RESUMO
Mouse double minute 2 (MDM2) is an oncoprotein that is frequently overexpressed in tumors and enhances cellular transformation. Owing to the important role of MDM2 in modulating p53 function, it is crucial to understand the mechanism underlying the regulation of MDM2 levels. We identified ribosomal protein S4X-linked (RPS4X) as a novel binding partner of MDM2 and showed that RPS4X promotes MDM2 stability. RPS4X suppressed polyubiquitination of MDM2 by suppressing homodimer formation and preventing auto-ubiquitination. Moreover, RPS4X inhibited the interaction between MDM2 and Cullin1, a scaffold protein of the Skp1-Cullin1-F-box protein (SCF) complex and an E3 ubiquitin ligase for MDM2. RPS4X expression in cells enhanced the steady-state level of MDM2 protein. RPS4X was associated not only with MDM2 but also with Cullin1 and then blocked the MDM2/Cullin1 interaction. This is the first report of an interaction between ribosomal proteins (RPs) and Cullin1. Our results contribute to the elucidation of the MDM2 stabilization mechanism in cancer cells, expanding our understanding of the new functions of RPs.
Assuntos
Proteínas Culina , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Ribossômicas , Ubiquitinação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Humanos , Proteínas Culina/metabolismo , Proteínas Culina/genética , Animais , Estabilidade Proteica , Camundongos , Ligação Proteica , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Células HEK293RESUMO
Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes' pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Leucemia-Linfoma de Células T do Adulto , Linfoma , Adulto , Animais , Humanos , Camundongos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas Adaptadoras de Sinalização CARD , Guanilato Ciclase , Leucemia-Linfoma de Células T do Adulto/genética , Camundongos Transgênicos , Mutação , NF-kappa B/genética , Proteínas dos RetroviridaeRESUMO
Urea (Ua) is produced in DNA as the result of oxidative damage to thymine and guanine. It was previously reported that Klenow fragment (Kf) exo- incorporated dATP opposite Ua, and that DNA polymerase ß was blocked by Ua. We report here the following nucleotide incorporations opposite Ua by various DNA polymerases: DNA polymerase α, dATP and dGTP (dATP > dGTP); DNA polymerase δ, dATP; DNA polymerase ζ, dATP; Kf exo-, dATP; Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), dGTP and dATP (dGTP > dATP); and DNA polymerase η, dCTP, dGTP, dATP, and dTTP (dCTP > dGTP > dATP > dTTP). DNA polymerases ß and ε were blocked by Ua. Elongation by DNA polymerases δ and ζ stopped after inserting dATP opposite Ua. Importantly, the elongation efficiency to full-length beyond Ua using DNA polymerase η and Dpo4 were almost the same as that of natural DNA.
RESUMO
Pulsed electric fields (PEFs), which are generated by pulsed power technologies, are being tested for their applicability in food processing through protein conformational change and the poration of cell membranes. In this article, enzyme activity change and the permeabilization of agricultural products using pulsed power technologies are reviewed as novel, nonthermal food processes. Compact pulsed power systems have been developed with repetitive operation and moderate output power for application in food processing. Firstly, the compact pulsed power systems for the enzyme activity change and permeabilization are outlined. Exposure to electric fields affects hydrogen bonds in the secondary and tertiary structures of proteins; as a result, the protein conformation is induced to be changed. The conformational change induces an activity change in enzymes such as α-amylase and peroxidase. Secondly, the conformational change in proteins and the induced protein functional change are reviewed. The permeabilization of agricultural products is caused through the poration of cell membranes by applying PEFs produced by pulsed discharges. The permeabilization of cell membranes can be used for the extraction of nutrients and health-promoting agents such as polyphenols and vitamins. The electrical poration can also be used as a pre-treatment for food drying and blanching processes. Finally, the permeabilization of cell membranes and its applications in food processing are reviewed.
