RESUMO
Within the past year considerable new insights have been gained into the roles the p53 and retinoblastoma tumour suppressors play in determining the fate of cells through their regulation of cell cycle progression, apoptosis and gene expression. Key advances have been achieved in the identification and characterization of functional domains and through functional knockout studies.
Assuntos
Divisão Celular/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Humanos , Proteína do Retinoblastoma/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The liver-specific F antigen, although not an autoimmunogen, can induce the production of autoantibodies in responder strains. The ability to respond is under the control of two genes, one linked to the H-2 locus of mice, the other not. Responders possessing both genes produce high anti-F titers, while the H-2-linked gene alone permits a significant but low antibody response. (Responder X nonresponder) F1 hybrids derived from parents possessing identical F molecules are nonresponders, in contrast with the dominance of responsiveness in Ir gene systems. The presence of the H-2 locus from nonresponders appears involved in the inability to respond. This is discussed in terms of self-tolerance and suppression.
Assuntos
Antígenos , Autoanticorpos , Genes Dominantes , Tolerância Imunológica , Fígado/imunologia , Animais , Feminino , Genes , Ligação Genética , Masculino , Camundongos , Camundongos Endogâmicos/imunologia , Especificidade da EspécieRESUMO
Seliciclib (CYC202, R-Roscovitine) is a 2, 6, 9-substituted purine analog that is currently in phase II clinical trials as an anticancer agent. We show in this study that R-Roscovitine can downregulate nuclear factor-kappa B (NF-kappaB) activation in response to tumor necrosis factor (TNF)alpha and interleukin 1. Activation of p53-dependent transcription is not compromised when R-Roscovitine is combined with TNFalpha. We characterize the molecular mechanism governing NF-kappaB repression and show that R-Roscovitine inhibits the IkappaB kinase (IKK) kinase activity, which leads to defective IkappaBalpha phosphorylation, degradation and hence nuclear function of NF-kappaB. We further show that the downregulation of the NF-kappaB pathway is also at the level of p65 modification and that the phosphorylation of p65 at Ser 536 is repressed by R-Roscovitine. Consistent with repression of canonical IKK signaling pathway, the induction of NF-kappaB target genes monocyte chemoattractant protein, intercellular adhesion molecule-1, cyclooxygenase-2 and IL-8 is also inhibited by R-Roscovitine. We further show that treatment of cells with TNFalpha and R-Roscovitine causes potentiation of cell death. Based on these results, we suggest the potential use of R-Roscovitine as a bitargeted anticancer drug that functions by simultaneously causing p53 activation and NF-kappaB suppression. This study also provides mechanistic insight into the molecular mechanism of action of R-Roscovitine, thereby possibly explaining its anti-inflammatory properties.
Assuntos
Apoptose/efeitos dos fármacos , NF-kappa B/metabolismo , Purinas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Interleucina-1/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Roscovitina , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after gamma-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis.
Assuntos
Apoptose/genética , Retículo Endoplasmático/metabolismo , Genes p53 , Membrana Nuclear/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Humanos , Proteínas de Membrana , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares , Proteínas/genética , Proteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In this report we describe the development of a Fluorescent Protein-Protein Interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and Mdm4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and Mdm4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Fluorescência , Humanos , Microscopia de Fluorescência/métodos , Neoplasias/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacosRESUMO
In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-associated protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro, suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6- and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo.
