RESUMO
Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
Assuntos
Ciclinas/deficiência , Ciclinas/metabolismo , Proteólise/efeitos dos fármacos , Purinas/química , Purinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Purinas/toxicidade , Piridinas/toxicidade , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinação/efeitos dos fármacosRESUMO
E-type cyclins (cyclins E1 and E2) are components of the core cell cycle machinery and are overexpressed in many human tumor types. E cyclins are thought to drive tumor cell proliferation by activating the cyclin-dependent kinase 2 (CDK2). The cyclin E1 gene represents the site of recurrent integration of the hepatitis B virus in the pathogenesis of hepatocellular carcinoma, and this event is associated with strong up-regulation of cyclin E1 expression. Regardless of the underlying mechanism of tumorigenesis, the majority of liver cancers overexpress E-type cyclins. Here we used conditional cyclin E knockout mice and a liver cancer model to test the requirement for the function of E cyclins in liver tumorigenesis. We show that a ubiquitous, global shutdown of E cyclins did not visibly affect postnatal development or physiology of adult mice. However, an acute ablation of E cyclins halted liver cancer progression. We demonstrated that also human liver cancer cells critically depend on E cyclins for proliferation. In contrast, we found that the function of the cyclin E catalytic partner, CDK2, is dispensable in liver cancer cells. We observed that E cyclins drive proliferation of tumor cells in a CDK2- and kinase-independent mechanism. Our study suggests that compounds which degrade or inhibit cyclin E might represent a highly selective therapeutic strategy for patients with liver cancer, as these compounds would selectively cripple proliferation of tumor cells, while sparing normal tissues.
Assuntos
Ciclina E/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina E/deficiência , Ciclina E/genética , Quinase 2 Dependente de Ciclina/metabolismo , Ciclinas/deficiência , Ciclinas/genética , Ciclinas/metabolismo , Progressão da Doença , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismoRESUMO
Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer.
Assuntos
Aurora Quinase B/metabolismo , Ciclinas/metabolismo , Neoplasias da Próstata/patologia , Apoptose/fisiologia , Aurora Quinase B/biossíntese , Aurora Quinase B/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Ciclinas/deficiência , Ciclinas/genética , Humanos , Masculino , Mitose/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
Uniparental disomy (UPD), in which an individual contains a pair of homologous chromosomes originating from only one parent, is a frequent phenomenon that is linked to congenital disorders and various cancers. UPD is thought to result mostly from pre- or post-zygotic chromosome missegregation. However, the factors that drive UPD remain unknown. Here we use the fission yeast Schizosaccharomyces pombe as a model to investigate UPD, and show that defects in the RNA interference (RNAi) machinery or in the YTH domain-containing RNA elimination factor Mmi1 cause high levels of UPD in vegetative diploid cells. This phenomenon is not due to defects in heterochromatin assembly at centromeres. Notably, in cells lacking RNAi components or Mmi1, UPD is associated with the untimely expression of gametogenic genes. Deletion of the upregulated gene encoding the meiotic cohesin Rec8 or the cyclin Crs1 suppresses UPD in both RNAi and mmi1 mutants. Moreover, overexpression of Rec8 is sufficient to trigger UPD in wild-type cells. Rec8 expressed in vegetative cells localizes to chromosomal arms and to the centromere core, where it is required for localization of the cohesin subunit Psc3. The centromeric localization of Rec8 and Psc3 promotes UPD by uniquely affecting chromosome segregation, causing a reductional segregation of one homologue. Together, these findings establish the untimely vegetative expression of gametogenic genes as a causative factor of UPD, and provide a solid foundation for understanding this phenomenon, which is linked to diverse human diseases.
