RESUMEN
Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.
Asunto(s)
Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caspasa 9/metabolismo , Proteínas Cullin/metabolismo , Ubiquitinación , Activación Enzimática/efectos de los fármacos , Etopósido/farmacología , Células HEK293 , Humanos , Leupeptinas/farmacología , Unión Proteica/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Proteína bcl-X/metabolismoRESUMEN
Olfaxin, which is a BNIP2 and Cdc42GAP homology (BCH) domain-containing protein, is predominantly expressed in mitral and tufted (M/T) cells in the olfactory bulb (OB). Olfaxin and Caytaxin, which share 56.3% amino acid identity, are similar in their glutamatergic terminal localization, kidney-type glutaminase (KGA) interaction, and caspase-3 substrate. Although the deletion of Caytaxin protein causes human Cayman ataxia and ataxia in the mutant mouse, the function of Olfaxin is largely unknown. In this study, we generated Prune2 gene mutant mice (Prune2Ex16-/-; knock out [KO] mice) using the CRISPR/Cas9 system, during which the exon 16 containing start codon of Olfaxin mRNA was deleted. Exon 16 has 80 nucleotides and is contained in four of five Prune2 isoforms, including PRUNE2, BMCC1, BNIPXL, and Olfaxin/BMCC1s. The levels of Olfaxin mRNA and Olfaxin protein in the OB and piriform cortex of KO mice significantly decreased. Although Prune2 mRNA also significantly decreased in the spinal cord, the gross anatomy of the spinal cord and dorsal root ganglion (DRG) was intact. Further, disturbance of the sensory and motor system was not observed in KO mice. Therefore, in the current study, we examined the role of Olfaxin in the olfactory system where PRUNE2, BMCC1, and BNIPXL are scarcely expressed. Odor preference was impaired in KO mice using opposite-sex urinary scents as well as a non-social odor stimulus (almond). Results of the odor-aversion test demonstrated that odor-associative learning was disrupted in KO mice. Moreover, the NMDAR2A/NMDAR2B subunits switch in the piriform cortex was not observed in KO mice. These results indicated that Olfaxin may play a critical role in odor preference and olfactory memory.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Neoplasias/fisiología , Percepción Olfatoria/fisiología , Olfato , Animales , Aprendizaje por Asociación/fisiología , Cerebelo/metabolismo , Exones , Femenino , Masculino , Ratones Noqueados , Proteínas de Neoplasias/genética , Odorantes , Bulbo Olfatorio/metabolismo , Corteza Piriforme/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Patients with Alzheimer's disease (AD) experience a wide array of cognitive deficits, which typically include the impairment of explicit memory. In previous studies, the authors reported that a flavonoid, quercetin, reduces the expression of ATF4 and delays memory deterioration in an early-stage AD mouse model. In the present study, the effects of long-term quercetin intake on memory recall were assessed using contextual fear conditioning in aged wild-type mice. In addition, the present study examined whether memory recall was affected by the intake of quercetin-rich onion (a new cultivar of hybrid onion 'Quergold') powder in early-stage AD patients. In-vivo analysis indicated that memory recall was enhanced in aged mice fed a quercetin-containing diet. Memory recall in early-stage AD patients, determined using the Revised Hasegawa Dementia Scale, was significantly improved by the intake of quercetin-rich onion (Quergold) powder for 4 weeks compared with the intake of control onion ('Mashiro' white onion) powder. These results indicate that quercetin might influence memory recall.
Asunto(s)
Antioxidantes/uso terapéutico , Condicionamiento Psicológico/efectos de los fármacos , Miedo/efectos de los fármacos , Trastornos de la Memoria/tratamiento farmacológico , Quercetina/uso terapéutico , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/diagnóstico por imagen , Compuestos de Anilina , Animales , Benzotiazoles/farmacocinética , Femenino , Humanos , Yofetamina/farmacocinética , Imagen por Resonancia Magnética , Masculino , Trastornos de la Memoria/diagnóstico por imagen , Trastornos de la Memoria/etiología , Recuerdo Mental/efectos de los fármacos , Pruebas de Estado Mental y Demencia , Ratones , Ratones Endogámicos C57BL , Pruebas Neuropsicológicas , Tomografía de Emisión de Positrones , TiazolesRESUMEN
The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax.
