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1.
Cell Mol Life Sci ; 81(1): 99, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38386071

RESUMO

Proneural genes play a crucial role in neuronal differentiation. However, our understanding of the regulatory mechanisms governing proneural genes during neuronal differentiation remains limited. RFX4, identified as a candidate regulator of proneural genes, has been reported to be associated with the development of neuropsychiatric disorders. To uncover the regulatory relationship, we utilized a combination of multi-omics data, including ATAC-seq, ChIP-seq, Hi-C, and RNA-seq, to identify RFX4 as an upstream regulator of proneural genes. We further validated the role of RFX4 using an in vitro model of neuronal differentiation with RFX4 knock-in and a CRISPR-Cas9 knock-out system. As a result, we found that RFX4 directly interacts with the promoters of POU3F2 and NEUROD1. Transcriptomic analysis revealed a set of genes associated with neuronal development, which are highly implicated in the development of neuropsychiatric disorders, including schizophrenia. Notably, ectopic expression of RFX4 can drive human embryonic stem cells toward a neuronal fate. Our results strongly indicate that RFX4 serves as a direct upstream regulator of proneural genes, a role that is essential for normal neuronal development. Impairments in RFX4 function could potentially be related to the development of various neuropsychiatric disorders. However, understanding the precise mechanisms by which the RFX4 gene influences the onset of neuropsychiatric disorders requires further investigation through human genetic studies.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Homeodomínio , Neurônios , Fatores do Domínio POU , Fatores de Transcrição de Fator Regulador X , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , RNA-Seq , Diferenciação Celular , Proteínas de Homeodomínio/genética , Fatores do Domínio POU/genética , Fatores de Transcrição de Fator Regulador X/genética
2.
Am J Cancer Res ; 13(9): 4446-4465, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37818060

RESUMO

Papillary thyroid cancer (PTC) is the most common type of endocrine cancer worldwide. Generally, PTC has an excellent prognosis; however, lymph node metastases and recurrences occur frequently. Over the last decade, circular RNAs (circRNAs), a large class of noncoding RNAs (ncRNAs), have emerged as key regulators of various tumor progression pathways. Here, we aimed to identify novel circRNAs as PTC biomarkers. Differentially expressed circRNAs and mRNAs were analyzed using public datasets from the Gene Expression Omnibus and Cancer Genome Atlas. In addition, we screened for target miRNAs using online prediction databases. Based on these results, we established a circRNA-miRNA-mRNA regulatory network associated with PTC, in which protein-protein interaction networks led to the identification of hub genes. Functional enrichment and survival analyses were performed to gain insights into the biological mechanisms of circRNA involvement. As a result, we found that two circRNAs (hsa_circ_0041829 and has_circ_0092299), four miRNAs (miR-369, miR-486, miR-574, and miR-665), and nine hub genes (BBC3, E2F1, FYN, MAG, SDC1, SDC3, SNAP25, TK1, and TYMS) play significant roles in PTC progression. This study provides a novel framework for understanding the roles of circRNA-miRNA-mediated gene regulation in PTC. It also introduces potential therapeutic targets and prognostic biomarkers, which may serve as a basis for developing targeted therapeutic interventions for PTC.

3.
J Autoimmun ; 133: 102940, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36323068

RESUMO

Primary biliary cholangitis (PBC) is an autoimmune disease that involves chronic inflammation and injury to biliary epithelial cells. To identify critical genetic factor(s) in PBC patients, we performed whole-exome sequencing of five female siblings, including one unaffected and four affected sisters, in a multi-PBC family, and identified 61 rare heterozygote variants that segregated only within the affected sisters. Among them, we were particularly interested in caspase-10, for although several caspases are involved in cell death, inflammation and autoimmunity, caspase-10 is little known from this perspective. We generated caspase-10 knockout macrophages, and then investigated the obtained phenotypes in comparison to those of its structurally similar protein, caspase-8. Unlike caspase-8, caspase-10 does not play a role during differentiation into macrophages, but after differentiation, it regulates the process of inflammatory cell deaths such as necroptosis and pyroptosis more strongly. Interestingly, caspase-10 displays better protease activity than caspase-8 in the process of RIPK1 cleavage, and an enhanced ability to form a complex with RIPK1 and FADD in human macrophages. Higher inflammatory cell death affected the fibrotic response of hepatic stellate cells; this effect could be recovered by treatment with UDCA and OCA, which are currently approved for PBC patients. Our findings strongly indicate that the defective roles of caspase-10 in macrophages contribute to the pathogenesis of PBC, thereby suggesting a new therapeutic strategy for PBC treatment.