Assuntos
Regulação Fúngica da Expressão Gênica , Células Germinativas/metabolismo , Modelos Biológicos , Mutação , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Dissomia Uniparental/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Ciclinas/deficiência , Ciclinas/genética , Diploide , Heterocromatina/metabolismo , Humanos , Meiose/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Tempo , Dissomia Uniparental/patologia , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
A new member of the cyclin family cyclin Y (CCNY) is involved in the regulation of various physiological processes. In this study, the role of CCNY in energy metabolism was characterized. We found that compared with wild-type (WT) mice, Ccny knockout (KO) mice had both lower body weight and lower fat content. The Ccny KO mice also had a higher metabolic rate, resisted the stress of a high-fat diet, and were sensitive to calorie restriction. The expression levels of UCP1 and PGC1α were significantly higher in the brown adipose tissue (BAT) of the Ccny KO mice than that of the WT littermate controls, whereas there was no significant difference in BAT weight between the WT and the Ccny KO mice. In addition, the down-regulation of Ccny resulted in suppression of white adipocyte differentiation both in vivo and in vitro, while the expression of Ccny was up-regulated by C/EBPα. Furthermore, both hepatocytes and HepG2 cells that were depleted of Ccny were insensitive to insulin stimulation, consistent with the significant inhibition of insulin sensitivity in the liver of the Ccny KO mice, but no significant changes in WAT and muscle, indicating that CCNY is involved in regulating the hepatic insulin signaling pathway. The hepatic insulin resistance generated by Ccny depletion resulted in down-regulation of the sterol-regulatory element-binding protein (SREBP1) and fatty acid synthase (FASN). Together, these results provide a new link between CCNY and lipid metabolism in mice, and suggest that inhibition of CCNY may offer a therapeutic approach to obesity and diabetes.
Assuntos
Adipogenia , Ciclinas/metabolismo , Lipídeos/biossíntese , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Restrição Calórica , Diferenciação Celular/efeitos dos fármacos , Ciclinas/deficiência , Dieta Hiperlipídica , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Insulina/metabolismo , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Termogênese/efeitos dos fármacos , Termogênese/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Cyclin J (CycJ) is a poorly characterized member of the Cyclin superfamily of cyclin-dependent kinase regulators, many of which regulate the cell cycle or transcription. Although CycJ is conserved in metazoans its cellular function has not been identified and no mutant defects have been described. In Drosophila, CycJ transcript is present primarily in ovaries and very early embryos, suggesting a role in one or both of these tissues. The CycJ gene (CycJ) lies immediately downstream of armitage (armi), a gene involved in the Piwi-associated RNA (piRNA) pathways that are required for silencing transposons in the germline and adjacent somatic cells. Mutations in armi result in oogenesis defects but a role for CycJ in oogenesis has not been defined. Here we assessed oogenesis in CycJ mutants in the presence or absence of mutations in armi or other piRNA pathway genes. CycJ null ovaries appeared normal, indicating that CycJ is not essential for oogenesis under normal conditions. In contrast, armi null ovaries produced only two egg chambers per ovariole and the eggs had severe axis specification defects, as observed previously for armi and other piRNA pathway mutants. Surprisingly, the CycJ armi double mutant failed to produce any mature eggs. The double null ovaries generally had only one egg chamber per ovariole and the egg chambers frequently contained an overabundance of differentiated germline cells. Production of these compound egg chambers could be suppressed with CycJ transgenes but not with mutations in the checkpoint gene mnk, which suppress oogenesis defects in armi mutants. The CycJ null showed similar genetic interactions with the germline and somatic piRNA pathway gene piwi, and to a lesser extent with aubergine (aub), a member of the germline-specific piRNA pathway. The strong genetic interactions between CycJ and piRNA pathway genes reveal a role for CycJ in early oogenesis. Our results suggest that CycJ is required to regulate egg chamber production or maturation when piRNA pathways are compromised.
Assuntos
Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Oogênese/genética , RNA Interferente Pequeno/genética , Animais , Animais Geneticamente Modificados , Ciclinas/deficiência , Proteínas de Drosophila/deficiência , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genes de Insetos , Mutação , Ovário/citologia , Ovário/crescimento & desenvolvimento , RNA Helicases/deficiência , RNA Helicases/genéticaRESUMO
UNLABELLED: The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G(0) /G(1) -S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. CONCLUSION: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo.
