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1.
J Biol Chem ; 295(48): 16280-16291, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-32934007

RESUMO

The Cdk8 kinase module (CKM) is a detachable Mediator subunit composed of cyclin C and one each of paralogs Cdk8/Cdk19, Med12/Med12L, and Med13/Med13L. Our previous RNA-Seq studies demonstrated that cyclin C represses a subset of hydrogen peroxide-induced genes under normal conditions but is involved in activating other loci following stress. Here, we show that cyclin C directs this transcriptional reprograming through changes in its promoter occupancy. Following peroxide stress, cyclin C promoter occupancy increased for genes it activates while decreasing at loci it represses under normal conditions. Promoter occupancy of other CKM components generally mirrored cyclin C, indicating that the CKM moves as a single unit. It has previously been shown that some cyclin C leaves the nucleus following cytotoxic stress to induce mitochondrial fragmentation and apoptosis. We observed that CKM integrity appeared compromised at a subset of repressed promoters, suggesting a source of cyclin C that is targeted for nuclear release. Interestingly, mTOR inhibition induced a new pattern of cyclin C promoter occupancy indicating that this control is fine-tuned to the individual stress. Using inhibitors, we found that Cdk8 kinase activity is not required for CKM movement or repression but was necessary for full gene activation. In conclusion, this study revealed that different stress stimuli elicit specific changes in CKM promoter occupancy correlating to altered transcriptional outputs. Finally, although CKM components were recruited or expelled from promoters as a unit, heterogeneity was observed at individual promoters, suggesting a mechanism to generate gene- and stress-specific responses.


Assuntos
Núcleo Celular/metabolismo , Ciclina C/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Transcrição Genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Transformada , Núcleo Celular/genética , Ciclina C/genética , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
2.
PLoS Genet ; 16(5): e1008832, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463833

RESUMO

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.


Assuntos
Ciclina C/genética , Quinase 8 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Haploinsuficiência , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Transdução de Sinais , Transcrição Genética
3.
J Am Heart Assoc ; 9(7): e014366, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32248761

RESUMO

Background Nuclear-to-mitochondrial communication regulating gene expression and mitochondrial function is a critical process following cardiac ischemic injury. In this study, we determined that cyclin C, a component of the Mediator complex, regulates cardiac and mitochondrial function in part by modifying mitochondrial fission. We tested the hypothesis that cyclin C functions as a transcriptional cofactor in the nucleus and a signaling molecule stimulating mitochondrial fission in response to stimuli such as cardiac ischemia. Methods and Results We utilized gain- and loss-of-function mouse models in which the CCNC (cyclin C) gene was constitutively expressed (transgenic, CycC cTg) or deleted (knockout, CycC cKO) in cardiomyocytes. The knockout and transgenic mice exhibited decreased cardiac function and altered mitochondria morphology. The hearts of knockout mice had enlarged mitochondria with increased length and area, whereas mitochondria from the hearts of transgenic mice were significantly smaller, demonstrating a role for cyclin C in regulating mitochondrial dynamics in vivo. Hearts from knockout mice displayed altered gene transcription and metabolic function, suggesting that cyclin C is essential for maintaining normal cardiac function. In vitro and in vivo studies revealed that cyclin C translocates to the cytoplasm, enhancing mitochondria fission following stress. We demonstrated that cyclin C interacts with Cdk1 (cyclin-dependent kinase 1) in vivo following ischemia/reperfusion injury and that, consequently, pretreatment with a Cdk1 inhibitor results in reduced mitochondrial fission. This finding suggests a potential therapeutic target to regulate mitochondrial dynamics in response to stress. Conclusions Our study revealed that cyclin C acts as a nuclear-to-mitochondrial signaling factor that regulates both cardiac hypertrophic gene expression and mitochondrial fission. This finding provides new insights into the regulation of cardiac energy metabolism following acute ischemic injury.