Assuntos
Permeabilidade da Membrana Celular/efeitos da radiação , Produtos Agrícolas/química , Eletroporação/métodos , Manipulação de Alimentos/métodos , Conformação Proteica/efeitos da radiação , Eletricidade , Proteínas/químicaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia (ATL). The viral protein HTLV-1 basic leucine-zipper factor (HBZ), which is constitutively expressed in all ATL patient cells, contributes toward the development of ATL; however, the underlying mechanism has not been elucidated yet. Here, we identified HS-1-associated protein X-1 (HAX-1) as a novel binding partner of HBZ. Interestingly, HAX-1 specifically associated with HBZ-US, but not HBZ-SI, in the cytoplasm. HBZ suppressed the polyubiquitination levels of HAX-1 protein by inhibiting the association HAX-1 with F-box protein 25 (FBXO25), which is a member of the SCF E3 ubiquitin ligase complex, and promoted the stabilization of HAX-1 levels. In fact, the protein levels of HAX-1 were significantly increased in HTLV-1 infected and the overexpressing HBZ in uninfected T-cell lines. Enhanced HAX-1 correlated well to suppression of caspase 9 processing, suggesting that HBZ may contribute to the enhancement of antiapoptotic function for HAX-1. Our results revealed a role for HBZ on HAX-1 stabilization by abrogating the ubiquitination-mediated degradation pathway, which may play an important role in understanding the potential mechanisms of HTLV-1 related pathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas dos Retroviridae/metabolismo , Linfócitos T/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Caspase 9/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Células Jurkat , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Proteínas dos Retroviridae/genética , Linfócitos T/virologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4ß (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.
Assuntos
Endocitose/genética , Endossomos/genética , Receptores ErbB/genética , Hepatite B/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Receptores ErbB/química , Células Hep G2 , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , MAP Quinase Quinase 1/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fatores de Transcrição STAT/genética , Simportadores , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Internalização do VírusRESUMO
In this work, the bacterial inactivation effects of shock waves, ultraviolet (UV) light, and electric field produced by high-voltage pulsed discharge in liquid with needle-plate configurations were studied. The contributions of each effect on the bacterial killing ratio in the discharge process were obtained individually by modifying reactor type and usage of glass, quartz, and black balloons. The results showed that the location from the discharge center axis significantly influenced the effects of shock waves and electric fields, although the effect of UV light was not affected by the location in the reactor. The effects of shock waves and electric fields were improved by decreasing the distance from the discharge center axis. Under this experimental condition, the effects of shock waves, UV light, and electric fields produced by discharges on bacterial inactivation were approximately 36.1%, 30.8%, 12.7%, respectively. Other contributions seemed to be due to activated species.
Assuntos
Técnicas Eletroquímicas/instrumentação , Escherichia coli/efeitos da radiação , Esterilização/instrumentação , Microbiologia da Água , Purificação da Água/instrumentação , Eletricidade , Desenho de Equipamento , Escherichia coli/citologia , Infecções por Escherichia coli/microbiologia , Humanos , Raios UltravioletaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper factor (HBZ), which is encoded by the minus strand of the provirus, is constitutively expressed in all ATL patient cells and likely contributes to the development and maintenance of ATL. Furthermore, the overexpression of the myeloid cell leukemia 1 (MCL1) protein is frequently observed in hematological cancers as well as several other types of cancers. Here, we found that the expression of HBZ in cells stabilized MCL1 protein expression and suppressed the MCL1-mediated release of cytochrome c from the mitochondria. This effect was mediated by inhibition of the ubiquitin-dependent degradation of MCL1. In a serial binding assay, HBZ interacted with cullin 1 (CUL1) through a head-to-tail interaction. The association between CUL1 and Skp1, which serves as the molecular scaffold for the components of SCF ubiquitin ligase complexes, was markedly repressed in the presence of HBZ. Mechanistic analysis indicated that HBZ abrogated the CUL1 association with Skp1, which in turn promoted the cellular expression of MCL1. This novel function of HBZ likely plays a role in the viral pathogenesis of HTLV-1 and provides important insights into our understanding of the development of ATL.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Culina/metabolismo , Infecções por HTLV-I/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas dos Retroviridae/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Citocromos c/metabolismo , Células HEK293 , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Células NIH 3T3 , Estabilidade Proteica , Proteínas Quinases Associadas a Fase S/metabolismo , UbiquitinaçãoRESUMO
Human T-cell leukaemia virus type-1 (HTLV-1) infection causes adult T-cell leukaemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in the pathogenesis of HTLV-1-associated disease. Here, we identified centromere protein B (CENP-B) as a novel interacting partner of HBZ. HBZ and CENP-B associate with their central regions in cells. Furthermore, overexpression of HBZ abrogated the DNA-binding activity of CENP-B to the α-satellite DNA region containing the CENP-B box motif, which in turn inhibited the CENP-B-mediated trimethylation of histone H3K9 in T-cells.