Assuntos
Papillomaviridae/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Hidrólise , CamundongosAssuntos
Genes p53 , Mutação , Animais , Feminino , Genes Dominantes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genéticaRESUMO
Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Domínios ProteicosRESUMO
p63, p73 and p53 compose a family of transcription factors involved in cell response to stress and development. p53 is the most frequently mutated gene in cancer (50%) and loss of p53 activity is considered to be ubiquitous to all cancers. Recent publications may have a profound impact on our understanding of p53 tumour suppressor activity. p63, p73 and p53 genes have a dual gene structure conserved in drosophila, zebrafish and man. They encode for multiple p63, p73 or p53 proteins containing different protein domains (isoforms) due to multiple splicing, alternative promoter and alternative initiation of translation. In this review, we describe the different isoforms of p63, p73, p53 and their roles in development and cancer. The changes in the interactions between p53, p63 and p73 isoforms are likely to be fundamental to our understanding in the transition between normal cell cycling and the onset of tumour formation.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Processamento Alternativo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Evolução Molecular , Humanos , Camundongos , Camundongos Knockout , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transativadores/genética , Fatores de Transcrição , Transcrição Gênica , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: The DNA-binding activity of p53 is essential to its function as a tumour suppressor. Point mutations that abolish this activity have been found to occur frequently in the p53 genes of human cancer cells. Wild-type p53 protein assembles into oligomers with latent DNA-binding activity that can be activated in vitro by phosphorylation of a carboxy-terminal regulatory region, catalyzed by protein kinase C or casein kinase II. We have investigated the mechanism underlying this post-translational regulation of p53. Specifically, we have asked the following questions. First, whether the carboxy-terminal regulatory site contributes to p53's ability to form tetramers. Second, whether the latent DNA-binding activity of p53 can be activated in vivo. And third, whether the activation of p53 is reversible. RESULTS: Biophysical molecular-sizing analysis shows that both latent and activated forms of p53 are tetramers. Using a novel method, we have further established that p53 remains tetrameric when bound to DNA. We have also found that p53 can indeed be activated in vivo: p53 prepared from cells can be separated into activated and latent forms. Finally, we generated a monoclonal antibody specific for the casein kinase II target site in the carboxy-terminal regulatory region of p53, and used it to demonstrate the allosteric inhibition of in vitro and in vivo activated forms of p53. CONCLUSIONS: p53 protein assembles naturally as a tetramer that can be converted between latent and activated forms by a concerted, allosteric transition. The highly purified, reconstituted system that we have developed, in which the DNA-binding activity of p53 can be reversibly regulated, should facilitate the discovery of agents that can modulate the DNA-binding activity of p53--particularly those that can activate mutant p53 proteins and that may have potential in the design of anti-cancer drugs.
Assuntos
Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , DNA/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Proteína Supressora de Tumor p53/isolamento & purificaçãoRESUMO
An increasing body of evidence indicates that p53, the product of a tumour suppressor gene, has a role in development - could this developmental role have provided the primary driving force in the evolution of a protein best known as a stress-response integrator?
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Neoplasias/genética , Proteína Supressora de Tumor p53/fisiologia , Anormalidades Induzidas por Radiação/genética , Adaptação Fisiológica/genética , Adolescente , Adulto , Distribuição por Idade , Idoso , Animais , Criança , Pré-Escolar , Morte Fetal/etiologia , Genes p53 , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Lesões Experimentais por Radiação/genética , Estresse Fisiológico/genética , Estresse Fisiológico/fisiopatologiaRESUMO
Overexpression of the translation initiation factor eIF4E leads to cell transformation and occurs in a number of human cancers [1]. mRNA translation and cell growth can be regulated through the availability of eIF4E to form initiation complexes by binding to eIF4G. The availability of eIF4E is blocked through the binding of members of a family of eIF4E-binding proteins (4E-BPs) [2] [3]. Indeed, cell transformation caused by the overexpression of eIF4E can be reversed by the overexpression of 4E-BPs [4] [5] [6] [7] [8]. To study the role of eIF4E in cell transformation, we developed a series of peptides based on the conserved eIF4E-binding motifs in 4E-BPs and eIF4G [9] linked to the penetratin peptide-carrier sequence, which mediates the rapid transport of peptides across cell membranes. Surprisingly, introduction of these eIF4E-binding peptides into MRC5 cells led to rapid, dose-dependent cell death, with characteristics of apoptosis. Single alanine substitutions at key positions in the peptides impair their binding to eIF4E and markedly reduce their ability to induce apoptosis. A triple alanine substitution, which abolishes binding to eIF4E, renders the peptide unable to induce apoptosis. Our data provide strong evidence that the peptides induce apoptosis through binding to eIF4E. They do not induce apoptosis through inhibition of protein synthesis, as chemical inhibitors of translation did not induce apoptosis or affect peptide-induced cell death. Thus these new data indicate that eIF4E has a direct role in controlling cell survival that is not linked to its known role in mRNA translation.
