RESUMO
D-type cyclin homologs have been found in the genomes of herpesviruses associated with neoplasias. They appear to exploit features of G(1) cyclins but extend their properties to allow for deregulation of the cell cycle. Advances in the study of the molecular basis for these novel features as well as the potential role of viral cyclins in tumorigenesis are addressed.
Assuntos
Ciclinas/genética , Herpesviridae/genética , Neoplasias/genética , Oncogenes , Proteínas Virais/genética , Animais , Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Herpesviridae/metabolismo , Humanos , Neoplasias/virologia , Proteínas Virais/metabolismoRESUMO
Previous studies have indicated that mutation of RAP1 (rap1s) or of the HMR-E silencer ARS consensus element leads to metastable repression of HMR. A number of extragenic suppressor mutations (sds, suppressors of defective silencing) that increase the fraction of repressed cells in rap1s hmr delta A strains have been identified. Here we report the cloning of three SDS genes. SDS11 is identical to SWI6, a transcriptional regulator of genes required for DNA replication and of cyclin genes. SDS12 is identical to RNR1, which encodes a subunit of ribonucleotide reductase. SDS15 is identical to CIN8, whose product is required for spindle formation. We propose that mutations in these genes improve the establishment of silencing by interfering with normal cell cycle progression. In support of this idea, we show that exposure to hydroxyurea, which increases the length of S phase, also restores silencing in rap1s hmr delta A strains. Mutations in different cyclin genes (CLN3, CLB5, and CLB2) and two cell cycle transcriptional regulators (SWI4 and MBP1) also suppress the silencing defect at HMR. The effect of these cell cycle regulators is not specific to the rap1s or hmr delta A mutation, since swi6, swi4, and clb5 mutations also suppress mutations in SIR1, another gene implicated in the establishment of silencing. Several mutations also improve the efficiency of telomeric silencing in wild-type strains, further demonstrating that disturbance of the cell cycle has a general effect on position effect repression in Saccharomyces cerevisiae. We suggest several possible models to explain this phenomenon.
Assuntos
Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Peptídeos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Clonagem Molecular , Ciclinas/genética , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Genes Fúngicos/genética , Genes Reguladores , Hidroxiureia/farmacologia , Cinesinas , Fator de Acasalamento , Proteínas Associadas aos Microtúbulos , Mutagênese , Mutação , Biossíntese Peptídica , Ribonucleotídeo Redutases/genética , Telômero/genética , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclinas/metabolismo , Replicação do DNA , Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Virais/metabolismo , Células 3T3 , Animais , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/antagonistas & inibidores , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Humanos , Camundongos , Microscopia de Fluorescência , Complexo de Reconhecimento de Origem , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/farmacologia , Roscovitina , Fase S , Transfecção , Células Tumorais CultivadasRESUMO
The impact of various disabilities may differ greatly from individual to individual. Therefore, for a full assessment of a rehabilitee, objective aspects of disability should be supplemented with disability perception. The purpose of this study was to construct a self-administered questionnaire of 39 abilities/activities based on the ICIDH D-code, with parallel questions about '(dis)ability' and 'impact'. The instrument was used to obtain data from 60 disabled people. Fifty forms (83%) were returned from 25 patients with spinal cord injury and 25 patients with rheumatoid arthritis. Mean time to fill out the questionnaire was 24 minutes. A first attempt to validate the 'weighted score' with an open question about the most negative aspects of the disease showed a (very) good correspondence in 74%. An information gain as a result of the impact question was noted in 44%. This instrument has attractive properties for clinical use and research purposes in rehabilitation medicine. It was developed for the identification of needs of individual patients. The 'weighted scores' allow the setting of priorities in an individually tailored rehabilitation programme. Future applications might include monitoring the progress of a rehabilitee and identification of group needs.
Assuntos
Avaliação da Deficiência , Qualidade de Vida , Reabilitação/psicologia , Atividades Cotidianas , Adolescente , Adulto , Idoso , Artrite Reumatoide/reabilitação , Feminino , Nível de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Planejamento de Assistência ao Paciente , Traumatismos da Medula Espinal/reabilitação , Inquéritos e QuestionáriosAssuntos
Metilação de DNA , Herpesvirus Humano 8/crescimento & desenvolvimento , N-Acetil-Muramil-L-Alanina Amidase , Sarcoma de Kaposi/virologia , Proteína de Ligação a CREB , Ilhas de CpG , Enzimas/genética , Regulação da Expressão Gênica , Herpesvirus Humano 8/genética , Modelos Genéticos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transativadores/metabolismo , Integração Viral , Latência ViralRESUMO
Viral cyclin/cdk6 complexes interact with and phosphorylate human Orc1, a component of the origin recognition complex (ORC) that functions in DNA replication. Here we assess the effect that viral cyclin has on the intracellular location of human Orc1, which is present in both nuclear and cytoplasmic pools. Overexpression of K cyclin or cyclin A results in Crm1-dependent export of Orc1 to the cytoplasm, and this process is dependent on the phosphorylation status of several cdk target sites in Orc1. These findings support a model where S phase promoting cyclin activity drives the export of a component of replication complexes.
