RESUMO
Early region 1A (E1A) of human adenovirus (HAdV) has been the focus of over 30 years of investigation and is required for the oncogenic capacity of HAdV in rodents. Alternative splicing of the E1A transcript generates mRNAs encoding multiple E1A proteins. The 55-residue (55R) E1A protein, which is encoded by the 9S mRNA, is particularly interesting due to the unique properties it displays relative to all other E1A isoforms. 55R E1A does not contain any of the conserved regions (CRs) present in the other E1A isoforms. The C-terminal region of the 55R E1A protein contains a unique sequence compared to all other E1A isoforms, which results from a frameshift generated by alternative splicing. The 55R E1A protein is thought to be produced preferentially at the late stages of infection. Here we report the first study to directly investigate the function of the species C HAdV 55R E1A protein during infection. Polyclonal rabbit antibodies (Abs) have been generated that are capable of immunoprecipitating HAdV-2 55R E1A. These Abs can also detect HAdV-2 55R E1A by immunoblotting and indirect immunofluorescence assay. These studies indicate that 55R E1A is expressed late and is localized to the cytoplasm and to the nucleus. 55R E1A was able to activate the expression of viral genes during infection and could also promote productive replication of species C HAdV. 55R E1A was also found to interact with the S8 component of the proteasome, and knockdown of S8 was detrimental to viral replication dependent on 55R E1A.
Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/genética , RNA Mensageiro/química , RNA Viral/química , Adenosina Trifosfatases/metabolismo , Proteínas E1A de Adenovirus/imunologia , Adenovírus Humanos/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Linhagem Celular , Núcleo Celular/metabolismo , Inibição de Contato , Citoplasma/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Transcrição Gênica , Replicação Viral/genéticaRESUMO
The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1α, TIF1ß, and TIF1γ-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.
Assuntos
Infecções por Adenoviridae/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Adenovírus Humanos/genética , Linhagem Celular , Humanos , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIbeta-binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Elonguina , Imunofluorescência , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Microscopia Confocal , Mutação , Proteínas Nucleares/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genéticaRESUMO
Adenovirus type 5 (Ad5) inactivates the host cell DNA damage response by facilitating the degradation of Mre11, DNA ligase IV, and p53. In the case of p53, this is achieved through polyubiquitylation by Ad5E1B55K and Ad5E4orf6, which recruit a Cul5-based E3 ubiquitin ligase. Recent evidence indicates that this paradigm does not apply to other adenovirus serotypes, since Ad12, but not Ad5, causes the degradation of TOPBP1 through the action of E4orf6 alone and a Cul2-based E3 ubiquitin ligase. We now have extended these studies to adenovirus groups A to E. While infection by Ad4, Ad5, and Ad12 (groups E, C, and A, respectively) cause the degradation of Mre11, DNA ligase IV, and p53, infection with Ad3, Ad7, Ad9, and Ad11 (groups B1, B1, D, and B2, respectively) only affects DNA ligase IV levels. Indeed, Ad3, Ad7, and Ad11 cause the marked accumulation of p53. Despite this, MDM2 levels were very low following infection with all of the viruses examined here, regardless of whether they increase p53 expression. In addition, we found that only Ad12 causes the degradation of TOPBP1, and, like Ad5, Ad4 recruits a Cul5-based E3 ubiquitin ligase to degrade p53. Surprisingly, Mre11 and DNA ligase IV degradation do not appear to be significantly affected in Ad4-, Ad5-, or Ad12-infected cells depleted of Cul2 or Cul5, indicating that E1B55K and E4orf6 recruit multiple ubiquitin ligases to target cellular proteins. Finally, although Mre11 is not degraded by Ad3, Ad7, Ad9, and Ad11, no viral DNA concatemers could be detected. We suggest that group B and D adenoviruses have evolved mechanisms based on the loss of DNA ligase IV and perhaps other unknown molecules to disable the host cell DNA damage response to promote viral replication.