Asunto(s)
Membrana Celular/fisiología , Membrana Celular/ultraestructura , Retículo Endoplásmico/fisiología , Fluidez de la Membrana/fisiología , Péptidos/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Células HEK293 , HumanosRESUMEN
Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasas/genética , Caspasas/metabolismo , MicroARNs/metabolismo , Ribosomas/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , MicroARNs/genética , Transporte de Proteínas/fisiología , ARN Mensajero , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The production of amyloid ß (Aß) in the brain from Aß precursor protein (APP) through γ-secretase is important for the pathogenesis of Alzheimer's disease (AD). Our previous studies have demonstrated that autophagy impairment and endoplasmic reticulum stress increase presenilin 1 expression and enhance γ-secretase activity through the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and the translation of activating transcription factor 4 (ATF4). However, the inhibitory molecules for γ-secretase are largely unknown. Here, we demonstrate that the levels of ATF4 expression are increased in the brain of APP23 AD model mice; furthermore, these levels enhanced in the brain of APP23 mice crossed with obese and diabetic db/db (Lepr(db/db)) mice. A polyhydroxylated flavonoid, quercetin, suppressed presenilin 1 expression and Aß secretion in autophagy-impaired cells by the induction of growth arrest and DNA damaged-inducible gene (GADD) 34, which mediates eIF2α dephosphorylation, leading to decreased ATF4 expression. GADD34 induction was observed in the brain of wild-type mice, and APP23 mice fed quercetin in their diet. After the long-term feeding of quercetin, deterioration in memory assessed by freezing behavior was delayed in APP23 mice. These results indicate that quercetin may reduce eIF2α phosphorylation and ATF4 expression through GADD34 induction in the brain, leading to the improvement of memory in aged mice and the delay of deterioration in memory at the early stage of AD in AD model mice.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Antioxidantes/farmacología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Fosfatasa 1/metabolismo , Quercetina/farmacología , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 4/metabolismo , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/genética , Animales , Antioxidantes/uso terapéutico , Proteína 5 Relacionada con la Autofagia , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Condicionamiento Clásico/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Fragmentos de Péptidos/farmacología , Fosforilación/efectos de los fármacos , Presenilina-1/metabolismo , Quercetina/uso terapéutico , Receptores de Leptina/genética , Receptores de Leptina/metabolismoRESUMEN
The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntington's disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.
Asunto(s)
Caspasa 7/metabolismo , Retículo Endoplásmico/metabolismo , Enfermedad de Huntington/metabolismo , Péptidos/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Retículo Endoplásmico/patología , Activación Enzimática , Células HEK293 , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Prune homolog 2 (Drosophila) (PRUNE2) encodes a BCH motif-containing protein that shares homology with the Cayman ataxia-related protein Caytaxin. Caytaxin is a substrate of caspase-3 and is specifically expressed at the presynapse of vesicular-type glutamate transporter (VGLUT)-positive neurons, where it plays a role in glutamate neurotransmission primarily in the cerebellum and hippocampus. Here, we showed that a novel Prune2 isoform contains a BCH motif and localizes predominantly to the synaptic cytosol, similar to Caytaxin. However, the isoform is expressed predominantly in the olfactory bulb and layer Ia of the piriform cortex, where Caytaxin is scarcely expressed. The isoform expression is upregulated during development, similar to that in the presynaptic-localizing proteins Synapsin I and Bassoon. Prune2 and its previously identified isoforms have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein, and an antagonist of cellular transformation. In addition, a novel isoform may develop new roles for Prune2 at the synapse in olfactory systems.
Asunto(s)
Proteínas de Neoplasias/genética , Bulbo Olfatorio/fisiología , Vías Olfatorias/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citosol/metabolismo , Estriol/análogos & derivados , Estriol/metabolismo , Exones/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/fisiologíaRESUMEN
The endoplasmic reticulum (ER) copes with unfolded proteins in the lumen (ER stress) by activating three distinct intracellular signaling pathways of unfolded protein response (UPR). ER stress contributes to the pathogenesis of obesity and diabetes, which are risk factors for Alzheimer's disease (AD) that accelerate the pathogenesis of AD. However, whether ER stress is involved in the development of AD remains unclear. In this study, we demonstrate that ER stress induces presenilin-1 expression through activating transcription factor 4 (ATF4), resulting in increased amyloid-ß (Aß) secretion by γ-secretase activity, which is suppressed by quercetin by modifying UPR signaling. This result suggests that ER stress may be stimulated in obesity and type 2 diabetes, thereby enhancing γ-secretase activity that is the underlying molecular mechanism affecting the pathogenesis of AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/enzimología , Factor de Transcripción Activador 4/antagonistas & inhibidores , Factor de Transcripción Activador 4/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Quercetina/farmacología , Receptor Notch1/metabolismoRESUMEN
A family of Bcl-2/adenovirus E1B 19kDa-interacting proteins (BNIPs) plays critical roles in several cellular processes such as cellular transformation, apoptosis, neuronal differentiation, and synaptic function, which are mediated by the BNIP2 and Cdc42GAP homology (BCH) domain. Prune homolog 2 (Drosophila) (PRUNE2) and its isoforms -C9orf65, BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1), and BNIP2 Extra Long (BNIPXL) - have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively. However, precise localization of PRUNE2 in the brain remains unclear. Here, we identified the distribution of Prune2 mRNA in the adult mouse brain. Prune2 mRNA is predominantly expressed in the neurons of the cranial nerve motor nuclei and the motor neurons of the spinal cord. The expression in the dorsal root ganglia (DRG) is consistent with the previously described reports. In addition, we observed the expression in another sensory neuron in the mesencephalic trigeminal nucleus. These results suggest that Prune2 may be functional in these restricted brain regions.