgia , Mitocôndrias Hepáticas/metabolismo , Veia Porta/cirurgia , Idoso , Estudos de Casos e Controles , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica , Hepatectomia/efeitos adversos , Humanos , Ligadura , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática/genética , Masculino , Pessoa de Meia-Idade , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
Hepatocellular carcinoma (HCC) is highly chemoresistant and therefore challenges both physicians and patients. Augmenter of liver regeneration (ALR), previously also known as 'hepatic stimulator substance', is reported to inhibit the epithelial-mesenchymal transition (EMT) in HCC, one of the frequent events that occur in cancer metastasis, suggesting that ALR is involved in HCC. In this study, we report for the first time that the transfection of ALR enhances the antitumor effect of chemotherapy with doxorubicin, a typical anticancer drug, on HCC in vitro and in vivo. The efflux of doxorubicin from ALR-transfected HCC cells is efficiently suppressed. This implies the intracellular retention of doxorubicin in tumor cells, which is at least partly attributable to the effective inhibition of ABCB1 and ABCG2 transporter expression in ALR-expressing cells. The downregulation of ALR expression by short hairpin RNA diminishes the antitumor effect of ALR. We further demonstrate that ALR inhibits the AKT/Snail signaling pathway, resulting in the downregulation of ABCB1 and ABCG2 expression. In conclusion, our results suggest that ALR is a potential chemotherapeutic agent against HCC.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Redutases do Citocromo , Doxorrubicina/farmacologia , Neoplasias Hepáticas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/análise , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Redutases do Citocromo/farmacologia , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The acute-phase response (APR) is an inflammatory process triggered mainly by IL-6 in response to neoplasm, tissue injury, infection or inflammation. Signaling of IL-6 is transduced by activating STAT3 which rapidly results in production of acute-phase proteins (APPs) such as fibrinogen ß (FGB) and haptoglobin (HP). Augmenter of liver regeneration (ALR), a hepatotrophic factor supporting liver regeneration, was reported to be upregulated after liver damage. In this study we analyzed the role of ALR for IL-6 signaling and APR. Thus, we investigated the expression and release of APPs in human liver cells under conditions of increased exogenous or endogenous ALR. HepG2 cells and ALR-reexpressing HepG2 cells were treated with IL-6 in the presence or absence of exogenous ALR for different time points. The mRNA expression and release of both FGB and HP were measured by RT-PCR and ELISA. We found that exogenously applied ALR attenuated the IL-6-induced mRNA expression and protein secretion of both FGB and HP. In contrast, IL-6 stimulation in HepG2 cells which re-express ALR, revealed elevated APR shown by increased mRNA expression and secretion of FGB and HP. Furthermore, we found that ALR-mediated regulation of IL-6-induced APP production is accompanied by altered STAT3 activity. While exogenous ALR reduced the IL-6-induced phosphorylation of STAT3, endogenous ALR enhanced STAT3 activity in liver cells. In conclusion, ALR, dependent on its localization, changes APR at least in part, by modifying STAT3 activation. This study shows a dual signaling of ALR and suggests that ALR is pivotal for the regulation of APR, a crucial event in liver injury and regeneration.
Assuntos
Reação de Fase Aguda/genética , Redutases do Citocromo/metabolismo , Hepatócitos/metabolismo , Fator de Transcrição STAT3/metabolismo , Reação de Fase Aguda/patologia , Redutases do Citocromo/genética , Fibrinogênio/genética , Fibrinogênio/metabolismo , Haptoglobinas/genética , Haptoglobinas/metabolismo , Células Hep G2 , Humanos , Interleucina-6/farmacologia , Fígado/metabolismo , Regeneração Hepática , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Fosforilação , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Regulação para CimaRESUMO
Autophagy may have protective effects in renal ischemia-reperfusion (I/R) injury, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR), a widely distributed multifunctional protein that is originally identified as a hepatic growth factor, may participate in the process of autophagy. To investigate the role of ALR in autophagy, ALR expression is knocked-down in human kidney 2 (HK-2) cells with short hairpin RNA lentivirals. Then, the level of autophagy is measured in the shRNA/ALR group and the shRNA/control group in an in vitro model of ischemia-reperfusion (I/R). The results indicate that the level of autophagy in two groups increase, accompanied by increased reactive oxygen species production, especially in the shRNA/ALR group. The AMPK/mTOR signaling pathway is hyperactive in the shRNA/ALR group. Inhibition of autophagy with the AMPK inhibitor compound C induce apoptosis, especially in the shRNA/ALR group. These findings collectively indicate that ALR negatively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.