Assuntos
Proteína B de Centrômero/metabolismo , Metilação de DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Infecções por HTLV-I/metabolismo , Histonas/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Células HEK293 , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Leucemia-Linfoma de Células T do Adulto/virologia , Linfócitos T/metabolismo , Linfócitos T/virologiaRESUMO
Adult T-cell leukemia (ATL) is caused by infection with human T-cell leukemia virus type-1 (HTLV-1). The tetrahydrotetramethylnaphthalene derivative TMNAA has recently been identified as a selective inhibitor of HTLV-1-infected T-cell lines and adult T-cell leukemia (ATL) cells but not of uninfected T-cell lines and peripheral blood mononuclear cells (PBMCs). In the present study, more than 100 derivatives of TMNAA were synthesized and examined for their inhibitory effects on the proliferation of various T-cell lines and PBMCs. Among the compounds, MN417 is a more potent inhibitor of ATL cells than TMNAA. This compound is a novel phenanthridinone derivative with the tetrahydrotetramethylnaphthalene structure. Interestingly, PN-H and MN314-B, which are also phenanthridinone derivatives but do not have the tetrahydrotetramethylnaphthalene structure, could not distinguish between HTLV-1-infected and uninfected T-cell lines in terms of their anti-proliferative activity. These results suggest that the tetrahydrotetramethylnaphthalene structure is required for the selective inhibition of HTLV-1-infected cells.
Assuntos
Antineoplásicos/farmacologia , Leucemia-Linfoma de Células T do Adulto , Norbornanos/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Norbornanos/químicaRESUMO
The coproduction of a useful material and electricity via a novel application of microbial fuel cell (MFC) technology to oxidative fermentation was investigated. We focused on vinegar production, i.e., acetic acid fermentation, as an initial and model useful material that can be produced by oxidative fermentation in combination with MFC technology. The coproduction of acetic acid and electricity by applying MFC technology was successfully demonstrated by the simultaneous progress of acetic acid fermentation and electricity generation through a series of repeated batch fermentations. Although the production rate of acetic acid was very small, it increased with the number of repeated batch fermentations that were conducted. We obtained nearly identical (73.1%) or larger (89.9%) acetic acid yields than that typically achieved by aerated fermentation (75.8%). The open-cycle voltages measured before and after fermentation increased with the total fermentation time and reached a maximum value of 0.521 V prior to the third batch fermentation. The maximum current and power densities measured in this study (19.1 µA/cm² and 2.47 µW/cm², respectively) were obtained after the second batch fermentation.
Assuntos
Ácido Acético/metabolismo , Fontes de Energia Bioelétrica , Fermentação , Acetobacter/metabolismo , EletricidadeRESUMO
Phenotypic transformation of mesangial cells (MCs) is implicated in the development of glomerular disease; however, the mechanisms underlying their altered genetic program is still unclear. α-smooth muscle actin (α-SMA) is known to be a crucial marker for phenotypic transformation of MCs. Recently, E-boxes and the class I basic helix-loop-helix proteins, such as E12 have been shown to regulateα-SMA expression. Therefore, we tried to identify a novel E12 binding protein in MCs and to examine its role in glomerulonephritis. We found that PIASy, one of the protein inhibitors of activated STAT family protein, interacted with E12 by yeast two-hybrid screens and coimmunopreciptation assays. Overexpression of E12 significantly enhanced theα-SMA promoter activity, and the increase was blocked by co-transfection of PIASy, but not by a PIASy RING mutant. In vivo sumoylation assays revealed that PIASy was a SUMO E3 ligase for E12. Furthermore, transforming growth factor-ß (TGF-ß) treatment induced expression of both PIASy and E12, consistent with α-SMA expression. Moreover, reduced expression of PIASy protein by siRNA specific for PIASy resulted in increased TGF-ß-mediated α-SMA expression. In vivo, PIASy and E12 were dramatically upregulated along with α-SMA and TGF-ß in the proliferative phase of Thy1 glomerulonephritis. Furthermore, an association between PIASy and E12 proteins was observed at day 6 by IP-western blotting, but not at day 0. These results suggest that TGF-ß up-regulates PIASy expression in MCs to down-regulateα-SMA gene transcription by the interaction with E12.
