RESUMO
Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Morte Celular , Membrana Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Morte Celular/genética , Feminino , Humanos , Macrófagos , Masculino , Camundongos , Mutação , Necrose , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Multimerização Proteica , Piroptose/genéticaRESUMO
Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Inflamassomos/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Caspases Iniciadoras , Linhagem Celular , Feminino , Bactérias Gram-Negativas/imunologia , Humanos , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Mutação/genética , Necrose , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sepse/microbiologia , Transdução de Sinais/genética , Análise de SobrevidaRESUMO
The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/deficiência , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Fibroblastos , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel/farmacologia , Farmacogenética , Fosforilação/efeitos dos fármacos , Poliploidia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Vincristina/farmacologiaRESUMO
MCL1 is essential for the survival of stem and progenitor cells of multiple lineages, and is unique among pro-survival BCL2 family members in that it is rapidly turned over through the action of ubiquitin ligases. B- and mantle-cell lymphomas, chronic myeloid leukaemia, and multiple myeloma, however, express abnormally high levels of MCL1, contributing to chemoresistance and disease relapse. The mechanism of MCL1 overexpression in cancer is not well understood. Here we show that the deubiquitinase USP9X stabilizes MCL1 and thereby promotes cell survival. USP9X binds MCL1 and removes the Lys 48-linked polyubiquitin chains that normally mark MCL1 for proteasomal degradation. Increased USP9X expression correlates with increased MCL1 protein in human follicular lymphomas and diffuse large B-cell lymphomas. Moreover, patients with multiple myeloma overexpressing USP9X have a poor prognosis. Knockdown of USP9X increases MCL1 polyubiquitination, which enhances MCL1 turnover and cell killing by the BH3 mimetic ABT-737. These results identify USP9X as a prognostic and therapeutic target, and they show that deubiquitinases may stabilize labile oncoproteins in human malignancies.
Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Poliubiquitina/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , Docetaxel , Etoposídeo/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Meia-Vida , Humanos , Lisina/metabolismo , Camundongos , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias/diagnóstico , Nitrofenóis/farmacologia , Fosforilação/efeitos da radiação , Piperazinas/farmacologia , Prognóstico , Ligação Proteica/efeitos da radiação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Sulfonamidas/farmacologia , Taxoides/farmacologia , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Ubiquitinação , Raios Ultravioleta , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.
Assuntos
Francisella tularensis/imunologia , Imunidade Inata , Proteínas Nucleares/imunologia , Tularemia/imunologia , Animais , Caspase 1/metabolismo , Células Cultivadas , Citosol/imunologia , DNA/genética , DNA/imunologia , Proteínas de Ligação a DNA , Ativação Enzimática , Interferon-alfa/imunologia , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Receptor de Interferon alfa e beta/imunologiaRESUMO
A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inflamação/metabolismo , Toxinas Biológicas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inflamação/enzimologia , Inflamação/imunologia , Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Listeria monocytogenes/fisiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Toxinas Marinhas/farmacologia , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nigericina/farmacologia , Proteína Adaptadora de Sinalização NOD2 , Oxocinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
INTRODUCTION: We assessed the effect on the food environments of public high schools of Maine's statewide nutrition policy (Chapter 51), which banned "foods of minimal nutritional value" (FMNV) in public high schools that participated in federally funded meal programs. We documented allowable exceptions to the policy and describe the school food environments. METHODS: We mailed surveys to 89 high school food-service directors to assess availability pre-Chapter 51 and post-Chapter 51 of soda, other sugar-sweetened beverages, and junk food. Frequency data were tabulated pre-Chapter 51 and post-Chapter 51, and Fisher exact test was used to assess significance in changes. We conducted food and beverage inventories at 11 high schools. RESULTS: The survey return rate was 61% (N = 54). Availability of soda in student vending significantly decreased pre-Chapter 51 versus post-Chapter 51 (P = .04). No significant changes were found for other sugar-sweetened beverages and junk food. Exceptions to Chapter 51 were permitted to staff (67%), to the public (86%), and in career and technical education programs (31%). Inventories in a subset of schools found no availability of soda for students, whereas other sugar-sweetened beverages and junk food were widely available in à la carte, vending machines, and school stores. Candy, considered a FMNV, was freely available. Soda advertisement on school grounds was common. CONCLUSION: Student vending choices improved after the implementation of Chapter 51; however, use of FMNV as the policy standard may be limiting, as availability of other sugar-sweetened beverages and junk food was pervasive. School environments were not necessarily supportive of the policy, as advertisement of soda was common and some FMNV were available. Furthermore, local exceptions to Chapter 51 likely reduced the overall effect of the policy.