Assuntos
Apoptose/genética , Redutases do Citocromo/genética , Rim/metabolismo , Traumatismo por Reperfusão/genética , Serina-Treonina Quinases TOR/genética , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Rim/lesões , Rim/patologia , Estresse Oxidativo/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Traumatismo por Reperfusão/patologia , Transdução de SinaisRESUMO
BACKGROUND: Alterations in GFER gene have been associated with progressive mitochondrial myopathy, congenital cataracts, hearing loss, developmental delay, lactic acidosis and respiratory chain deficiency in 3 siblings born to consanguineous Moroccan parents by homozygosity mapping and candidate gene approach (OMIM#613076). Next generation sequencing recently confirmed this association by the finding of compound heterozygous variants in 19-year-old girl with a strikingly similar phenotype, but this ultra-rare entity remains however unknown from most of the scientific community. MATERIALS AND METHODS: Whole exome sequencing was performed as part of a "diagnostic odyssey" for suspected mitochondrial condition in 2 patients, presenting congenital cataracts, progressive encephalomyopathy and hypotrophy and detected unreported compound heterozygous variants in GFER. RESULTS: Thanks to an international data sharing, we found 2 additional patients carrying compound heterozygous variants in GFER. Reverse phenotyping confirmed the phenotypical similarities between the 4 patients. Together with the first literature reports, the review of these 8 cases from 4 unrelated families enables us to better describe this apparently homogeneous disorder, with the clinical and biological stigmata of mitochondrial disease. CONCLUSION: This report highlights the clinical utility of whole exome sequencing and reverse phenotyping for the diagnosis of ultra-rare diseases and underlines the importance of a broad data sharing for accurate clinical delineation of previously unrecognized entities.
Assuntos
Redutases do Citocromo/genética , Sequenciamento do Exoma , Predisposição Genética para Doença , Encefalomiopatias Mitocondriais/genética , Adolescente , Adulto , Criança , Feminino , Heterozigoto , Humanos , Masculino , Encefalomiopatias Mitocondriais/fisiopatologia , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Linhagem , Adulto JovemRESUMO
Family Hominidae, which includes humans and great apes, is recognized for unique complex social behavior and intellectual abilities. Despite the increasing genome data, however, the genomic origin of its phenotypic uniqueness has remained elusive. Clade-specific genes and highly conserved noncoding sequences (HCNSs) are among the high-potential evolutionary candidates involved in driving clade-specific characters and phenotypes. On this premise, we analyzed whole genome sequences along with gene orthology data retrieved from major DNA databases to find Hominidae-specific (HS) genes and HCNSs. We discovered that Down syndrome critical region 4 (DSCR4) is the only experimentally verified gene uniquely present in Hominidae. DSCR4 has no structural homology to any known protein and was inferred to have emerged in several steps through LTR/ERV1, LTR/ERVL retrotransposition, and transversion. Using the genomic distance as neutral evolution threshold, we identified 1,658 HS HCNSs. Polymorphism coverage and derived allele frequency analysis of HS HCNSs showed that these HCNSs are under purifying selection, indicating that they may harbor important functions. They are overrepresented in promoters/untranslated regions, in close proximity of genes involved in sensory perception of sound and developmental process, and also showed a significantly lower nucleosome occupancy probability. Interestingly, many ancestral sequences of the HS HCNSs showed very high evolutionary rates. This suggests that new functions emerged through some kind of positive selection, and then purifying selection started to operate to keep these functions.
Assuntos
Evolução Molecular , Hominidae/genética , Fases de Leitura Aberta , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência Conservada , Redutases do Citocromo/genética , Genoma Humano , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Polimorfismo Genético , Proteínas da Gravidez/genética , RNA Longo não Codificante , Seleção GenéticaRESUMO
GFER-related mitochondrial encephalomyopathy has been previously described only in 3 siblings of a consanguineous Moroccan family. Their phenotype included congenital cataracts, hypotonia, developmental delay, and sensorineural hearing loss. Multiple mitochondrial respiratory chain complex deficiencies were identified on muscle biopsy. We describe a now-19-year-old woman with adrenal insufficiency, lactic acidosis, congenital cataracts, and respiratory insufficiency secondary to mitochondrial disorder, who was reported by North et al (1996) as a toddler. Compound heterozygous GFER mutations c.373C>T (Q125X) and c.581G>A (R194 H) were recently discovered in this patient. The purpose of this report is (1) to expand the phenotype this ultra-rare disorder and (2) to provide a review of the literature describing the unique finding of adrenal insufficiency in patients with molecularly confirmed disorders of mitochondrial metabolism.