Assuntos
Ciclina C/metabolismo , Metabolismo Energético , Mitocôndrias Cardíacas/metabolismo , Dinâmica Mitocondrial , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Ciclina C/deficiência , Ciclina C/genética , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Ratos Wistar , Transdução de Sinais
4.
Mol Biol Cell ; 31(10): 1015-1031, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32160104

RESUMO

Environmental stress elicits well-orchestrated programs that either restore cellular homeostasis or induce cell death depending on the insult. Nutrient starvation triggers the autophagic pathway that requires the induction of several Autophagy (ATG) genes. Cyclin C-cyclin-dependent kinase (Cdk8) is a component of the RNA polymerase II Mediator complex that predominantly represses the transcription of stress-responsive genes in yeast. To relieve this repression following oxidative stress, cyclin C translocates to the mitochondria where it induces organelle fragmentation and promotes cell death prior to its destruction by the ubiquitin-proteasome system (UPS). Here we report that cyclin C-Cdk8, together with the Ume6-Rpd3 histone deacetylase complex, represses the essential autophagy gene ATG8. Similar to oxidative stress, cyclin C is destroyed by the UPS following nitrogen starvation. Removing this repression is important as deleting CNC1 allows enhanced cell growth under mild starvation. However, unlike oxidative stress, cyclin C is destroyed prior to its cytoplasmic translocation. This is important as targeting cyclin C to the mitochondria induces both mitochondrial fragmentation and cell death following nitrogen starvation. These results indicate that cyclin C destruction pathways are fine tuned depending on the stress and that its terminal subcellular address influences the decision between initiating cell death or cell survival pathways.


Assuntos
Ciclina C/metabolismo , Nitrogênio/deficiência , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quinase 8 Dependente de Ciclina/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Estresse Fisiológico/efeitos dos fármacos
5.
Plant Physiol ; 182(3): 1375-1386, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31964799

RESUMO

Control of flowering time has been a major focus of comparative genetic analyses in plant development. This study reports on a forward genetic approach to define previously uncharacterized components of flowering control pathways in the long-day legume, pea (Pisum sativum). We isolated two complementation groups of late-flowering mutants in pea that define two uncharacterized loci, LATE BLOOMER3 (LATE3) and LATE4, and describe their diverse effects on vegetative and reproductive development. A map-based comparative approach was employed to identify the underlying genes for both loci, revealing that that LATE3 and LATE4 are orthologs of CYCLIN DEPENDENT KINASE8 (CDK8) and CYCLIN C1 (CYCC1), components of the CDK8 kinase module of the Mediator complex, which is a deeply conserved regulator of transcription in eukaryotes. We confirm the genetic and physical interaction of LATE3 and LATE4 and show that they contribute to the transcriptional regulation of key flowering genes, including the induction of the florigen gene FTa1 and repression of the floral repressor LF Our results establish the conserved importance of the CDK8 module in plants and provide evidence for the function of CYCLIN C1 orthologs in the promotion of flowering and the maintenance of normal reproductive development.


Assuntos
Flores/metabolismo , Complexo Mediador/metabolismo , Ervilhas/metabolismo , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica de Plantas
6.
Proc Natl Acad Sci U S A ; 117(6): 2894-2905, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988137

RESUMO

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.


Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Complexo Mediador/metabolismo , Domínio Catalítico , Ciclina C/genética , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/química , Quinase 8 Dependente de Ciclina/genética , Ativação Enzimática , Humanos , Complexo Mediador/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos
7.
EMBO Rep ; 20(9): e47425, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31385392

RESUMO

Intrinsic apoptosis requires mitochondrial outer membrane disruption triggered by recruitment, activation, and oligomerization of the Bcl-2 homology protein Bax. Following oxidative stress, we demonstrated that the transcriptional regulator cyclin C is released into the cytosol where it directs mitochondrial fragmentation and efficient apoptotic induction. This study reveals that cytoplasmic cyclin C is required for both normal Bax activation and its efficient mitochondrial localization. This activity appears direct as cyclin C co-immunoprecipitates with active Bax in stressed cells and binds recombinant Bax in vitro. In addition, stable cyclin C-Bax association requires the fission complex. Pharmacologically stimulating cyclin C nuclear release is sufficient for Bax association and their mitochondrial localization in the absence of any stress signals. However, these cells do not undergo cell death as Bax fails to oligomerize. These data support a model that cyclin C association defines an initial step in Bax mitochondrial recruitment and provides a physical connection between the fission and apoptotic factors. This strategy allows the cell to discriminate stress-induced fission able to recruit Bax from other types of mitochondrial divisions.