Assuntos
Infecções por Adenoviridae/genética , Infecções por Adenoviridae/metabolismo , Adenoviridae/fisiologia , Dano ao DNA , Adenoviridae/classificação , Adenoviridae/genética , Infecções por Adenoviridae/enzimologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade da Espécie , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3.
Assuntos
Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Adenoviridae/classificação , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenoviridae/patogenicidade , Infecções por Adenoviridae/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/virologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Imunofluorescência , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation.
Assuntos
Proteína de Ligação a CREB/metabolismo , Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Transcrição Gênica , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc7 do Ciclossomo-Complexo Promotor de Anáfase , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/genética , Linhagem Celular , Transformação Celular Neoplásica , Sequência Conservada , Humanos , Mitose , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Sequência de Aminoácidos , Linhagem Celular , Sequência Conservada , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Humanos , Mutação , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismoRESUMO
C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.
Assuntos
Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/genética , Oxirredutases do Álcool/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Carcinoma de Células Pequenas/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Células HT29 , Células HeLa , Humanos , Luciferases/análise , Neoplasias Pulmonares/metabolismo , Dados de Sequência Molecular , Mutação , Osteossarcoma/metabolismo , Plasmídeos , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/classificação , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.
Assuntos
Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Histonas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Proteína de Replicação A/metabolismo , Transdução de SinaisRESUMO
Adenovirus early region 1B-associated protein 5, E1B-AP5, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, was originally isolated on the basis of its ability to bind to the adenovirus 5 early region1B55K protein. Here, it has been demonstrated that E1B-AP5 interacts with mutant and wild-type p53 from human cells in pull-down assays using GST-E1B-AP5. This interaction has been confirmed by co-immunoprecipitation studies and pull-down experiments with in vitro translated E1B-AP5 and GST-p53. The binding site for E1B-AP5 has been mapped to the C-terminal region of p53. In reciprocal experiments, it has been shown that several regions of E1B-AP5 bound to p53 although it is probable that a major site of interaction is located between amino acids 395 and 732 of E1B-AP5. In reporter assays, E1B-AP5 inhibited p53 transcriptional activity although not as efficiently as the Ad5E1B55K protein. Transfection of E1B-AP5 into human tumour cells affected the cellular response to UV radiation, such that, although p53 expression was induced, little change in the level of p53-inducible genes could be observed.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Raios UltravioletaAssuntos
Infecções por Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Transformação Celular Viral , Proteínas E1B de Adenovirus/química , Proteínas E1B de Adenovirus/genética , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Peso Molecular , Processamento de Proteína Pós-TraducionalRESUMO
Proteasomes represent the major non-lysosomal mechanism responsible for the degradation of proteins. Following interferon γ treatment 3 proteasome subunits are replaced producing immunoproteasomes. Adenovirus E1A interacts with components of the 20S and 26S proteasome and can affect presentation of peptides. In light of these observations we investigated the relationship of AdE1A to the immunoproteasome. AdE1A interacts with the immunoproteasome subunit, MECL1. In contrast, AdE1A binds poorly to the proteasome ß2 subunit which is replaced by MECL1 in the conversion of proteasomes to immunoproteasomes. Binding sites on E1A for MECL1 correspond to the N-terminal region and conserved region 3. Furthermore, AdE1A causes down-regulation of MECL1 expression, as well as LMP2 and LMP7, induced by interferon γ treatment during Ad infections or following transient transfection. Consistent with previous reports AdE1A reduced IFNγ-stimulated STAT1 phosphorylation which appeared to be responsible for its ability to reduce expression of immunoproteasome subunits.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/patogenicidade , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Sítios de Ligação , Linhagem Celular Tumoral , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/metabolismo , Regulação para Baixo , Humanos , Interferon gama/farmacologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Fator de Transcrição STAT1/metabolismo , Transdução de SinaisRESUMO
Adenovirus early region 1A (AdE1A) binds to the C-terminal binding protein 1 (CtBP1) primarily through a highly conserved PXDLS motif located close to its C-terminus. Purified synthetic peptides equivalent to this region of AdE1A have been shown to form a series of beta-turns. In this present study the effect of CtBP1 binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional (1)H NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I(241)-V(260) and E(247)-N(266) of Ad12E1A were examined in the absence of CtBP1. Whilst the latter peptide forms a series of beta-turns in its C-terminal half as reported previously, the former peptide is alpha-helical over the region D(243)-Q(253). Upon interaction with CtBP1 the conformation of the backbone in the region (255)PVDLCVK(261) of the Ad12E1A E(247)-N(266) peptide reorganises from a predominately beta-turn to an alpha-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 ppm for the delta-CH(3) proton resonance of V(256). 2-D NOESY experiments showed new signals in the amide-alpha region which correlate to transferred NOEs from the protein to the peptide residues E(251), V(256) and K(261). In further analyses the contribution of individual amino acids within the sequence (254)VPVDLS(259) was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues (255)P-V(260) serve initially as a recognition site for CtBP and then as an anchor through a beta-turns-->alpha-helix conformational rearrangement. In addition it has been predicted that regions N-terminal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form alpha-helices.
