RESUMEN
Adult neural stem cells (NSCs) in the hippocampal dentate gyrus continuously proliferate and generate new neurons throughout life. Although various functions of organelles are closely related to the regulation of adult neurogenesis, the role of endoplasmic reticulum (ER)-related molecules in this process remains largely unexplored. Here we show that Derlin-1, an ER-associated degradation component, spatiotemporally maintains adult hippocampal neurogenesis through a mechanism distinct from its established role as an ER quality controller. Derlin-1 deficiency in the mouse central nervous system leads to the ectopic localization of newborn neurons and impairs NSC transition from active to quiescent states, resulting in early depletion of hippocampal NSCs. As a result, Derlin-1-deficient mice exhibit phenotypes of increased seizure susceptibility and cognitive dysfunction. Reduced Stat5b expression is responsible for adult neurogenesis defects in Derlin-1-deficient NSCs. Inhibition of histone deacetylase activity effectively induces Stat5b expression and restores abnormal adult neurogenesis, resulting in improved seizure susceptibility and cognitive dysfunction in Derlin-1-deficient mice. Our findings indicate that the Derlin-1-Stat5b axis is indispensable for the homeostasis of adult hippocampal neurogenesis.
Asunto(s)
Hipocampo , Homeostasis , Proteínas de la Membrana , Células-Madre Neurales , Neurogénesis , Factor de Transcripción STAT5 , Animales , Neurogénesis/genética , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Hipocampo/metabolismo , Hipocampo/citología , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT5/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Noqueados , Convulsiones/metabolismo , Convulsiones/genética , Giro Dentado/metabolismo , Giro Dentado/citología , Transducción de Señal , Proliferación CelularRESUMEN
Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic endoplasmic reticulum (ER) stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and wild-type SOD1 (SOD1(WT)) comprises a masked Derlin-1 binding region (DBR). Here, we found that, under zinc-deficient conditions, SOD1(WT) adopts a mutant-like conformation that exposes the DBR and induces the homeostatic ER stress response, including the inhibition of protein synthesis and induction of a zinc transporter. We conclude that SOD1 has a function as a molecular switch that activates the ER stress response, which plays an important role in cellular homeostasis under zinc-deficient conditions.
Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/enzimología , Superóxido Dismutasa/metabolismo , Zinc/deficiencia , Sitios de Unión , Proteínas de Transporte de Catión/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Homeostasis , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Conformación Proteica , Interferencia de ARN , Transducción de Señal , Relación Estructura-Actividad , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.
Asunto(s)
Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Prealbúmina/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Ácido Azetidinocarboxílico/farmacología , Proteínas de Unión al Calcio , Retículo Endoplásmico/metabolismo , Glicosilación/efectos de los fármacos , Células HEK293 , Hexosiltransferasas/genética , Humanos , Immunoblotting , Manosidasas , Proteínas de la Membrana/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Polisacáridos/metabolismo , Prealbúmina/química , Prealbúmina/genética , Estructura Terciaria de Proteína , Desplegamiento Proteico , Interferencia de ARN , Vías Secretoras/efectos de los fármacos , Homología de Secuencia de Aminoácido , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , alfa-ManosidasaRESUMEN
We have developed three types of lipid droplet (LD)-specific fluorescent probes for live-cell imaging, Lipi-Blue, Lipi-Green, and Lipi-Red, which exhibit fluorescence upon being incorporated into LDs both of living and of fixed cells. These Lipi-probes are LD-specific probes that contain a pyrene or perylene group as a fluorescent scaffold and can be used to observe dynamics of LD in live cells and also interrelations with other organelles by simultaneous staining with multiple organelle-specific probes. Additionally, Lipi-Blue and Lipi-Green allow monitoring LDs in live cells even for 48 h after the staining. Here we show that newly formed LDs and previously existed LDs can be separately monitored in a single cell by using these probes and that intercellular transfer of whole LDs is observed in KB cells, but not in HepG2 cells under the same culturing condition. These findings indicate that newly developed LD-specific probes are useful to analyze the dynamics of LDs in live cells.