Assuntos
Ciclina C/metabolismo , Mitocôndrias/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
8.
J Cell Sci ; 132(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31331961

RESUMO

The cyclin C-Cdk8 kinase has been identified as both a tumor suppressor and an oncogene depending on the cell type. The genomic locus encoding cyclin C (Ccnc) is often deleted in aggressive anaplastic thyroid tumors. To test for a potential tumor suppressor role for cyclin C, Ccnc alone, or Ccnc in combination with a previously described thyroid tumor suppressor Pten, was deleted late in thyroid development. Although mice harboring individual Pten or Ccnc deletions exhibited modest thyroid hyperplasia, the double mutant demonstrated dramatic thyroid expansion resulting in animal death by 22 weeks. Further analysis revealed that Ccncthyr-/- tissues exhibited a reduction in signal transducer and activator of transcription 3 (Stat3) phosphorylation at Ser727. Further analysis uncovered a post-transcriptional requirement of both Pten and cyclin C in maintaining the levels of the p21 and p53 tumor suppressors (also known as CDKN1A and TP53, respectively) in thyroid tissue. In conclusion, these data reveal the first tumor suppressor role for cyclin C in a solid tumor model. In addition, this study uncovers new synergistic activities of Pten and cyclin C to promote quiescence through maintenance of p21 and p53.


Assuntos
Ciclina C/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Linhagem Celular Tumoral , Ciclina C/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia
9.
G3 (Bethesda) ; 9(6): 1901-1908, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036676

RESUMO

The transcriptional changes that occur in response to oxidative stress help direct the decision to maintain cell viability or enter a cell death pathway. Cyclin C-Cdk8 is a conserved kinase that associates with the RNA polymerase II Mediator complex that stimulates or represses transcription depending on the locus. In response to oxidative stress, cyclin C, but not Cdk8, displays partial translocation into the cytoplasm. These findings open the possibility that cyclin C relocalization is a regulatory mechanism governing oxidative stress-induced transcriptional changes. In the present study, the cyclin C-dependent transcriptome was determined and compared to transcriptional changes occurring in oxidatively stressed Mus musculus embryonic fibroblasts. We observed a similar number (∼2000) of genes up or downregulated in oxidatively stressed cells. Induced genes include cellular repair/survival factors while repressed loci were generally involved in proliferation or differentiation. Depleting cyclin C in unstressed cells produced an approximately equal number of genes (∼2400) that were repressed by, or whose transcription required, cyclin C. Consistent with the possibility that cyclin C nuclear release contributes to transcriptional remodeling in response to oxidative stress, we found that 37% cyclin C-dependent genes were downregulated following stress. Moreover, 20% of cyclin C- repressed genes were induced in response to stress. These findings are consistent with a model that cyclin C relocalization to the cytoplasm, and corresponding inactivation of Cdk8, represents a regulatory mechanism to repress and stimulate transcription of stress-responsive genes.


Assuntos
Ciclina C/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Estresse Oxidativo/genética , Transcriptoma , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Camundongos , Reprodutibilidade dos Testes
10.
J Biol Chem ; 294(23): 9076-9083, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31028171

RESUMO

The Mediator complex plays a critical role in the regulation of transcription by linking transcription factors to RNA polymerase II. By examining mouse livers, we have found that in the fasted state, the Mediator complex exists primarily as an approximately 1.2-MDa complex, consistent with the size of the large Mediator complex, whereas following feeding, it converts to an approximately 600-kDa complex, consistent with the size of the core Mediator complex. This dynamic change is due to the dissociation and degradation of the kinase module that includes the MED13, MED12, cyclin-dependent kinase 8 (CDK8), and cyclin C (CCNC) subunits. The dissociation and degradation of the kinase module are dependent upon nutrient activation of mTORC1 that is necessary for the induction of lipogenic gene expression because pharmacological or genetic inhibition of mTORC1 in the fed state restores the kinase module. The degradation but not dissociation of the kinase module depends upon the E3 ligase, SCFFBW7 In addition, genetically insulin-resistant and obese db/db mice in the fasted state displayed elevated lipogenic gene expression and loss of the kinase module that was reversed following mTORC1 inhibition. These data demonstrate that the assembly state of the Mediator complex undergoes physiologic regulation during normal cycles of fasting and feeding in the mouse liver. Furthermore, the assembly state of the Mediator complex is dysregulated in states of obesity and insulin resistance.