Assuntos
Actinas/metabolismo , Células Mesangiais/metabolismo , Fator 3 de Transcrição/metabolismo , Actinas/genética , Animais , Western Blotting , Células COS , Linhagem Celular , Proliferação de Células , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Ligação Proteica , Proteínas Inibidoras de STAT Ativados , Interferência de RNA , Ratos , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 3 de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Four kinds of transporters, HXT1 and HXY7 from Saccharomyces cerevisiae, and GXF1 and GXS1 from Candida intermedia, were overexpressed in xylose-metabolizing S. cerevisiae harboring a xyloseisomerase-based pathway. Overexpression of transporter enhanced sugar consumption and ethanol production, and GXF1 was efficient for ethanol fermentation from both glucose and xylose.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Etanol/metabolismo , Fermentação , Proteínas de Transporte de Monossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Candida/genética , Candida/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Human T-cell leukemia virus type-1 (HTLV-1) causes ATL in 2.5% of carriers after a long period of latent infection. Moreover, half of adult T-cell leukemia (ATL) patients succumb to this disease within 1year of onset. HTLV-1 bZIP factor (HBZ) is constitutively expressed in all the ATL cells. Thus, suggesting that HBZ may play a key role in cellular leukemogenesis. Herein we present evidence that interferon regulatory factor IRF-1, which is a member of IRF transcription family, interacts with HBZ. The N-terminal of HBZ interacted with IRF-1. HBZ reduced both IRF-1 DNA-binding activity and stability via a proteasome-dependent pathway. In addition, IRF-1-mediated apoptosis is significantly reduced by ectopic production of the HBZ. These results suggested that HBZ has dual suppressive effects on IRF-1 function, which may contribute to HTLV-1 related pathogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Transcrição Gênica , Animais , Regulação da Expressão Gênica , Células HEK293 , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Camundongos , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.
Assuntos
Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Neocallimastigales/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Xilose/metabolismo , Aldeído Redutase/genética , Metabolismo Energético/genética , Etanol/isolamento & purificação , Etanol/metabolismo , Fermentação/efeitos dos fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Amplificação de Genes , Expressão Gênica , Engenharia Genética , Glucose/metabolismo , Lignina/metabolismo , Via de Pentose Fosfato/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Transformação Genética , Xilitol/farmacologiaRESUMO
The transition from latent to lytic phases of the Epstein-Barr virus life cycle is triggered by expression of a viral transactivator, BZLF1, that then induces expression of the viral immediate-early and early genes. The BZLF1 protein is post-translationally modified by a small ubiquitin-related modifier-1 (SUMO-1). Here we found that BZLF1 is conjugated at lysine 12 not only by SUMO-1 but also by SUMO-2 and 3. The K12R mutant of BZLF1, which no longer becomes sumoylated, exhibits stronger transactivation than the wild-type BZLF1 in a reporter assay system as well as in the context of virus genome with nucleosomal structures. Furthermore, exogenous supply of a SUMO-specific protease, SENP, caused de-sumoylation of BZLF1 and enhanced BZLF1-mediated transactivation. Immunoprecipitation experiments proved that histone deacetylase 3 preferentially associated with the sumoylated form of BZLF1. Levels of the sumoylated BZLF1 increased as lytic replication progressed. Based on these observations, we conclude that sumoylation of BZLF1 regulates its transcriptional activity through histone modification during Epstein-Barr virus productive replication.