Assuntos
Acidose Láctica/genética , Insuficiência Adrenal/genética , Catarata/genética , Redutases do Citocromo/genética , Doenças Mitocondriais/genética , Acidose Láctica/patologia , Acidose Láctica/fisiopatologia , Insuficiência Adrenal/patologia , Insuficiência Adrenal/fisiopatologia , Catarata/patologia , Catarata/fisiopatologia , Consanguinidade , Família , Feminino , Humanos , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Marrocos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Fenótipo , Adulto JovemRESUMO
OBJECTIVE: To investigate the effects of over-expression of 23 kDa augmenter of liver regeneration (ALR) on cell proliferation and apoptosis in the normal human hepatic cell line LO2. METHODS: The recombinant plasmid expressing 23 kDa ALR (pcDNA6/23 kDa ALR) was constructed and transfected into LO2 cells with MegaTran 1.0 transfection reagent. The expressions of ALR mRNA and protein in LO2 cells were detected by real-time quantitative PCR and Western blotting, respectively; MTS assay was used to detect the cell proliferation of LO2 cells; cell cycle and apoptosis of LO2 cells were measured by flow cytometry. RESULTS: The recombinant expression plasmid pcDNA6/23 kDa ALR was constructed successfully, and the expression of the target protein 23 kDa ALR increased significantly in the transfected cells. Compared with pcDNA6-myc/HisA group, the transient transfection of pcDNA6/23 kDa ALR into LO2 cells promoted cell proliferation and inhibited cell apoptosis induced by H2O2, however, no significant differences were detected in G0 phase and S phase. CONCLUSION: The over-expression of 23 kDa ALR in LO2 cells promoted the cell proliferation and enhanced cell resistance to H2O2.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células , Redutases do Citocromo/metabolismo , Peróxido de Hidrogênio/farmacologia , Apoptose/genética , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Redutases do Citocromo/genética , Citometria de Fluxo , Expressão Gênica , Células Hep G2 , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Oxidantes/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). METHODS: Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. RESULTS: Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. CONCLUSION: ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.
Assuntos
Autofagia , Redutases do Citocromo/metabolismo , Hepatócitos/enzimologia , Adenina/análogos & derivados , Adenina/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Redutases do Citocromo/genética , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Transdução de Sinais , Fatores de Tempo , Transfecção , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
OBJECTIVE: Augmenter of liver regeneration (ALR) is a growth factor that is ubiquitously expressed in multiple forms among eukaryotes. The present study focused on the role of endogenous ALR on the hypoxia/reoxygenation (H/R)-induced inflammatory response in human kidney 2 (HK-2) cells, and the underlying molecular mechanisms. METHODS: To determine the relationship between exogenous and endogenous ALR, exogenous ALR was administrated to HK-2 cells, and endogenous ALR protein and mRNA expression was examined by Western blotting and quantitative real-time polymerase chain reaction (qPCR), respectively. In order to knockdown endogenous ALR expression, HK-2 cells were infected with lentiviral shRNA/ALR, after which cell viability was determined by the MTS cell viability assay. Cells were subjected to hypoxia for 6 h and reoxygenation for 12 h. Levels of monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR. Cells were harvested, and nuclear and phosphorylated protein extracts were prepared from the HK-2 cell lysates. Nuclear factor κB (NF-κB), and phosphorylated extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were analyzed by Western blotting. The translocation of NF-κB was detected by immunofluorescence. RESULTS: Exogenous ALR inhibited the expression of endogenous ALR. Lentiviral shRNA/ALR markedly downregulated endogenous ALR expression, whereas there were no changes in ALR expression in lentiviral shRNA/control HK-2 cells. The results of the MTS assay showed that silencing ALR expression did not influence cell viability. H/R led to increased production of MCP-1, IL-6, and TNF-α. However, knockdown of ALR attenuated the inflammatory response via inhibition of ERK, p38, and JNK phosphorylation. The translocation of NF-κB into the nucleus was also decreased. CONCLUSIONS: These results suggest that there is a negative feedback loop involving ALR in HK-2 cells. Knockdown of ALR exerts anti-inflammatory actions via suppression of the mitogen-activated protein kinase signaling pathway.