Assuntos
Adenoviridae/química , Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Alinhamento de SequênciaRESUMO
C-terminal binding protein (CtBP) has been shown to bind to a highly conserved five-amino-acid motif (PXDLS) located very close to the C-terminus of adenovirus early region 1A proteins. It has also been demonstrated that amino acids C-terminal and N-terminal to this original proposed binding site contribute to the interaction. However, conflicting evidence has been presented to show that acetylation of an adjacent lysine residue in Ad5E1A may or may not influence binding. It has now been demonstrated here that acetylation of a lysine, equivalent to position 261 in Ad12 E1A and position 285 in Ad5E1A, in a synthetic peptide disrupts the binding to CtBP1 and CtBP2 and alters the K(i) of the peptide, indicative of a reduction in the affinity of the peptide for CtBP1 and CtBP2, but only to a rather limited extent (less than 2-fold). The solution structures of synthetic peptides equivalent to wild-type and acetylated forms of the Ad12 E1A peptide have been determined by proton NMR spectroscopy. The wild-type form of the peptide adopts a series of beta-turns over the region Val(254)-Arg(262). Within the acetylated isoform, the beta-turn conformation is less extensive, Val(260)-Arg(262) adopting a random confirmation. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains over an extended binding site (PXDLSXK) are necessary for recognition by CtBP, acetylation of lysine interfering with both of these features, but not to such an extent as to totally inhibit interaction. Moreover, it is possible that the beta-turn conformation at the C-terminus of AdE1A contributes to binding to alpha importin and nuclear import. Acetylation of lysine (261) could disrupt interaction through structural destabilization as well as charge neutralization and subsequent nuclear localization.
Assuntos
Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mucoproteínas/metabolismo , Estrutura Secundária de Proteína , Acetilação , Motivos de Aminoácidos , Sítios de Ligação , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Espectroscopia de Ressonância Magnética , Microesferas , Modelos Moleculares , Ligação ProteicaRESUMO
We have determined distinct roles for different proteasome complexes in adenovirus (Ad) E1A-dependent transcription. We show that the 19S ATPase, S8, as a component of 19S ATPase proteins independent of 20S (APIS), binds specifically to the E1A transactivation domain, conserved region 3 (CR3). Recruitment of APIS to CR3 enhances the ability of E1A to stimulate transcription from viral early gene promoters during Ad infection of human cells. The ability of CR3 to stimulate transcription in yeast is similarly dependent on the functional integrity of yeast APIS components, Sug1 and Sug2. The 20S proteasome is also recruited to CR3 independently of APIS and the 26S proteasome. Chromatin immunoprecipitation reveals that E1A, S8 and the 20S proteasome are recruited to both Ad early region gene promoters and early region gene sequences during Ad infection, suggesting their requirement in both transcriptional initiation and elongation. We also demonstrate that E1A CR3 transactivation and degradation sequences functionally overlap and that proteasome inhibitors repress E1A transcription. Taken together, these data demonstrate distinct roles for APIS and the 20S proteasome in E1A-dependent transactivation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transcrição Gênica , Adenoviridae/fisiologia , Células HCT116 , Meia-Vida , Humanos , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Termodinâmica , Ativação Transcricional/genéticaRESUMO