Assuntos
Ciclina E/deficiência , Quinase 2 Dependente de Ciclina/deficiência , Replicação do DNA/fisiologia , Hepatócitos/patologia , Hepatócitos/fisiologia , Regeneração Hepática/fisiologia , Proteínas Oncogênicas/deficiência , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Células Cultivadas , Cromatina/fisiologia , Ciclina E/genética , Ciclina E/fisiologia , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/fisiologia , Ciclinas/deficiência , Ciclinas/genética , Ciclinas/fisiologia , Feminino , Homeostase/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/fisiologia , Fase S/fisiologiaRESUMO
Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 â¼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.
Assuntos
Agenesia do Corpo Caloso/genética , Proteínas de Transporte/genética , Ciclinas/deficiência , Ciclinas/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Éxons , Estudos de Associação Genética , Homozigoto , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Insercional , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Mitotic catastrophe occurs when cells enter mitosis with damaged DNA or excess centrosomes. Cells overexpressing the centrosome protein CP110 or depleted of cyclin F, which targets CP110 for destruction, have more than two centrosomes and undergo mitotic catastrophe. Our studies show centrosome reduplication and mitotic catastrophe in osteosarcoma cells inducibly expressing a p27Kip1 mutant (termed p27K) that binds cyclins but not cyclin-dependent kinases (CDKs). p27K inhibited cell proliferation but not CDK activity or cell cycle progression. It did not induce apoptosis; however, cells expressing p27K had more than two centrosomes and, indicative of mitotic catastrophe, irregularly shaped nuclei or multiple micronuclei. p27K interacted with cyclin F in vivo (as did endogenous p27Kip1) and displaced cyclin F from CP110. Depletion of CP110 rescued p27K-expressing cells from centrosome reduplication and mitotic catastrophe. Collectively, our data show that p27Kip1 can perturb mitosis and suggest that it does so by sequestering cyclin F, which prevents its interaction with and the subsequent degradation of CP110, ultimately resulting in centrosome reduplication, mitotic catastrophe and abrogation of cell proliferation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Fosfoproteínas/metabolismo , Apoptose/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células , Ciclinas/deficiência , Dano ao DNA , Humanos , Osteossarcoma/genética , Osteossarcoma/patologiaRESUMO
Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.
Assuntos
Portadores de Fármacos/química , Levivirus/química , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclinas/deficiência , Ciclinas/genética , Portadores de Fármacos/metabolismo , Endocitose , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Viral/metabolismo , Ricina/metabolismo , Ricina/farmacologiaRESUMO
Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the approximately 40-amino-acid domain required for binding to Skp1) was first described. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs). However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein that is essential for centrosome duplication, as an interactor and substrate of Cyclin F. Using a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated by the SCF(Cyclin F) ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that cannot bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCF(Cyclin F)-mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Ciclinas/metabolismo , Homeostase , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Centríolos/metabolismo , Centrossomo/química , Ciclinas/química , Ciclinas/deficiência , Ciclinas/genética , Fase G2 , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Fenótipo , Ligação Proteica , Proteínas Ligases SKP Culina F-Box/metabolismo , Especificidade por Substrato , UbiquitinaçãoRESUMO
The Drosophila gene CG14939 encodes a member of a highly conserved family of cyclins, the Y-type cyclins, which have not been functionally characterized in any organism. Here we report the generation and phenotypic characterization of a null mutant of CG14939, which we rename Cyclin Y (CycY). We show that the null mutant, CycY(E8), is homozygous lethal with most mutant animals arresting during pupal development. The mutant exhibits delayed larval growth and major developmental defects during metamorphosis, including impaired gas bubble translocation, head eversion, leg elongation, and adult tissue growth. Heat-shock-induced expression of CycY at different times during development resulted in variable levels of rescue, the timing of which suggests a key function for zygotic CycY during the transition from third instar larvae to prepupae. CycY also plays an essential role during embryogenesis since zygotic null embryos from null mothers fail to hatch into first instar larvae. We provide evidence that the CycY protein (CycY) interacts with Eip63E, a cyclin-dependent kinase (Cdk) for which no cyclin partner had previously been identified. Like CycY, the Eip63E gene has essential functions during embryogenesis, larval development, and metamorphosis. Our data suggest that CycY/Eip63E form a cyclin/Cdk complex that is essential for several developmental processes.