Assuntos
Herpesvirus Humano 4/metabolismo , Histona Desacetilases/metabolismo , Proteína SUMO-1/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Modelos Genéticos , Mutação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Neoplasias Gástricas/metabolismo , Ativação Transcricional , Proteínas Virais/genéticaRESUMO
HTLV-1 infection causes adult T-cell leukemia (ATL). The development of ATL is thought to be associated with disruption of transcriptional control of cellular genes. HTLV-1 basic leucine-zipper (bZIP) factor, HBZ, is encoded by the complementary strand of the provirus. We previously reported that HBZ interacts with c-Jun and suppresses its transcriptional activity. To identify the cellular factor(s) that interact with HBZ, we conducted a yeast two-hybrid screen using full-length HBZ as bait and identified MafB. HBZ heterodimerizes with MafB via each bZIP domain. Luciferase analysis revealed a significant decrease in transcription through Maf recognition element (MARE) in a manner dependent on the bZIP domain of HBZ. Indeed, production of full-length HBZ in cells decreased the MARE-bound MafB protein, indicating that HBZ abrogates the DNA-binding activity of MafB. In addition, HBZ reduced the steady-state levels of MafB, and the levels were restored by treatment with a proteasome inhibitor. These results suggest a suppressive effect of HBZ on Maf function, which may have a significant role in HTLV-1 related pathogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação Viral da Expressão Gênica , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Fator de Transcrição MafB/metabolismo , Elementos Reguladores de Transcrição , Transcrição Gênica , Proteínas Virais/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células HEK293 , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Zíper de Leucina , Fator de Transcrição MafB/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas dos Retroviridae , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genéticaRESUMO
Several nucleoside analogs containing a methylene group instead of a 5'-O atom were synthesized to study the effect of the 5'-modification of nucleoside analogs on their anti-HCV activity. Among the analogs, a 5'-phenacyl analog exhibited good anti-HCV activity with an EC(50) of 15.1 muM. This compound is hypothesized to function via a novel type of mechanism that does not involve the conventional 5'-O-triphosphorylation process.
Assuntos
Adenosina/análogos & derivados , Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Adenosina/síntese química , Adenosina/farmacologia , Antivirais/química , Antivirais/farmacologia , Desoxirribonucleosídeos/síntese química , Desoxirribonucleosídeos/química , Desoxirribonucleosídeos/farmacologiaRESUMO
Reactivation of the Epstein-Barr virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found here that Transducer of Regulated cAMP-response Element-binding Protein (CREB) (TORC) 2 enhances Zp activity 10-fold and more than 100-fold with co-expression of the BZLF1 protein. Mutational analysis of Zp revealed that the activation by TORC is dependent on ZII and ZIII cis elements, binding sites for CREB family transcriptional factors and the BZLF1 protein, respectively. Immunoprecipitation, chromatin immunoprecipitation, and reporter assay using Gal4-luc and Gal4BD-BZLF1 fusion protein indicate that TORC2 interacts with BZLF1, and that the complex is efficiently recruited onto Zp. These observations clearly indicate that TORC2 activates the promoter through interaction with the BZLF1 protein as well as CREB family transcriptional factors. Induction of the lytic replication resulted in the translocation of TORC2 from cytoplasm to viral replication compartments in nuclei, and furthermore, activation of Zp by TORC2 was augmented by calcium-regulated phosphatase, calcineurin. Silencing of endogenous TORC2 gene expression by RNA interference decreased the levels of the BZLF1 protein in response to 12-O-tetradecanoylphorbol-13-acetate/ionophore. Based on these results, we conclude that Epstein-Barr virus exploits the calcineurin-TORC signaling pathway through interactions between TORC and the BZLF1 protein in reactivation from latency.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Herpesvirus Humano 4/fisiologia , Elementos de Resposta/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Carcinógenos/farmacologia , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Mutação , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/genética , Fatores de Transcrição/genética , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacosRESUMO
OBJECTIVE: TGFbeta and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) alpha-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFbeta activates SM alpha-actin through PIAS1. METHODS AND RESULTS: An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFbeta-induced expression of SM alpha-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM alpha-actin promoter activity in a TGFbeta control element (TCE)-dependent manner. Because the TCE within the SM alpha-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM alpha-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. CONCLUSIONS: These results provide evidence that PIAS1 promotes TGFbeta-induced activation of SM alpha-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.