The N-terminal region of the adenovirus (Ad) 12S E1A gene product targets several cellular proteins that are essential for the induction of S phase, cellular immortalization, cellular transformation, transcriptional repression, and transcriptional activation. The precise binding sites for these proteins, however, remain to be resolved. We therefore undertook an extensive site-directed mutagenesis approach to generate specific point mutants and to precisely map the binding sites for CBP, p300, TATA-binding protein (TBP), S4, S8, hGcn5, P/CAF, and Ran within the first 30 amino acids of the Ad5 12S E1A protein. We determined that although common residues within the N-terminal region can form partial binding sites for these proteins, point mutants were also generated that could discriminate between binding sites. These data indicate that AdE1A can target each of these proteins individually through distinct binding sites. It was evident, however, that the mutation of specific hydrophobic residues typically had the greatest effect upon AdE1A's ability to bind individual partners. Indeed, the mutation of L at positions 19 and 20 eliminated the ability of AdE1A to interact with any of the N-terminal binding proteins studied here. Interestingly, although TBP and S8 or CBP/p300 can exist as functional complexes, RNA interference revealed that the recruitment of either TBP, S8, or CBP/p300 to AdE1A was not dependent upon the expression of the other proteins. These data further indicate that AdE1A can target individual partner proteins in vivo and that it does not necessarily recruit these proteins indirectly as components of larger macromolecular complexes. Finally, we took advantage of the fine-mapping data to ascertain which proteins were targeted during the transformation process. Consistent with previous studies, CBP/p300 was found to be targeted by AdE1A during this process, although our data suggest that binding to other N-terminal proteins is also important for transformation.
Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Proteínas E1A de Adenovirus/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/metabolismo , Transcrição GênicaRESUMO
Many tumor cells are resistant to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. Adenovirus early region 1A (AdE1A) sensitizes the otherwise resistant cells to TNFalpha. AdE1A also stabilizes the p53 protein. The present study demonstrates a correlation between AdE1A-induced sensitization and stabilization of p53 in TNFalpha-induced apoptosis since the N-terminal and CR2 regions, the binding sites for CBP/p300, Rb and 26S proteasome regulatory components, are required for both these actions of AdE1A. TNFalpha does not induce apoptosis and AdE1A fails to sensitize TNFalpha cytotoxicity in p53-negative cells. However, introduction of exogenous p53 overcomes the cellular resistance to TNFalpha toxicity and enhances AdE1A sensitization, demonstrating that AdE1A sensitizes TNFalpha-induced apoptosis by its stabilization of p53. A proteasome inhibitor, lactacystin, enhances TNFalpha cytotoxicity in p53-positive and -negative cells, suggesting that accumulation of cellular proteins other than p53 might also regulate the cellular response to TNFalpha signaling.