Assuntos
Cirrose Hepática Biliar , Humanos , Feminino , Caspase 10 , Caspase 8/genética , Cirrose Hepática Biliar/genética , Morte Celular/genética
4.
Exp Mol Med ; 54(2): 156-168, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35169223

RESUMO

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.


Assuntos
Neoplasias Colorretais , Metilação de DNA , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ilhas de CpG/genética , Proteínas de Homeodomínio , Humanos , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Proteínas do Tecido Nervoso , Oncogenes , Transativadores
5.
Biochem Biophys Res Commun ; 590: 125-131, 2022 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-34974300

RESUMO

Embryonic stem cells (ESCs) are derived from the inner cell mass of developing blastocysts, which have self-renewal ability and have the potential to develop or reconstitute into all embryonic lineages. Selenophosphate synthetase 1 (SEPHS1) is an essential protein in mouse early embryo development. However, the role of SEPHS1 in mouse ESCs remains to be elucidated. In this study, we generated Sephs1 KO ESCs and found that deficiency of SEPSH1 has little effect on pluripotency maintenance and proliferation. Notably, SEPHS1 deficiency impaired differentiation into three germ layers and gastruloid aggregation in vitro. RNA-seq analysis showed SEPHS1 is involved in cardiogenesis, verified by no beating signal in Sephs1 KO embryoid body at d10 and low expression of cardiac-related and contraction markers. Taken together, our results suggest that SPEHS1 is dispensable in ESC self-renewal, but indispensable in subsequent germ layer differentiation especially for functional cardiac lineage.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miocárdio/citologia , Fosfotransferases/metabolismo , Animais , Diferenciação Celular/genética , Corpos Embrioides/citologia , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases/deficiência , Transcrição Gênica
6.
Oncol Rep ; 42(6): 2686-2693, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31578581

RESUMO

In recent years, efforts to treat cancer by improving the immune function of patients have received a great deal of attention. As part of the immune system, complement is also under such evaluation. Among the many components of the complement system, complement decay accelerating factor (CD55 or DAF) is known to inhibit complement­mediated cell lysis. However, little is known about the role of CD55 in terms of cancer therapy. The present study aimed to demonstrate that increased levels of CD55 are strongly correlated with the progression of colorectal cancer. A novel CD55 chimeric monoclonal antibody was developed that may boost the immune response, thereby suppressing cancer. The CD55 antibody treatment activated complement and therefore suppressed the proliferation, invasion and migration of colorectal cancer cells. This tumoricidal activity is partly explained by the inflammatory response via the activation of proinflammatory cytokines. In addition, the CD55 antibody treatment synergistically enhanced the tumoricidal activity of 5­FU in colorectal cancer cells, suggesting that combined treatment may be a better strategy in colorectal cancer therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD55/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Antígenos CD55/imunologia , Antígenos CD55/farmacologia , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Proteínas do Sistema Complemento/genética , Citotoxicidade Imunológica/efeitos dos fármacos , Sinergismo Farmacológico , Fluoruracila/farmacologia , Células HT29 , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica
7.
Sci Rep ; 8(1): 8960, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895866

RESUMO

Decay-accelerating factor (CD55 or DAF) inhibits complement-dependent cytotoxicity. We determined that CD55 is overexpressed in 76.47% of human non-small cell lung cancer tissue specimens. We therefore developed a lutetium-177-labeled chimeric monoclonal antibody against CD55. CD55-specific single-chain variable fragment (scFv) was selected from a naïve chicken scFv phage-display library, converted to IgG, and radiolabeled with lutetium-177 to generate a 177Lu-anti-CD55 antibody. We then charaterized the biodistribution of this antibody in a mouse model of pleural metastatic lung cancer. The 177Lu-anti-CD55 antibody was primarily retained in tumor tissue rather than normal tissue. Treatment of the mice with 177Lu-anti-CD55 reduced the growth of lung tumors and improved median survival in vivo by two-fold compared to controls. Finally, 177Lu-anti-CD55 also enhanced the antitumor activity of cisplatin both in vitro and in vivo. These data suggest 177Lu-anti-CD55 antibody is a promising theranostic agent for pleural metastatic lung cancer.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígenos CD55/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/radioterapia , Lutécio/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Pleurais/radioterapia , Radioimunoterapia , Radioisótopos/farmacologia , Nanomedicina Teranóstica , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias Pleurais/patologia , Neoplasias Pleurais/secundário , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Biochim Biophys Acta Mol Cell Res ; 1865(7): 995-1001, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29694915

RESUMO

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.