Assuntos
Resistência à Insulina , Complexo Mediador/metabolismo , Obesidade/patologia , Animais , Núcleo Celular/metabolismo , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Fígado/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Nutrientes/administração & dosagem , Obesidade/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Ligases SKP Culina F-Box/deficiência , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia
11.
Biol Pharm Bull ; 42(5): 692-702, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30828041

RESUMO

Endoplasmic reticulum (ER) stress-mediated apoptosis pathway is considered to play a vital role in mediating stroke and other cerebrovascular diseases. Previous studies have showed that vascular endothelial growth factor (VEGF) antagonism reduced cerebral ischemic-reperfusion (CI/R) damage, but whether attenuation of ER stress-induced apoptosis is contributing to its mechanisms remains elusive. Our study aimed to investigate the protective effect of VEGF antagonism on CI/R-induced injury. First, oxygen-glucose deprivation and re-oxygenation (OGD/R) BEND3 cell model was constructed to estimate small interfering RNA (siRNA)-VEGF on damage of endothelial cells. Next, in animal model, CI/R mice were induced by middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h reperfusion to investigate cerebral tissue damage. For treatment group, mice received 100 µg/kg anti-VEGF antibodies at 30 min before MCAO, followed by 24 h reperfusion. Our findings demonstrated that pre-administration of siRNA-VEGF before OGD/R changed the biological characteristics of BEND3 cells, reversed the levels of X-box binding protein-1 (XBP-1) and glucose-regulated protein 78 (GRP78), showing siRNA-VEGF attenuated, at least in part, the oxidative damage in OGD/R cell by down-regulating ER stress. In mice experiment, pre-administration of anti-VEGF antibody reduced the brain infarct volume and edema extent and improved neurological scores outcome of CI/R injury mice. Pathological and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining results also confirmed this protective effect. The expressions of VEGF, CATT/EBP homologous protein (CHOP), inositol requiring enzyme 1α (IRE-1α), and cleaved-caspase12 and c-jun N-terminal kinase (JNK) phosphorylation were also prominently decreased. These results suggested that inhibition of endogenous VEGF attenuates CI/R-induced injury via inhibiting ER stress-mediated apoptosis.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carbamatos/metabolismo , Caspase 12/metabolismo , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina C/metabolismo , Células Endoteliais , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
mBio ; 10(1)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755515

RESUMO

Cryptococcus neoformans kills 200,000 people worldwide each year. After inhalation, this environmental yeast proliferates either extracellularly or within host macrophages. Under conditions of immunocompromise, cryptococci disseminate from the lungs to the brain, causing a deadly meningoencephalitis that is difficult and expensive to treat. Cryptococcal adaptation to the harsh lung environment is a critical first step in its pathogenesis, and consequently a compelling topic of study. This adaptation is mediated by a complex transcriptional program that integrates cellular responses to environmental stimuli. Although several key regulators in this process have been examined, one that remains understudied in C. neoformans is the Mediator complex. In other organisms, this complex promotes transcription of specific genes by increasing assembly of the RNA polymerase II preinitiation complex. We focused on the Kinase Module of Mediator, which consists of cyclin C (Ssn801), cyclin-dependent kinase 8 (Cdk8), Med12, and Med13. This module provides important inhibitory control of Mediator complex assembly and activity. Using transcriptomics, we discovered that Cdk8 and Ssn801 together regulate cryptococcal functions such as the ability to grow on acetate and the response to oxidative stress, both of which were experimentally validated. Deletion of CDK8 yielded altered mitochondrial morphology and the dysregulation of genes involved in oxidation-reduction processes. This strain exhibited increased susceptibility to oxidative stress, resulting in an inability of mutant cells to proliferate within phagocytes, decreased lung burdens, and attenuated virulence in vivo These findings increase our understanding of cryptococcal adaptation to the host environment and its regulation of oxidative stress resistance and virulence.IMPORTANCE Cryptococcus neoformans is a fungal pathogen that primarily affects severely immunocompromised patients, resulting in 200,000 deaths every year. This yeast occurs in the environment and can establish disease upon inhalation into the lungs of a mammalian host. In this harsh environment it must survive engulfment by host phagocytes, including the oxidative stresses it experiences inside them. To adapt to these challenging conditions, C. neoformans deploys a variety of regulatory proteins to alter gene expression levels and enhance its ability to survive. We have elucidated the role of a protein complex that regulates the cryptococcal response to oxidative stress, survival within phagocytes, and ability to cause disease. These findings are important because they advance our understanding of cryptococcal disease, which we hope will help in the efforts to control this devastating infection.