Assuntos
Abastecimento de Alimentos/normas , Política Nutricional , Instituições Acadêmicas , Bebidas , Coleta de Dados , Ingestão de Alimentos , Serviços de Alimentação/normas , Humanos , Maine , Inquéritos Nutricionais , Valor NutritivoRESUMO
COP1 (constitutively photomorphogenic 1) is a RING-finger-containing protein that functions to repress plant photomorphogenesis, the light-mediated programme of plant development. Mutants of COP1 are constitutively photomorphogenic, and this has been attributed to their inability to negatively regulate the proteins LAF1 (ref. 1) and HY5 (ref. 2). The role of COP1 in mammalian cells is less well characterized. Here we identify the tumour-suppressor protein p53 as a COP1-interacting protein. COP1 increases p53 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent fashion, independently of MDM2 or Pirh2, which are known to interact with and negatively regulate p53. Moreover, COP1 serves as an E3 ubiquitin ligase for p53 in vitro and in vivo, and inhibits p53-dependent transcription and apoptosis. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Furthermore, we identify COP1 as a p53-inducible gene, and show that the depletion of COP1 and MDM2 by siRNA cooperatively sensitizes U2-OS cells to ionizing-radiation-induced cell death. Overall, these results indicate that COP1 is a critical negative regulator of p53 and represents a new pathway for maintaining p53 at low levels in unstressed cells.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Apoptose , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Fase G1 , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The NF-kappaB family of transcription factors is activated in response to many stimuli, including pro-inflammatory cytokines, environmental stresses and, in the case of B and T lymphocytes, by antigenic stimulation. Bcl10 is essential for NF-kappaB activation by T- and B-cell receptors. T and B lymphocytes from Bcl10-deficient mice fail to activate NF-kappaB in response to antigen-receptor stimulation and, as a consequence, are unable to proliferate. Bcl10 overexpression is sufficient to activate NF-kappaB, a process that requires the NF-kappaB essential modulator NEMO (also known as IKK-gamma), which is the regulatory subunit of the IkappaB kinase complex. However, the cellular mechanism by which Bcl10 activates the NF-kappaB pathway remains unclear. Here we show that Bcl10 targets NEMO for lysine-63-linked ubiquitination. Notably, a mutant form of NEMO that cannot be ubiquitinated inhibited Bcl10-induced NF-kappaB activation. Paracaspase and a ubiquitin-conjugating enzyme (UBC13) were both required for Bcl10-induced NEMO ubiquitination and subsequent NF-kappaB activation. Furthermore, short interfering RNAs that reduced the expression of paracaspase and UBC13 abrogated the effects of Bcl10. Thus, the adaptor protein Bcl10 promotes activation of NF-kappaB transcription factors through paracaspase- and UBC13-dependent ubiquitination of NEMO.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Proteína 10 de Linfoma CCL de Células B , Caspases , Linhagem Celular , Deleção de Genes , Humanos , Quinase I-kappa B , Células Jurkat , Linfoma de Zona Marginal Tipo Células B/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta. Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.
Assuntos
NF-kappa B/metabolismo , Proteínas/química , Proteínas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA , Regulação para Baixo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Estrutura Terciária de Proteína , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Fator 2 Associado a Receptor de TNF , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.
Assuntos
Fator Regulador 2 de Interferon/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/genética , Piroptose/genética , Transcrição Gênica/genética , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/genéticaRESUMO
More than half of all children in the United States aged 3 to 6 years are enrolled in child care centers. Maine received funds from the U.S. Department of Health and Human Services' Communities Putting Prevention to Work to promote the adoption of Nutrition and Physical Activity Self-Assessment for Child Care (NAPSACC), an evidence-based program for the child care setting. We evaluated the rollout and adoption of NAP SACC in Maine using multiple methods. Our findings suggest that the NAP SACC program has been successfully adopted in Maine. Nutrition and physical activity policies and offerings have improved, especially with regard to purchasing healthier options in the child care setting.