Assuntos
Redutases do Citocromo/genética , Redutases do Citocromo/fisiologia , Inflamação/genética , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quimiocinas/biossíntese , Citocinas/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Lentivirus/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , RNA Interferente Pequeno/genéticaRESUMO
ALR is a mystic protein. It has a so called "long" 22 kDa and a "short" 15 kDa forms. It has been described after partial hepatectomy and it has just been considered as a key protein of liver regeneration. At the beginning of the 21st century it has been revealed that the "long" form is localized in the mitochondrial intermembrane space and it is an element of the mitochondrial protein import and disulphide relay system. Several proteins of the substrates of the mitochondrial disulphide relay system are necessary for the proper function of the mitochondria, thus any mutation of the ALR gene leads to mitochondrial diseases. The "short" form of ALR functions as a secreted extracellular growth factor and it promotes the protection, regeneration and proliferation of hepatocytes. The results gained on the recently generated conditional ALR mutant mice suggest that ALR can play an important role in the pathogenesis of alcoholic and non-alcoholic steatosis. Since the serum level of ALR is modified in several liver diseases it can be a promising marker molecule in laboratory diagnostics.
Assuntos
Redutases do Citocromo/fisiologia , Hepatócitos/metabolismo , Regeneração Hepática , Mitocôndrias Hepáticas/metabolismo , Animais , Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Hepatectomia , Humanos , Mutação , Oxirredutases atuantes sobre Doadores de Grupo EnxofreRESUMO
Oxidative stress plays an important role in cellular destruction. Augmenter of liver regeneration (ALR) is an anti-apoptotic factor that is expressed in all mammalian cells and functions as an anti-oxidant by stimulating the expression of a secretory isoform of clusterin and inhibiting reactive oxygen species (ROS) generation. Previous work from our group showed that ALR expression is upregulated in acute kidney injury (AKI) rats, and recombinant human ALR reduces tubular injury. In the present study, we used small interfering RNA (siRNA) silencing of ALR to examine its role in H2O2 induced mitochondrial injury and apoptosis. Knockdown of ALR increased ROS levels, reduced mitochondrial membrane potential, and increased the release of mitochondrial proteins and the rate of apoptosis in response to H2O2. In addition, the ratio of Bax/Bcl-2 was increased in siRNA/ALR groups treated with H2O2. These data confirm the protective role of ALR against oxidative stress-induced mitochondrial injury and suggest a potential mechanism underlying the protective role of ALR in AKI.
Assuntos
Injúria Renal Aguda/enzimologia , Apoptose , Redutases do Citocromo/metabolismo , Peróxido de Hidrogênio/metabolismo , Túbulos Renais Proximais/enzimologia , Estresse Oxidativo , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Redutases do Citocromo/genética , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/fisiopatologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo EnxofreRESUMO
Erv1 (essential for respiration and viability 1) is an FAD-dependent thiol oxidase of the Erv/ALR (augmenter of liver regeneration) sub-family. It is an essential component of the mitochondrial import and assembly (MIA) pathway, playing an important role in the oxidative folding of the mitochondrial intermembrane space (IMS) proteins and linking the MIA pathway to the mitochondrial respiratory chain via cytochrome c (cyt c). The importance of the Erv/ALR enzymes was also demonstrated in a recent study where a single mutation in the human ALR (R194H) leads to autosomal recessive myopathy [Di Fonzo, Ronchi, Lodi, Fassone, Tigano, Lamperti, Corti, Bordoni, Fortunato, Nizzardo et al. (2009) Am. J. Hum. Genet. 84, 594-604]. However, the molecular mechanism of the disease is still unclear. In the present study, we use yeast Erv1 as a model to provide clear evidence for a progressive functional defect in the catalytic activity of the corresponding Erv1 R182H mutant. We show that the FAD cofactor was released from Erv1 R182H during its catalytic cycle, which led to the inactivation of the enzyme. We also characterized the effects of the mutation on the folding and stability of Erv1 and tested our in vitro findings in vivo using a yeast genetic approach. The results of the present study allow us to provide a model for the functional defect in Erv1 R182H, which could potentially be extended to human ALR R194H and provides insights into the molecular basis of autosomal recessive myopathy.
Assuntos
Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Doenças Musculares/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Substituição de Aminoácidos , Catálise , Domínio Catalítico/genética , Coenzimas/metabolismo , Redutases do Citocromo/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Ligação Proteica , Estrutura Terciária de Proteína/genética , Homologia de Sequência de AminoácidosRESUMO
Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s(-1). However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s(-1) at air saturation) and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While ß-mercaptoethanol is a very poor substrate of sfALR (â¼0.3 min(-1) at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be physiologically important in preventing the oxidase from catalyzing the potentially harmful oxidation of intracellular glutathione.