RESUMO
Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.
Assuntos
Proteínas E1B de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas E1B de Adenovirus/genética , Infecções por Adenovirus Humanos/metabolismo , Proteínas Correpressoras , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão Intranuclear , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteína SUMO-1/genética , Sumoilação , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Replicação ViralRESUMO
UNLABELLED: Promyelocytic leukemia nuclear bodies (PML-NBs) are nuclear structures that accumulate intrinsic host factors to restrict viral infections. To ensure viral replication, these must be limited by expression of viral early regulatory proteins that functionally inhibit PML-NB-associated antiviral effects. To benefit from the activating capabilities of Sp100A and simultaneously limit repression by Sp100B, -C, and -HMG, adenoviruses (Ads) employ several features to selectively and individually target these isoforms. Ads induce relocalization of Sp100B, -C, and -HMG from PML-NBs prior to association with viral replication centers. In contrast, Sp100A is kept at the PML tracks that surround the newly formed viral replication centers as designated sites of active transcription. We concluded that the host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression. Ad-dependent loss of Sp100 SUMOylation is another crucial part of the virus repertoire to counteract intrinsic immunity by circumventing Sp100 association with HP1, therefore limiting chromatin condensation. We provide evidence that Ad selectively counteracts antiviral responses and, at the same time, benefits from PML-NB-associated components which support viral gene expression by actively recruiting them to PML track-like structures. Our findings provide insights into novel strategies for manipulating transcriptional regulation to either inactivate or amplify viral gene expression. IMPORTANCE: We describe an adenoviral evasion strategy that involves isoform-specific and active manipulation of the PML-associated restriction factor Sp100. Recently, we reported that the adenoviral transactivator E1A targets PML-II to efficiently activate viral transcription. In contrast, the PML-associated proteins Daxx and ATRX are inhibited by early viral factors. We show that this concept is more intricate and significant than originally believed, since adenoviruses apparently take advantage of specific PML-NB-associated proteins and simultaneously inhibit antiviral measures to maintain the viral infectious program. Specifically, we observed Ad-induced relocalization of the Sp100 isoforms B, C, and HMG from PML-NBs juxtaposed with viral replication centers. In contrast, Sp100A is retained at Ad-induced PML tracks that surround the newly formed viral replication centers, acting as designated sites of active transcription. The host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression.
Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Regulação Viral da Expressão Gênica/genética , Imunidade Inata/imunologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adenovírus Humanos/genética , Linhagem Celular , Primers do DNA/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting , Hibridização In Situ , Luciferases , Proteína da Leucemia Promielocítica , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SumoilaçãoRESUMO
Data-intensive research depends on tools that manage multidimensional, heterogeneous datasets. We built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices and tables. OMERO's design and flexibility have enabled its use for light-microscopy, high-content-screening, electron-microscopy and even non-image-genotype data. OMERO is open-source software, available at http://openmicroscopy.org/.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Interpretação de Imagem Assistida por Computador/métodos , Armazenamento e Recuperação da Informação/métodos , Modelos Biológicos , Software , Interface Usuário-Computador , Animais , Biologia/métodos , Simulação por Computador , HumanosRESUMO
The NF-κB pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule IκBα, the IKK subunit γ/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of IκBα and activation of NF-κB mediated by TNFα. We found that under conditions of deficient SUMOylation, an important delay in both TNFα-mediated proteolysis of IκBα and NF-κB dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs), revealed the formation of chains on IκBα containing SUMO-2/3 and ubiquitin after TNFα stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on IκBα after activation of the TNFα signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of IκBα by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of IκBα modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified IκBα to the action of 26S proteasome, contributing to the optimal control of NF-κB activity after TNFα-stimulation.
Assuntos
Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais/efeitos dos fármacos , Sumoilação , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, PsiKXE (where Psi represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of beta-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the beta2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína SUMO-1/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Humanos , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de SinaisRESUMO
A consistent feature of the Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is the constitutive activation of NF-kappaB transcription factors. In Epstein-Barr virus (EBV)-associated cases of cHL, expression of viral antigens most probably leads to NF-kappaB activation but for non-EBV-associated cases, the mechanism is not clear. Previous small studies have demonstrated deleterious mutations of NFKBIA, the gene encoding IkappaB alpha, in HRS cells. In the present study, we aimed to establish the frequency of NFKBIA mutation in cHL by investigating a larger series of cases and to determine whether these mutations are a characteristic feature of non-EBV-associated cHL. Single HRS cells from 20 cases of cHL were analysed by PCRs covering all 6 exons of the gene. Clonal deleterious mutations were detected in 3 cases and in 1 case both alleles of the gene were shown to harbour mutations. NFKBIA mutations were detected only in non-EBV-associated cases but the majority of these cases had wild-type NFKBIA. It remains possible that defects in genes encoding other inhibitors of NF-kappaB, such as TNFAIP3 (A20) and CYLD, are involved in the latter cases, as described for one case in this series.