Assuntos
Compartimento Celular/genética , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Transporte Proteico/genética , Fase S/genética , Sequência de Aminoácidos/genética , Antineoplásicos/farmacologia , Ácido Aspártico/genética , Núcleo Celular/genética , Ciclina A/genética , Ciclina E/genética , Citoplasma/genética , Ácidos Graxos Insaturados/farmacologia , Regulação da Expressão Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Mutação/fisiologia , Complexo de Reconhecimento de Origem , Fosforilação , Células Tumorais Cultivadas , Proteínas ViraisRESUMO
Synthesis of urease by Klebsiella species is known to be induced when the nitrogen source of the growth medium is limiting, suggesting that urease gene expression is controlled by the nitrogen regulatory (ntr) system. This study showed that K. pneumoniae with mutations in either ntrA or ntrC, two integral components of the ntr system, were phenotypically urease-negative. These mutants could be complemented back to a urease positive phenotype with recombinant plasmids encoding the corresponding ntr gene. A series of ure-lacZYA transcriptional fusions, in conjunction with primer extension analysis, identified a DNA region that encoded a nitrogen-regulated promoter. This promoter region controlled transcription of ureD, the first gene in the Klebsiella pneumoniae urease gene cluster, and ureA, a gene that resides immediately downstream of ureD. A high level of transcription from the ureD promoter required NAC, a recently characterized member of the nitrogen regulatory cascade. NAC is a Lys R-like transcriptional regulator that can act at sigma 70 promoters; expression from nac itself is dependent upon NTRA. Therefore, expression of K. pneumoniae urease was dependent upon the nitrogen regulatory cascade, and transcription of at least two urease genes was from a promoter that was positively regulated by NAC.
Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/genética , Nitrogênio/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Urease/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/fisiologia , Genes Bacterianos , Klebsiella pneumoniae/metabolismo , Dados de Sequência Molecular , Transdução de Sinais , Fatores de Transcrição/fisiologia , Transcrição Gênica , Urease/genéticaRESUMO
Several gamma-herpesviruses encode proteins related to the mammalian cyclins, regulatory subunits of cyclin-dependent kinases (cdks) essential for cell cycle progression. We report a 2.5 A crystal structure of a full-length oncogenic viral cyclin from gamma-herpesvirus 68 complexed with cdk2. The viral cyclin binds cdk2 with an orientation different from cyclin A and makes several novel interactions at the interface, yet it activates cdk2 by triggering conformational changes similar to cyclin A. Sequences within the viral cyclin N-terminus lock part of the cdk2 T-loop within the core of the complex. These sequences and others are conserved amongst the viral and cellular D-type cyclins, suggesting that this structure has wider implications for other cyclin-cdk complexes. The observed resistance of this viral cyclin-cdk complex to inhibition by the p27(KIP:) cdk inhibitor is explained by sequence and conformational variation in the cyclin rendering the p27(KIP:)-binding site on the cyclin subunit non-functional.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclina A/química , Quinases Ciclina-Dependentes/química , Ciclinas/química , Gammaherpesvirinae/química , Proteínas Serina-Treonina Quinases/química , Proteínas Supressoras de Tumor , Proteínas Virais/química , Sequência de Aminoácidos , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistência a Medicamentos , Proteínas Associadas aos Microtúbulos/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Ligação ProteicaRESUMO
DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposi's sarcoma-associated herpesvirus) which is able to resist the actions of p16(Ink4a), p21(Cip1) and p27(Kip1) cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27(Kip1). We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin-cdk2 complexes but not cyclin D1-cdk6. Included amongst these substrates is p27(Kip1) which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27(Kip1) downregulation, this being consistent with previous studies indicating that phosphorylation of p27(Kip1) on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27(Kip1) in which Thr187 is mutated to non-phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27(Kip1)-imposed G1 arrest by facilitating phosphorylation and downregulation of p27(Kip1) to enable activation of endogenous cyclin-cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus-encoded cyclin.