Assuntos
Apoptose/efeitos dos fármacos , Fatores de Iniciação em Eucariotos , Oligopeptídeos/farmacologia , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Fator de Iniciação 4E em Eucariotos , Fator de Iniciação Eucariótico 4G , Humanos , Marcação In Situ das Extremidades Cortadas , Oligopeptídeos/metabolismo , Pactamicina/farmacologia , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/farmacologia , Fosfoproteínas/química , Fosfoproteínas/farmacologia , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
BACKGROUND: The CDKN2/INK4A tumour suppressor gene is deleted or mutated in a large number of human cancers. Overexpression of its product, p16, has been shown to block the transition through the G1/S phase of the cell cycle in a pRb-dependent fashion by inhibiting the cyclin D-dependent kinases cdk4 and cdk6. Reconstitution of p16 function in transformed cells is therefore an attractive target for anti-cancer drug design. RESULTS: We have identified a 20-residue synthetic peptide--corresponding to amino acids 84-103 of p16--that interacts with cdk4 and cdk6, and inhibits the in vitro phosphorylation of pRb mediated by cdk4-cyclin D1. The amino-acid residues of p16 important for its interaction with cdk4 and cdk6 and for the inhibition of pRb phosphorylation were defined by an alanine substitution series of peptides. In normal proliferating human HaCaT cells and in cells released from serum starvation, entry into S phase was blocked by the p16-derived peptide when it was coupled to a small peptide carrier molecule and applied directly to the tissue culture medium. This cell-cycle block was associated with an inhibition of pRb phosphorylation in vivo. CONCLUSIONS: These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.
Assuntos
Proteínas de Transporte/farmacologia , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas , Proteína do Retinoblastoma/antagonistas & inibidores , Sequência de Aminoácidos , Ciclo Celular , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fase SRESUMO
BACKGROUND: A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway. RESULTS: We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity. CONCLUSIONS: We have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.
Assuntos
Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Sequência de Aminoácidos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/farmacologia , Humanos , Dados de Sequência MolecularRESUMO
BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ciclinas/química , Ciclinas/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Ciclo Celular , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , Escherichia coli , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin. RESULTS: When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity. CONCLUSIONS: These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.
Assuntos
Proteínas Nucleares , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/farmacologia , Proteína Supressora de Tumor p53/biossíntese , Sequência de Aminoácidos , Animais , Ligação Competitiva , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Próstata/citologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/fisiologia , Tiorredoxinas/genética , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Vast libraries of random peptides displayed on the surface of bacteriophage potentially allow identification of specific ligands for any peptide receptor of interest. The sequence specificity of antibody-peptide interactions allows these libraries to be used to define and localize continuous epitopes. Remarkably clear-cut results have been obtained with some antibodies; however, the technique has not proved to be generally applicable.
Assuntos
Bacteriófagos/genética , Epitopos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Bacteriófagos/imunologia , Capsídeo/genética , Epitopos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.
Assuntos
Adenovírus Humanos/genética , Diferenciação Celular , Proteínas Oncogênicas Virais/fisiologia , Proteínas Precoces de Adenovirus , Antígenos de Superfície/análise , Antígenos Virais de Tumores/genética , Divisão Celular , Queratinas/biossíntese , Vírus 40 dos Símios/genética , Teratoma/patologia , TransfecçãoRESUMO
The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.
Assuntos
RNA Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Compartimento Celular , Ciclo Celular , Nucléolo Celular/enzimologia , Clonagem Molecular , Códon , Imunofluorescência , Genes Fúngicos , Humanos , Íntrons , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , RNA Helicases , Homologia de Sequência do Ácido NucleicoRESUMO
In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.