Assuntos
Creches/organização & administração , Dieta , Exercício Físico , Política de Saúde , Promoção da Saúde/organização & administração , Creches/normas , Pré-Escolar , Comportamentos Relacionados com a Saúde , Promoção da Saúde/normas , Humanos , Entrevistas como Assunto , MaineRESUMO
OBJECTIVE: To examine the relationship between stores selling calorie-dense food near schools and student obesity risk, with the hypothesis that high availability predicts increased risk. METHODS: Mail surveys determined height, weight, and calorie-dense food consumption for 552 students at 11 Maine high schools. Driving distance from all food stores within 2 km (1.24 miles) of schools (or the closest store) was computed, and the impact of food store density and proximity to schools on student body mass index was determined by logistic regression. RESULTS: Ten schools had ≥ 1 store selling soda, and 8 schools had ≥1 fast-food restaurant within 1 km (0.62 miles). There were no significant relationships between the proximity or density of food stores around schools and student obesity risk. Students obtained sugar-sweetened beverages in many locations including at school. CONCLUSIONS AND IMPLICATIONS: Unhealthful food choices are ubiquitous. Consequently, stores selling these food items near schools have no significant affect on student obesity.
Assuntos
Obesidade/epidemiologia , Restaurantes/estatística & dados numéricos , Instituições Acadêmicas/estatística & dados numéricos , Estudantes/estatística & dados numéricos , Adolescente , Estatura , Peso Corporal , Feminino , Humanos , Maine/epidemiologia , Masculino , Características de Residência , Fatores de RiscoRESUMO
The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Endopeptidases/metabolismo , Oncogenes , Processamento de Proteína Pós-Traducional , Fosfatases cdc25/metabolismo , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Dano ao DNA , Endopeptidases/genética , Estabilidade Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Genes ras , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Fatores de Tempo , Transfecção , Transplante Heterólogo , Carga Tumoral , Ubiquitinação , Fosfatases cdc25/genéticaRESUMO
Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 beta-propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1(C) subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1(C), suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G(1)-S transition.
Assuntos
Fator C1 de Célula Hospedeira/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Proliferação de Células , Interfase/genética , Camundongos , Ligação ProteicaRESUMO
OBJECTIVE: To evaluate the effect of a pediatric primary care-based intervention, on improved clinical decision support and family management of risk behaviors for childhood overweight. METHODS: An experimental field trial was conducted with 12 intervention sites in urban and rural areas of Maine and nonrandomized control sites. Change was assessed by using clinical and parent measures from 9 intervention and 10 control sites before and during the Maine Youth Overweight Collaborative intervention. Longitudinal information was collected from chart audits of patients aged 5-18 years (n = 600), systematic samples of parents collected before (n = 346) and during (n = 386) the intervention in 12 sites, and systematic samples of parents in 9 intervention (n = 235) and 10 control (n = 304) sites collected during the intervention. Surveys of health care providers (n = 14 and 17) before and during the intervention were also collected. Teams worked over 18 months to implement improvements in clinical decision support, including tracking BMI percentiles, identification of overweight patients, appropriate laboratory tests, counseling of families and patients use of a behavioral screening tool, and other improvements following the chronic-care model targeting patients aged 5 to 18 and their families. RESULTS: Large changes occurred in clinical practice from before to during the Maine Youth Overweight Collaborative: increases in assessment of BMI (38%-94%), BMI percentile for age and gender (25%-89%), use of the 5-2-1-0 behavioral screening tool (0%-82%), and weight classification (19%-75%). Parent surveys indicated improvements in providers' behavior and rates of counseling. Intervention providers reported improvements in knowledge, attitudes, self-efficacy, and practice. CONCLUSIONS: The Maine Youth Overweight Collaborative intervention improved clinical decision support and family management of risk behaviors, indicating a promising primary care-based approach to address overweight risk among children and youth.
Assuntos
Promoção da Saúde/organização & administração , Estilo de Vida , Sobrepeso/prevenção & controle , Padrões de Prática Médica , Atenção Primária à Saúde/métodos , Assunção de Riscos , Adolescente , Fatores Etários , Atitude Frente a Saúde , Terapia Comportamental , Índice de Massa Corporal , Criança , Pré-Escolar , Estudos de Coortes , Intervalos de Confiança , Relações Familiares , Feminino , Humanos , Maine , Masculino , Relações Médico-Paciente , Probabilidade , Sensibilidade e Especificidade , Fatores SexuaisRESUMO
In the fall of 2003, Maine underwent a rigorous assessment of the diabetes public health system using a modified version of the State Public Health System Performance Instrument developed by the Centers for Disease Control and Prevention's National Public Health Performance Standards Program and other national partners. The assessment was intended to serve as the impetus for the development of a statewide improvement plan. This article details the assessment process and provides a case study highlighting the successful application of systems-based model standards for a categorical issue.