Assuntos
Adaptação Fisiológica , Cryptococcus neoformans/fisiologia , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Estresse Oxidativo , Estresse Fisiológico , Animais , Células Cultivadas , Contagem de Colônia Microbiana , Criptococose/microbiologia , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Quinase 8 Dependente de Ciclina/genética , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Humanos , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Virulência
13.
PLoS Biol ; 17(1): e2006767, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30695077

RESUMO

Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional/métodos , Ciclina C/genética , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismo , Quinase 8 Dependente de Ciclina/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
14.
J Cell Biochem ; 120(1): 182-191, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30230586

RESUMO

Fibroadenoma is the most common type of benign breast tumor, accounting for 90% of benign lesions in India. Somatic mutations in the mediator complex subunit 12 (MED12) gene play a critical role in fibroepithelial tumorigenesis. The current study evaluated the hotspot region encompassing exon 2 of the MED12 gene, in benign and malignant breast tumor tissue from women who presented for breast lump evaluation. A total of 100 (80 fibroadenoma and 20 breast cancer) samples were analyzed by polymerase chain reaction-Sanger sequencing. Sequence variant analysis showed that 68.75% of nucleotide changes were found in exon 2 and the remaining in the adjacent intron 1. Codon 44 was implicated as a hotspot mutation in benign tumors, and 86.36% of the identified mutations involved this codon. An in silico functional analysis of missense mutations using consensus scoring sorting intolerant from tolerant (SIFT), SIFT seq, Polyphen2, Mutation Assessor, SIFT transFIC, Polyphen2 transFIC, Mutation Assesor transFIC, I-Mutant, DUET, PON-PS, SNAP2, and protein variation effect analyzer] revealed that apart from variants involving codon 44 (G44S; G44H), others like V41A and E55D were also predicted to be deleterious. Most of the missense mutations appeared in the loop region of the MED12 protein, which is expected to affect its functional interaction with cyclin C-CDK8/CDK19, causing loss of mediator-associated cyclin depended kinase (CDK) activity. These results suggest a key role of MED12 somatic variations in the pathogenesis of fibroadenoma. For the first time, it was demonstrated that MED12 sequence variations are present in benign breast tumors in the south Indian population.


Assuntos
Neoplasias da Mama/genética , Éxons/genética , Fibroadenoma/genética , Complexo Mediador/química , Complexo Mediador/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Idoso , Sequência de Bases/genética , Criança , Códon/genética , Simulação por Computador , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Humanos , Índia , Íntrons/genética , Aprendizado de Máquina , Pessoa de Meia-Idade , Fenótipo , Polimorfismo Genético , Estrutura Secundária de Proteína , Adulto Jovem
15.
ChemMedChem ; 14(1): 107-118, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30403831

RESUMO

Selective inhibition of cyclin-dependent kinase 8 and cyclin C (CDK8/CycC) has been suggested as a promising strategy for decreasing mitogenic signals in cancer cells with reduced toxicity toward normal cells. We developed a novel virtual screening protocol for drug development and applied it to the discovery of new CDK8/CycC type II ligands, which is likely to achieve long residence time and specificity. We first analyzed the binding thermodynamics of 11 published pyrazolourea ligands using molecular dynamics simulations and a free-energy calculation method, VM2, and extracted the key binding information to assist virtual screening. The urea moiety was found to be the critical structural contributor of the reference ligands. Starting with the urea moiety, we conducted substructure-based searches with our newly developed superposition and single-point energy evaluation method, followed by free-energy calculations, and singled out three purchasable compounds for bioassay testing. The ranking from the experimental results is completely consistent with the predicted rankings. A potent drug-like compound was found to have a Kd value of 42.5 nm, which is similar to those of the most potent reference ligands; this provided a good starting point for further improvement. This study shows that our novel virtual screening protocol is an accurate and efficient tool for drug development.