Assuntos
Sequência Conservada , Ciclinas/química , Ciclinas/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Alelos , Animais , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/deficiência , Ciclinas/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Metamorfose Biológica/genética , Fenótipo , Fosforilação , Pupa/genética , Pupa/crescimento & desenvolvimento , Deleção de Sequência , Zigoto/metabolismoRESUMO
The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.
Assuntos
Ciclinas/deficiência , Hipocampo/citologia , Memória/fisiologia , Neurogênese/genética , Neurônios/fisiologia , Análise de Variância , Animais , Ansiedade/genética , Bromodesoxiuridina/metabolismo , Condicionamento Clássico/fisiologia , Condicionamento Operante/fisiologia , Ciclina D2 , Proteínas do Domínio Duplacortina , Comportamento Exploratório/fisiologia , Medo/fisiologia , Locomoção/genética , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Transtornos do Olfato/genética , Desempenho Psicomotor/fisiologiaRESUMO
The positive transcription elongation factor b (P-TEFb) is essential for the elongation of transcription and cotranscriptional processing by RNA polymerase II. In mammals, it contains predominantly the C-type cyclin cyclin T1 (CycT1) or CycT2 and cyclin-dependent kinase 9 (Cdk9). To determine if these cyclins have redundant functions or affect distinct sets of genes, we genetically inactivated the CycT2 gene (Ccnt2) using the beta-galactosidase-neomycin gene (beta-geo) gene trap technology in the mouse. Visualizing beta-galactosidase during mouse embryogenesis revealed that CycT2 is expressed abundantly during embryogenesis and throughout the organism in the adult. This finding was reflected in the expression of CycT2 in all adult tissues and organs. However, despite numerous matings of heterozygous mice, we observed no CycT2(-/-) embryos, pups, or adult mice. This early lethality could have resulted from decreased expression of critical genes, which were revealed by short interfering RNAs against CycT2 in embryonic stem cells. Thus, CycT1 and CycT2 are not redundant, and these different P-TEFb complexes regulate subsets of distinct genes that are important for embryonic development.
Assuntos
Ciclinas/metabolismo , Desenvolvimento Embrionário/fisiologia , Animais , Sequência de Bases , Ciclina T/antagonistas & inibidores , Ciclina T/deficiência , Ciclina T/genética , Ciclina T/metabolismo , Ciclinas/antagonistas & inibidores , Ciclinas/deficiência , Ciclinas/genética , Primers do DNA/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Fator B de Elongação Transcricional Positiva/metabolismo , Gravidez , RNA Interferente Pequeno/genética , Distribuição TecidualRESUMO
Peripheral nerve injury is followed by a wave of Schwann cell proliferation in the distal nerve stumps. To resolve the role of Schwann cell proliferation during functional recovery of the injured nerves, we used a mouse model in which injury-induced Schwann cell mitotic response is ablated via targeted disruption of cyclin D1. In the absence of distal Schwann cell proliferation, axonal regeneration and myelination occur normally in the mutant mice and functional recovery of injured nerves is achieved. This is enabled by pre-existing Schwann cells in the distal stump that persist but do not divide. On the other hand, in the wild type littermates, newly generated Schwann cells of injured nerves are culled by apoptosis. As a result, distal Schwann cell numbers in wild type and cyclin D1 null mice converge to equivalence in regenerated nerves. Therefore, distal Schwann cell proliferation is not required for functional recovery of injured nerves.