Assuntos
Histidina/química , Bombas de Íon/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismo , Células 3T3 , Animais , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Desaminação , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteína bcl-X/genética
9.
Immune Netw ; 18(1): e11, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29503741

RESUMO

The complement is a part of the immune system that plays several roles in removing pathogens. Despite the importance of the complement system, the exact role of each component has been overlooked because the complement system was thought to be a nonspecific humoral immune mechanism that worked against pathogens. Decay-accelerating factor (DAF or CD55) is a known inhibitor of the complement system and has recently attracted substantial attention due to its role in various diseases, such as cancer, protein-losing enteropathy, and malaria. Some protein-losing enteropathy cases are caused by CD55 deficiency, which leads to complement hyperactivation, malabsorption, and angiopathic thrombosis. In addition, CD55 has been reported to be an essential host receptor for infection by the malaria parasite. Moreover, CD55 is a ligand of the seven-span transmembrane receptor CD97. Since CD55 is present in various cells, the functional role of CD55 has been expanded by showing that CD55 is associated with a variety of diseases, including cancer, malaria, protein-losing enteropathy, paroxysmal nocturnal hemoglobinuria, and autoimmune diseases. This review summarizes the current understanding of CD55 and the role of CD55 in these diseases. It also provides insight into the development of novel drugs for the diagnosis and treatment of diseases associated with CD55.

10.
Exp Cell Res ; 351(1): 51-58, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28034671

RESUMO

NADPH oxidase (NOX) generates reactive oxygen species (ROS) and has been suggested to mediate cell proliferation in some cancers. Here, we show that an increase in the expression of NOX5 long form (NOX5-L) is critical for tumor progression in breast tumor tissues. Immunostaining of clinical samples indicated that NOX5 was overexpressed in 41.1% of breast ductal carcinoma samples. NOX5-L depletion consistently suppressed cell proliferation, invasion, and migration in vitro. Antibody-mediated neutralization of NOX5-L attenuated tumor progression in a mouse xenograft model. Promoter analysis revealed that NOX5-L expression is regulated by STAT5A in breast cancer cells. Based on our novel findings, we suggest that inhibition of NOX5-L may be a promising therapeutic strategy that exerts anti-cancer effects via the modulation of ROS-mediated cell signaling.


Assuntos
Proliferação de Células , Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NADPH Oxidase 5 , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Metástase Neoplásica , Regiões Promotoras Genéticas , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética
11.
Oncotarget ; 7(7): 7856-65, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26760963

RESUMO

G protein-coupled receptors (GPCRs) are among the most significant therapeutic targets and some of them promote the growth and metastasis of cancer. Here, we show that an increase in the levels of GPR171 is crucial for lung cancer tumor progression in vitro and in vivo. Immunostaining of clinical samples indicated that GPR171 was overexpressed in 46.8% of lung carcinoma tissues. Depletion of GPR171 with an anti-GPR171 antibody decreased proliferation of lung carcinoma cells and attenuated tumor progression in a mouse xenograft model. Knockdown of GPR171 also inhibited migration and invasion of the lung cancer cell lines. Notably, inhibition of GPR171 synergistically enhanced the tumoricidal activity of an epidermal growth factor receptor (EGFR) inhibitor in lung cancer cells. These results indicate that GPR171 blockade is a promising antineoplastic strategy and provide a preclinical rationale for combined inhibition of GPR171 and EGFR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Neoplasias Pulmonares/patologia , Receptores Acoplados a Proteínas G/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estadiamento de Neoplasias , Prognóstico , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Oncotarget ; 6(36): 39235-46, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26513170

RESUMO

The NADPH oxidase, NOX5, is known to stimulate cell proliferation in some cancers by generating reactive oxygen species (ROS). We show here that the long form of NOX5 (NOX5-L) also promotes cell death, and thus determines the balance of proliferation and death, in skin, breast and lung cancer cells. Moderate expression of NOX5-L induced cell proliferation accompanied by AKT and ERK phosphorylation, whereas an increase in NOX5-L above a certain threshold promoted cancer cell death accompanied by caspase-3 activation. Notably, cisplatin treatment increased NOX5-L levels through CREB activation and enhanced NOX5-L activity through augmentation of Ca2+ release and c-Abl expression, ultimately triggering ROS-mediated cancer cell death-a distinct pathway absent in normal cells. These results indicate that NOX5-L determines cellular responses in a concentration- and context-dependent manner.


