RESUMO
c-Myc is a proto-oncoprotein that regulates various cellular processes and whose abnormal expression leads to tumorigenesis. c-Myc protein stability has been shown to be predominantly controlled by the ubiquitin ligase (E3) CRL1Fbxw7 in a manner dependent on glycogen synthase kinase 3 (GSK3)-mediated phosphorylation. Here we show that, in some types of cancer cells, c-Myc degradation is largely insensitive to the GSK3 inhibitor (GSK3i) CHIR99021, suggesting the existence of an E3 other than CRL1Fbxw7 for c-Myc degradation. Mass spectrometry identified CRL2KLHDC3 as such an E3. In GSK3i-insensitive cancer cells, combined depletion of Fbxw7 and KLHDC3 resulted in marked stabilization of c-Myc, suggestive of a cooperative action of Fbxw7 and KLHDC3. Furthermore, transplantation of such cells deficient in both Fbxw7 and KLHDC3 into immunodeficient mice gave rise to larger tumors compared with those formed by cells lacking only Fbxw7. GSK3i-insensitive pancreatic cancer cells expressed lower levels of SHISA2, a negative regulator of the Wnt signaling pathway, than did GSK3i-sensitive cells. KLHDC3 mRNA abundance was associated with prognosis in pancreatic cancer patients with a low level of SHISA2 gene expression. These results suggest that KLHDC3 cooperates with Fbxw7 to promote c-Myc degradation in a subset of cancer cells with low GSK3 activity.
Assuntos
Proteína 7 com Repetições F-Box-WD , Proteólise , Proteínas Proto-Oncogênicas c-myc , Ubiquitina-Proteína Ligases , Humanos , Proteína 7 com Repetições F-Box-WD/metabolismo , Proteína 7 com Repetições F-Box-WD/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Linhagem Celular Tumoral , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Reactive oxygen species (ROS) posed a risk for the transition of vertebrates from aquatic to terrestrial life. How ancestral organisms adapted to such ROS exposure has remained a mystery. Here, we show that attenuation of the activity of the ubiquitin ligase CRL3Keap1 for the transcription factor Nrf2 during evolution was key to development of an efficient response to ROS exposure. The Keap1 gene was duplicated in fish to give rise to Keap1A and the only remaining mammalian paralog Keap1B, the latter of which shows a lower affinity for Cul3 and contributes to robust Nrf2 induction in response to ROS exposure. Mutation of mammalian Keap1 to resemble zebrafish Keap1A resulted in an attenuated Nrf2 response, and most knock-in mice expressing such a Keap1 mutant died on exposure as neonates to sunlight-level ultraviolet radiation. Our results suggest that molecular evolution of Keap1 was essential for adaptation to terrestrial life.
Assuntos
Fator 2 Relacionado a NF-E2 , Peixe-Zebra , Animais , Camundongos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Espécies Reativas de Oxigênio , Raios Ultravioleta , Evolução Molecular , Mamíferos/metabolismo , Proteínas de Transporte/genética , Proteínas de Peixe-ZebraRESUMO
Inochinohaha White (IHW) is a Japanese herbal medicine for treating women with anxiety associated with premenstrual syndrome (PMS). In this study, we examined the effects of IHW on anxiety-like behavior in rats undergoing progesterone withdrawal (PWD), a model for PMS. Female rats were injected daily with progesterone for 21 days. Water and ethanol extracts of IHW (WE-IHW and EE-IHW, respectively) were administered orally 15 days after the initiation of progesterone injections. Anxiety-like behavior in an elevated plus maze was evaluated 48 h after the final injection of progesterone. PWD induced anxiety-like behavior, and EE-IHW (300 mg/kg), but not WE-IHW, significantly attenuated this behavior. Administration of the GABA agonists, diazepam or muscimol, significantly attenuated PWD-induced anxiety-like behavior. To investigate the underlying mechanisms of IHW action, we analyzed GABAA receptor expression in the amygdala of these rats. EE-IHW ameliorated the PWD-induced decrease in GABAA receptor ß2-subunit mRNA, although ß2-subunit protein was unchanged. Brain-derived neurotrophic factor (BDNF) has been reported to have anxiolytic effects and enhance GABAergic synaptic transmission. We found that EE-IHW increased BDNF levels in a dose-dependent manner. Our results suggest that EE-IHW attenuates PWD-induced anxiety-like behavior by increasing GABAA receptor-mediated signaling via increases in ß2-subunit and BDNF in the amygdala.
Assuntos
Ansiedade/tratamento farmacológico , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Expressão Gênica/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Síndrome Pré-Menstrual/tratamento farmacológico , Síndrome Pré-Menstrual/psicologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Administração Oral , Tonsila do Cerebelo/metabolismo , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Injeções , Síndrome Pré-Menstrual/genética , Progesterona/administração & dosagem , Ratos WistarRESUMO
Pyruvate is the key substance controlling the formation of diacetyl, acetaldehyde, and acetate during alcoholic fermentation. Here we report the breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport. Mitochondrial function was involved in resistance to this substance and in the production of pyruvate by the mutants.