Assuntos
Apoptose/fisiologia , Axônios/fisiologia , Proliferação de Células , Regeneração Nervosa/fisiologia , Células de Schwann/fisiologia , Degeneração Walleriana/patologia , Animais , Apoptose/genética , Axônios/patologia , Ciclina D , Ciclinas/deficiência , Ciclinas/genética , Camundongos , Camundongos Knockout , Fibras Nervosas Mielinizadas/patologia , Fibras Nervosas Mielinizadas/fisiologia , Regeneração Nervosa/genética , Nervos Periféricos/citologia , Nervos Periféricos/patologia , Nervos Periféricos/fisiologia , Células de Schwann/citologia , Degeneração Walleriana/genéticaRESUMO
Recent studies have shown that cell cycle inhibitors encoded by the Ink4a gene locus constrain the self-renewing activity of adult stem cells of the hematopoietic and nervous systems. Here we report that knockout (KO) of the Cables1 [cyclin-dependent kinase (CDK)-5 and ABL enzyme substrate 1] cell cycle-regulatory gene in mice has minimal to no effect on hematopoietic stem cell (HSC) dynamics. However, female Cables1-null mice exhibit a significant expansion of germ cell (oocyte) numbers throughout adulthood. This is accompanied by a dramatic elevation in the number of atretic immature oocytes within the ovaries and an increase in the incidence of degenerating oocytes retrieved following superovulation of CABLES1-deficient females. These outcomes are not observed in mice lacking p16INK4a alone or both p16INK4a and p19ARF. These data support recent reports that adult female mice can generate new oocytes and follicles but the enhancement of postnatal oogenesis by Cables1 KO appears offset by a reduction in oocyte quality, as reflected by increased elimination of these additional germ cells via apoptosis. This work also reveals cell lineage specificity with respect to the role that specific CDK-interacting proteins play in restraining the activity of adult germline versus somatic stem cells.
Assuntos
Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/deficiência , Ciclinas/metabolismo , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , Fatores Etários , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Contagem de Células , Quinases Ciclina-Dependentes/deficiência , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/fisiologia , Feminino , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Óvulo/citologia , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/fisiologiaRESUMO
AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1-/- mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Fator B de Elongação Transcricional Positiva/metabolismo , Timo/citologia , Timo/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Ciclinas/deficiência , Células Epiteliais/enzimologia , Células HeLa , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismo , Timo/enzimologia , Ativação Transcricional , Proteína AIRERESUMO
Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Ciclinas/metabolismo , Citocininas/metabolismo , Envelhecimento/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Ciclo Celular , Proliferação de Células , Tamanho Celular , Ciclinas/classificação , Ciclinas/deficiência , Ciclinas/genética , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Mutação/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-Delta) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1-Delta phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1-Delta limit the synthesis of nitric oxide (.NO) in both physiological and oxidative stress conditions. This defect in .NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1-Delta phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize .NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease.
Assuntos
Lipofuscinoses Ceroides Neuronais/metabolismo , Óxido Nítrico/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclinas/deficiência , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vitamina K 3/farmacologiaRESUMO
Peritoneal B-1a cells differ from splenic B-2 cells in the molecular mechanisms that control G(0)-S progression. In contrast to B-2 cells, cyclin D2 is up-regulated in a rapid and transient manner in phorbol ester (PMA)-stimulated B-1a cells, whereas cyclin D3 does not accumulate until late G(1) phase. This nonoverlapping expression of cyclins D2 and D3 suggests distinct functions for these proteins in B-1a cells. To investigate the contribution of cyclin D3 in the proliferation of B-1a cells, we transduced p16(INK4a) peptidyl mimetics (TAT-p16) into B-1a cells before cyclin D3 induction to specifically block cyclin D3-cyclin-dependent kinase 4/6 assembly. TAT-p16 inhibited DNA synthesis in B-1a cells stimulated by PMA, CD40L, or LPS as well as endogenous pRb phosphorylation by cyclin D-cyclin-dependent kinase 4/6. Unexpectedly, however, cyclin D3-deficient B-1a cells proliferated in a manner similar to wild-type B-1a cells following PMA or LPS stimulation. This was due, at least in part, to the compensatory sustained accumulation of cyclin D2 throughout G(0)-S progression. Taken together, experiments in which cyclin D3 was inhibited in real time demonstrate the key role this cyclin plays in normal B-1a cell mitogenesis, whereas experiments with cyclin D3-deficient B-1a cells show that cyclin D2 can compensate for cyclin D3 loss in mutant mice.