Assuntos
Cisplatino/farmacologia , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NADPH Oxidase 5 , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Transfecção , Regulação para Cima/efeitos dos fármacos
13.
Brain Res ; 1590: 97-106, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25451090

RESUMO

The role of astrocytes in glutamate toxicity has been controversial. Here, we show that astrocytes in neuron-astrocyte co-cultures increased neuronal sensitivity to chronic glutamate exposure but not to acute exposure. Enhanced neuronal toxicity by chronic exposure was dependent on astrocyte cell numbers. A reduced generation of extracellular H2O2 induced by glutamate was observed in co-cultures. Further, neuronal glutamate toxicity was not suppressed by NADPH oxidase (Nox) inhibitors, catalase or Nox4 knockdown in co-cultures, whereas these compounds effectively reduced the toxicity in pure neuron cultures. Instead, the intracellular scavenger of reactive oxygen species, N-acetylcysteine (NAC), reduced neuronal cytotoxicity in co-cultures, whereas catalase worked in pure neuron cultures. Lipoxygenase (LOX) inhibitors attenuated neuronal glutamate toxicity in co-cultures but not in pure neuron cultures. Neuronal 5-LOX activity was increased only in co-cultures, whereas 12-LOX activity was increased in both types of cultures. The cyclooxygenase (COX) inhibitors, indomethacin and NS-398, and the phospholipase A2 (PLA2) inhibitors, LY311727 and MAFP, more effectively reduced neuronal glutamate toxicity in co-cultures than in pure neuron cultures. However, in co-cultures, pre-treating neurons and astrocytes with the same inhibitors generated opposite results. COX inhibitors suppressed neuronal glutamate toxicity in pre-treated neurons rather than astrocytes, whereas PLA2 inhibitors reduced the toxicity in pre-treated astrocytes rather than neurons. Gene-specific knockdown of PLA2 confirmed these results. Knockdown of cPLA2α and/or sPLA2-V in astrocytes rather than in neurons more effectively reduced glutamate toxicity in co-cultures. These findings suggest that astrocytic PLA2 activity increases neuronal sensitivity to chronic glutamate exposure in neuron-astrocyte co-cultures.


Assuntos
Astrócitos/enzimologia , Ácido Glutâmico/toxicidade , Neurônios/efeitos dos fármacos , Fosfolipases A2/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Técnicas de Silenciamento de Genes , Peróxido de Hidrogênio/toxicidade , Lipoxigenase/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Oxidantes/toxicidade , Fosfolipases A2/genética , Cultura Primária de Células , Prostaglandina-Endoperóxido Sintases/metabolismo
14.
Aging (Albany NY) ; 6(7): 524-44, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25063768

RESUMO

Skeletal muscle degenerates progressively, losing mass (sarcopenia) over time, which leads to reduced physical ability and often results in secondary diseases such as diabetes and obesity. The regulation of gene expression by microRNAs is a key event in muscle development and disease. To understand genome­wide changes in microRNAs and mRNAs during muscle aging, we sequenced microRNAs and mRNAs from mouse gastrocnemius muscles at two different ages (6 and 24 months). Thirty­four microRNAs (15 up­regulated and 19 down­regulated) were differentially expressed with age, including the microRNAs miR­206 and ­434, which were differentially expressed in aged muscle in previous studies. Interestingly, eight microRNAs in a microRNA cluster at the imprinted Dlk1­Dio3 locus on chromosome 12 were coordinately down­regulated. In addition, sixteen novel microRNAs were identified. Integrative analysis of microRNA and mRNA expression revealed that microRNAs may contribute to muscle aging through the positive regulation of transcription, metabolic processes, and kinase activity. Many of the age­related microRNAs have been implicated in human muscular diseases. We suggest that genome­wide microRNA profiling will expand our knowledge of microRNA function in the muscle aging process.


