RESUMEN
Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar/intralysosomal autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here, we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity. The activity of CtAtg15(73-475) was markedly elevated by limited digestion. We inserted the human rhinovirus 3C protease recognition sequence and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). Our molecular dynamics simulation suggested that the cleavage facilitated conformational change around the active center of CtAtg15, resulting in an exposed state. We confirmed that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC of S. cerevisiae were disintegrated by CtAtg15. This study suggests Atg15 plays a role in disrupting any organelle membranes delivered to vacuoles by autophagy.
Asunto(s)
Proteínas Fúngicas , Membranas Intracelulares , Fosfolipasas , Chaetomium/enzimología , Chaetomium/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfolipasas/química , Fosfolipasas/genética , Fosfolipasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dominios Proteicos , Simulación de Dinámica Molecular , Mitocondrias/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estructura Terciaria de Proteína , Modelos Moleculares , Activación EnzimáticaRESUMEN
In our previous study, the vaginal opening (VO) day of C57BL/6 mice was accelerated several days by chronic exposure to a 0.05% isoflavone (IF) fortified diet. The purpose of this study was to investigate whether the acceleration of VO by IF (1) has a critical window, (2) is modified by IF exposure combined with 17beta-estradiol (E2), and (3) has any relation with gene expressions of estrogen-related receptors (ERRs). As a result, we determined that the critical window for the acceleration of VO was between 15 and 21 days postnatal. The combined effect of E2 and IF was thought to be additional in the acceleration of VO. The gene expression of ERRgamma was significantly decreased in vagina by IF. The reduction of ERRgamma may have two possible sequelae: disarrangement of vaginal development and high risk of vaginal cancer. In conclusion, IF exposure has a critical window for acceleration of VO and may have adverse effect on mouse vagina.
Asunto(s)
Suplementos Dietéticos/toxicidad , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Isoflavonas/toxicidad , Efectos Tardíos de la Exposición Prenatal , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Maduración Sexual/efectos de los fármacos , Vagina/efectos de los fármacos , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Estradiol/toxicidad , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Embarazo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Estrógenos/genética , Maduración Sexual/genética , Factores de Tiempo , Útero/efectos de los fármacos , Útero/metabolismo , Vagina/crecimiento & desarrollo , Vagina/metabolismo , Neoplasias Vaginales/inducido químicamente , Neoplasias Vaginales/genéticaRESUMEN
Isoflavone (IF), a type of phytoestrogen, has multiple beneficial effects, but too much phytoestrogen can have adverse effects on offspring. To examine whether chronic exposure to high IF has adverse effects on reproductive development, mice offspring were exposed to IF through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning until sacrifice. In male offspring, there was no difference between the IF group and controls; however, in female offspring in the IF group, remarkably earlier puberty and induction of multioocyte follicles on postnatal day (PND) 21 were observed. Gene expression levels of estrogen receptor beta decreased in the ovary and vagina on PND 21. These results suggest that chronic exposure to higher than normal levels of IF induces alterations in the reproductive development of female mice through an estrogenic effect.