Assuntos
Ciclina C/antagonistas & inibidores , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ligantes , Inibidores de Proteínas Quinases/farmacologia , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Termodinâmica
16.
Mol Biol Cell ; 30(3): 302-311, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30516433

RESUMO

Mitochondria exist in an equilibrium between fragmented and fused states that shifts heavily toward fission in response to cellular damage. Nuclear-to-cytoplasmic cyclin C relocalization is essential for dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in response to oxidative stress. This study finds that cyclin C directly interacts with the Drp1 GTPase domain, increases its affinity to GTP, and stimulates GTPase activity in vitro. In addition, the cyclin C domain that binds Drp1 is contained within the non-Cdk binding second cyclin box domain common to all cyclin family members. This interaction is important, as this domain is sufficient to induce mitochondrial fission when expressed in mouse embryonic fibroblasts in the absence of additional stress signals. Using gel filtration chromatography and negative stain electron microscopy, we found that cyclin C interaction changes the geometry of Drp1 oligomers in vitro. High-molecular weight low-GTPase activity oligomers in the form of short filaments and rings were diminished, while dimers and elongated filaments were observed. Our results support a model in which cyclin C binding stimulates the reduction of low-GTPase activity Drp1 oligomers into dimers capable of producing high-GTPase activity filaments.


Assuntos
Ciclina C/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Estresse Fisiológico , Citoesqueleto de Actina/metabolismo , Animais , Fibroblastos/metabolismo , Humanos , Camundongos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
17.
Dev Biol ; 444(2): 62-70, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30352217

RESUMO

The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, we have previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. Here we report that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. We observed that cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on lipid metabolism and developmental timing in Drosophila larvae.


Assuntos
Quinase 8 Dependente de Ciclina/fisiologia , Proteínas de Drosophila/fisiologia , Larva/metabolismo , Necessidades Nutricionais/fisiologia , Animais , Ciclina C/metabolismo , Ciclina C/fisiologia , Quinase 8 Dependente de Ciclina/metabolismo , Dieta , Proteínas na Dieta/metabolismo , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Expressão Gênica , Reprodutibilidade dos Testes
18.
J Biol Chem ; 293(13): 4870-4882, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29440396

RESUMO

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs.


Assuntos
Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Éxons , Leiomioma/metabolismo , Complexo Mediador/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Regulação Alostérica , Ciclina C/genética , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Feminino , Humanos , Leiomioma/genética , Leiomioma/patologia , Complexo Mediador/genética , Miométrio/metabolismo , Miométrio/patologia , Proteínas de Neoplasias/genética
19.
Mol Biol Cell ; 29(3): 363-375, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29212878

RESUMO

In response to oxidative stress, cells decide whether to mount a survival or cell death response. The conserved cyclin C and its kinase partner Cdk8 play a key role in this decision. Both are members of the Cdk8 kinase module, which, with Med12 and Med13, associate with the core mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which thereafter releases cyclin C into the cytoplasm. Cytoplasmic cyclin C associates with mitochondria, where it induces hyperfragmentation and regulated cell death. In this report, we show that residues 742-844 of Med13's 600-amino acid intrinsic disordered region (IDR) both directs cyclin C-Cdk8 association and serves as the degron that mediates ubiquitin ligase SCFGrr1-dependent destruction of Med13 following oxidative stress. Here, cyclin C-Cdk8 phosphorylation of Med13 most likely primes the phosphodegron for destruction. Next, pro-oxidant stimulation of the cell wall integrity pathway MAP kinase Slt2 initially phosphorylates cyclin C to trigger its release from Med13. Thereafter, Med13 itself is modified by Slt2 to stimulate SCFGrr1-mediated destruction. Taken together, these results support a model in which this IDR of Med13 plays a key role in controlling a molecular switch that dictates cell fate following exposure to adverse environments.


Assuntos
Ciclina C/metabolismo , Complexo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Núcleo Celular/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Proteínas F-Box , Complexo Mediador/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/fisiologia , Fosforilação , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases
20.
Hum Mol Genet ; 26(22): 4367-4374, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28973654

RESUMO

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the first time in an animal model that overexpression of PRDM13 alone causes a severe abnormal retinal development. It is noteworthy that mutations associated with this autosomal dominant foveal developmental disorder are frequently duplications always including an entire copy of PRDM13, or variants in one DNase 1 hypersensitivity site at this locus.


Assuntos
Distrofias Hereditárias da Córnea/genética , Ciclina C/genética , Histona-Lisina N-Metiltransferase/genética , Adulto , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 6 , Distrofias Hereditárias da Córnea/metabolismo , Ciclina C/metabolismo , Drosophila melanogaster , Proteínas do Olho/genética , Feminino , Ligação Genética , Haplótipos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Domínios PR-SET , Linhagem , Sequenciamento Completo do Genoma
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