Assuntos
Proteínas de Ligação a DNA/genética , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/fisiologia , Doença de Hodgkin/genética , Proteínas I-kappa B/genética , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Adulto , Idoso , Criança , Hibridização Genômica Comparativa , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Perfilação da Expressão Gênica , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa , Análise de Sequência com Séries de Oligonucleotídeos , Adulto JovemRESUMO
We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.
Assuntos
Adenosina Desaminase/metabolismo , Nucléolo Celular/metabolismo , Edição de RNA , Proteína SUMO-1/fisiologia , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA , Proteína SUMO-1/metabolismoRESUMO
A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.
Assuntos
Proteínas de Ligação a DNA/química , Proteína SUMO-1/fisiologia , Fatores de Transcrição/química , Transcrição Gênica , Adenoviridae/genética , Animais , Arginina/química , Sítios de Ligação , Western Blotting , Células COS , Cisteína Endopeptidases , DNA/química , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/genética , Endopeptidases/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Células HeLa , Humanos , Imunoprecipitação , Molécula 1 de Adesão Intercelular/metabolismo , Lisina/química , Microscopia de Fluorescência , Modelos Genéticos , Plasmídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Coelhos , Proteína SUMO-1/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/químicaRESUMO
The TFIID complex is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) and is the only component of the general RNA polymerase II (RNAP II) transcription machinery with intrinsic sequence-specific DNA-binding activity. Binding of transcription factor (TF) IID to the core promoter region of protein-coding genes is a key event in RNAP II transcription activation and is the first and rate-limiting step of transcription initiation complex assembly. Intense research efforts in the past have established that TFIID promoter-binding activity as well as the function of TFIID-promoter complexes is tightly regulated through dynamic TFIID interactions with positive- and negative-acting transcription regulatory proteins. However, very little is known about the role of post-translational modifications in the regulation of TFIID. Here we show that the human TFIID subunits hsTAF5 and hsTAF12 are modified by the small ubiquitin-related modifier SUMO-1 in vitro and in human cells. We identify Lys-14 in hsTAF5 and Lys-19 in hsTAF12 as the primary SUMO-1 acceptor sites and show that SUMO conjugation has no detectable effect on nuclear import or intranuclear distribution of hsTAF5 and hsTAF12. Finally, we demonstrate that purified human TFIID complex can be SUMO-1-modified in vitro at both hsTAF5 and hsTAF12. We find that SUMO-1 conjugation at hsTAF5 interferes with binding of TFIID to promoter DNA, whereas modification of hsTAF12 has no detectable effect on TFIID promoter-binding activity. Our observations suggest that reversible SUMO modification at hsTAF5 contributes to the dynamic regulation of TFIID promoter-binding activity in human cells.
Assuntos
Proteína SUMO-1/metabolismo , Fator de Transcrição TFIID/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , Eletroforese em Gel de Poliacrilamida , Epitopos/química , Células HeLa , Humanos , Immunoblotting , Lisina/química , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA Polimerase II/química , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Fatores Associados à Proteína de Ligação a TATA/química , Transcrição Gênica , TransfecçãoRESUMO
SUMO modification of proteins is being increasingly linked with transcriptional repression. We recently demonstrated that SUMO modification also downregulates the transcriptional activity of the ETS-domain transcription factor Elk-1. However, as Elk-1 becomes activated through MAP kinase-mediated phosphorylation, the SUMO modification is lost, providing an elegant molecular switch that promotes the loss of repressive activities at the same time as permitting the recruitment of coactivator proteins. However, the mechanism by which SUMO promotes transcriptional repression remains enigmatic.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição , Transcrição Gênica , Animais , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Elk-1 do Domínio etsRESUMO
The ETS domain transcription factor Elk-1 is a direct target of the MAP kinase pathways. Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation. However, Elk-1 also contains two domains with repressive activities. One of these, the R motif, appears to function by suppressing the activity of the activation domain. Here, we demonstrate that SUMO modification of the R motif is required for this repressive activity. A dynamic interplay exists between the activating ERK MAP kinase pathway and the repressive SUMO pathway. ERK pathway activation leads to both phosphorylation of Elk-1 and loss of SUMO conjugation and, hence, to the loss of the repressive activity of the R motif. Thus, the reciprocal regulation of the activation and repressive activities are coupled by MAP kinase modification of Elk-1.
Assuntos
Proteínas de Ligação a DNA , Células Eucarióticas/enzimologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Proteína SUMO-1/genética , Fatores de Transcrição , Transcrição Gênica/genética , Animais , Linhagem Celular , Genes Reguladores/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Elk-1 do Domínio etsRESUMO
The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-kappa B, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.