Assuntos
Envelhecimento/fisiologia , Perfilação da Expressão Gênica , MicroRNAs/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Genoma , Masculino , Camundongos , MicroRNAs/genética , Proteínas Musculares/genética
15.
PLoS Biol ; 11(6): e1001588, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23823868

RESUMO

The cellular concentration of Bcl-xL is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-xL undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-xL is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-xL for degradation. Additionally, we show that degradation of deamidated Bcl-xL is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-xL, underscoring its importance in Bcl-xL regulation. Our findings strongly suggest that deamidation-regulated Bcl-xL degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.


Assuntos
Amidas/metabolismo , Proteólise , Proteína bcl-X/metabolismo , Sequência de Aminoácidos , Animais , Calpaína/metabolismo , Linhagem Celular , Sequência Conservada , Dano ao DNA , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteína bcl-X/química
16.
Biogerontology ; 13(5): 525-36, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22971926

RESUMO

Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated ß-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.


Assuntos
Senescência Celular , Citosol/metabolismo , Malato Desidrogenase/metabolismo , Sequência de Bases , Western Blotting , Metabolismo dos Carboidratos , Primers do DNA , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Malato Desidrogenase/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Biol Chem ; 278(34): 31902-8, 2003 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-12807881

RESUMO

The Ret finger protein (RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger, a B-box, and a coiled-coil domain (together called RBCC). Although RFP is known to become oncogenic when its RBCC moiety is connected to a tyrosine kinase domain by DNA rearrangement, its biological function is not well defined. Here we show that ectopic expression of RFP in human embryonic kidney 293 cells causes extensive apoptosis, as assessed by multiple criteria. RFP expression activates Jun N-terminal kinase and p38 kinase and also increases caspase-3-like activity. However, RFP failed to release cytochrome c and, therefore, to increase caspase-9-like activity. RFP-induced apoptosis could be blocked by the caspase-8 inhibitor crmA and dominant negative ASK1 but not by Bcl-2. These results reveal a novel RFP death pathway that recruits mitogen-activated protein kinase and caspases independently of mitochondrial events. Domain mapping showed that the intact RBCC moiety is necessary for the pro-apoptotic function of RFP. Moreover, expression of the RBCC moiety further potentiated the pro-apoptotic activity and resulted in a 7-fold increase of caspase activation compared with that induced by full-length RFP. This suggests that a large number of tripartite motif family members sharing the RBCC moiety may participate in the control of cell survival.


Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA , Proteínas Nucleares/fisiologia , Motivos de Aminoácidos , Caspases/metabolismo , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/química , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno
18.
Blood ; 101(12): 5033-8, 2003 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12586629

RESUMO

Peroxiredoxins (Prxs) are a family of antioxidant proteins that reduce peroxide levels by using reducing agents such as thioredoxin. These proteins were characterized to have a number of cellular functions, including cell proliferation and differentiation and protection of specific proteins from oxidative damage. However, the physiological roles of the peroxiredoxins have not been determined. To clarify the physiological relevance of this protein type, we generated a mouse model deficient in Prx II, which is abundantly expressed in all types of cells. The Prx II-/- mice were healthy in appearance and fertile. However, they had splenomegaly caused by the congestion of red pulp with hemosiderin accumulation. Heinz bodies were detected in their peripheral blood, and morphologically abnormal cells were elevated in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). The Prx II-/- mice had significantly decreased hematocrit levels, but increased reticulocyte counts and erythropoietin levels, indicative of a compensatory action to maintain hematologic homeostasis in the mice. In addition, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prx II-/- mice revealed that a variety of RBC proteins were highly oxidized. Our results suggest that Prx II-/- mice have hemolytic anemia and that Prx II plays a major role in protecting RBCs from oxidative stress in mice.


Assuntos
Envelhecimento Eritrocítico , Peroxidases/fisiologia , Anemia Hemolítica/etiologia , Animais , Proteínas Sanguíneas/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Eritropoese , Eritropoetina/sangue , Corpos de Heinz , Hematócrito , Hemossiderina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Peroxidases/deficiência , Peroxirredoxina III , Peroxirredoxinas , Espécies Reativas de Oxigênio/sangue , Contagem de Reticulócitos , Esplenomegalia
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