Assuntos
Proteínas E1A de Adenovirus/genética , Apoptose/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/genética , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Proteínas E1A de Adenovirus/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares , Proteínas Recombinantes/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/toxicidade , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although adenovirus early region 1A (AdE1A) can modulate protein expression through its interaction with transcriptional regulators it can also influence the ability of the cell to degrade proteins by binding to components of the 26 S proteasome. We demonstrate here that AdE1A interacts with the S2 subunit of the 19 S regulatory complex in addition to the ATPase subunits S4 and S8 previously identified. S2 forms complexes with both the 13 and 12 S AdE1A proteins both in vivo and in vitro. Mutational analysis has shown direct binding through a short sequence toward the N terminus of conserved region 2 of AdE1A, which encompasses the LXCXE motif, involved in interaction with the pRb family of proteins. In vivo, additional contacts are made between AdE1A and proteasomal components, as well as within the proteasome, such that deletion of the N-terminal region of E1A as well as part of conserved region 2 is required to completely disrupt S2 binding. Mutation of AdE1A, which disrupts complex formation with S2, results in the loss of its ability to stabilize the p53 protein. Similarly down-regulation of S2 expression using small interfering RNAs leads to the inhibition of p53 degradation. These effects were observed in normally growing cells and those subjected to UV irradiation. Furthermore, AdE1A had no effect on the Mdm2-mediated ubiquitination of p53. We suggest therefore that interaction of AdE1A with S2, as well as with the ATPases S4 and S8, directly causes inhibition of proteasomal activity and consequent increase in the protein levels of p53.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas Repressoras , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Camundongos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Testes de Precipitina , Complexo de Endopeptidases do Proteassoma , Subunidades Proteicas , Interferência de RNA , Proteína Supressora de Tumor p53/análise , Ubiquitina/metabolismoRESUMO
We have investigated the functional interactions between adenovirus early region 1A (AdE1A) protein, the co-activators cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and SUG1, and the transcriptional repressor retinoblastoma (Rb) in mediating T3-dependent repression. Utilizing the human glycoprotein hormone common alpha-subunit (alpha-subunit) promoter and AdE1A mutants with selective binding capacity to these molecules we have determined an essential role for CBP/p300. In normal circumstances, wild-type 12 S AdE1A inhibited alpha-subunit activity. In contrast, adenovirus mutants that retain both the SUG1- and Rb-binding sites, but lack the CBP/p300-binding site, were unable to repress promoter activity. We have also identified a role for the tumour-suppressor gene product p53 in regulation of the alpha-subunit promoter. Akin to 12 S AdE1A, exogenous p53 expression repressed alpha-subunit activity. This function resided in the ability of p53 to interact with CBP/p300; an N-terminal mutant incapable of interacting with CBP/p300 did not inhibit alpha-subunit activity. Stabilization of endogenous p53 by UV irradiation also correlated positively with reduced alpha-subunit activity. Intriguingly, T3 stimulated endogenous p53 transcriptional activity, implicating p53 in T3-dependent signalling pathways. These data indicate that CBP/p300 and p53 are key regulators of alpha-subunit activity.
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/genética , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Adenoviridae/genética , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Transativadores/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Adenovirus 2 and 12 early region 1A (Ad2 and Ad12 E1A) proteins were cleaved during cisplatin-induced apoptosis of Ad-transformed rat and human cells. Cleavage was inhibited in the presence of caspase inhibitors such as Z-VAD-FMK. In Ad12 transformants both 13S and 12S E1A proteins were cleaved at a similar rate. In Ad2 transformants the E1A 13S component was appreciably less stable than the 12S component. In in vitro studies Ad2 and Ad12 E1A 13S and Ad2 12S proteins were rapidly cleaved by caspase 3 whereas Ad12 12S E1A and Ad12 13S E1A were rapidly degraded by caspase 7. Cleavage sites in Ad12 13S proteins for caspase 3 have been determined. Initial cleavage occurred at D24 and D150; this was followed by cleavage at D204 and D242. Caspase-3-mediated cleavage of Ad12 13S E1A destroyed its ability to bind to CBP and TBP but interaction between C terminal E1A polypeptides and CtBP was observed. During viral infection Ad5 and Ad12 E1A 12S proteins were markedly more stable than 13S proteins but no difference was observed in Ad E1A levels in the absence or presence of the caspase inhibitors Z-VAD-FMK or Z-D(OMe)-E(OMe)-V-D(OMe)-CH(2)F. Limited caspase 3 and 10 activation occurred during infection with the E1B 19K(-) virus Ad2 pm1722 but little or no activation of caspase 3 was observed during wt virus infection. Examination of protein cleavage during viral infection of A549 cells showed proteolysis of lamin B and PARP in response to Ad5 wt and Ad2 pm1722. Protein degradation in response to both viruses was partially inhibited by Z-VAD-FMK. Following infection of human skin fibroblasts lamin B was degraded, although only limited changes in PARP levels were observed. We have concluded that Ad E1A is cleaved by caspases during apoptosis but not during viral infection. However, some of the processes commonly associated with apoptosis occur during viral infection, particularly with E1B 19K(-) mutants, although apoptosis per se is not evident.