Asunto(s)
Colorantes Fluorescentes/química , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Imagen Molecular/métodos , Células Hep G2 , Humanos , Metabolismo de los LípidosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure. To develop effective treatments for this devastating disease, an appropriate strategy for targeting the molecule responsible for the pathogenesis of ALS is needed. We previously reported that mutant SOD1 protein causes motor neuron death through activation of ASK1, a mitogen-activated protein kinase kinase kinase. Additionally, we recently developed K811 and K812, which are selective inhibitors for ASK1. Here, we report the effect of K811 and K812 in a mouse model of ALS (SOD1(G93A) transgenic mice). Oral administration of K811 or K812 significantly extended the life span of SOD1(G93A) transgenic mice (1.06 and 1.08% improvement in survival). Moreover, ASK1 activation observed in the lumbar spinal cord of mice at the disease progression stage was markedly decreased in the K811- and K812-treated groups. In parallel, immunohistochemical analysis revealed that K811 and K812 treatment inhibited glial activation in the lumbar spinal cord of SOD1(G93A) transgenic mice. These results reinforce the importance of ASK1 as a therapeutic target for ALS treatment.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Modelos Animales de Enfermedad , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Masculino , Ratones , Ratones Transgénicos , Resultado del TratamientoRESUMEN
RbAp46/48, histone chaperone, is a family of evolutionarily conserved WD40 repeat-containing proteins, which are involved in various chromatin-metabolizing processes, but their in vivo functional relevance is yet unclear. In order to examine the biological role of pRbAp48 in chicken DT40 cells, we generated a tetracycline-inducible system for conditional RbAp48-knockout cells. Depletion of RbAp48 led to delayed S phase progression associated with slow DNA synthesis and nascent nucleosome formation, followed by accumulation in G2/M phase, finally leading to cell death. Prior to cell death, these cells exhibited aberrant mitosis such as highly condensed and abnormal chromosome alignment on the metaphase plate, leading to chromosome missegregation. Depletion of RbAp48 also caused dissociation of heterochromatin protein 1 (HP1) from pericentromeric heterochromatin. Furthermore, depletion of RbAp48 from cells led to elevated levels of acetylation and slightly decreased levels of methylation, specifically at Lys-9 residue of histone H3. These results suggest that RbAp48 plays an important role in chromosome stability for proper organization of heterochromatin structure through the regulation of epigenetic mark.
Asunto(s)
Supervivencia Celular/genética , Pollos/genética , Inestabilidad Cromosómica/genética , Proteína 4 de Unión a Retinoblastoma/genética , Acetilación , Animales , Línea Celular , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Fase G2/genética , Técnicas de Inactivación de Genes , Heterocromatina/metabolismo , Histonas/metabolismo , Metilación , Proteína 4 de Unión a Retinoblastoma/metabolismo , Fase S/genéticaRESUMEN
Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca(2+) influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.
Asunto(s)
Apoptosis , Regulación de la Expresión Génica , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Osteoblastos/fisiología , Receptores del Factor de Necrosis Tumoral/genética , Estrés Mecánico , Células 3T3 , Animales , Sistema de Señalización de MAP Quinasas , Ratones , Osteoblastos/metabolismo , Receptor de TWEAKRESUMEN
Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1(mut)) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1(mut), the DBR-neighboring region [SOD1(30-40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30-40). We found that MS27 could distinguish SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785-MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Anticuerpos/inmunología , Inmunoprecipitación/métodos , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/patología , Animales , Anticuerpos/metabolismo , Escherichia coli , Células HEK293 , Células HeLa , Humanos , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Proteínas Recombinantes/inmunología , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa/inmunología , Superóxido Dismutasa-1 , TransfecciónRESUMEN
Histone acetyltransferase p300/CBP-associated factor (PCAF) belonging to GCN5 family regulates various epigenetic events for transcriptional regulation through alterations in the chromatin structure. During normal development of B cells, gene expressions of numerous transcription factors are strictly regulated by epigenetic mechanisms including histone acetylation and deacetylation to complete their development pathways. Here, by analyzing PCAF-deficient DT40 mutants, ΔPCAF, we report that PCAF takes part in transcriptional activation of B cell lymphoma-6 (Bcl-6) and Paired box gene 5 (Pax5), which are essential transcription factors for normal development of B cells. PCAF-deficiency caused drastic decrease in mRNA levels of Bcl-6 and Pax5, and remarkable increase in that of B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, chromatin immunoprecipitation assay showed that PCAF-deficiency caused remarkable decrease in acetylation levels of both H3K9 and H3K14 residues within chromatin surrounding the 5'-flanking regions of Bcl-6 and Pax5 genes in vivo, suggesting that their gene expressions may be regulated by PCAF. These results revealed that PCAF is involved in transactivation of Bcl-6 and Pax5 genes, resulting in down-regulation of Blimp-1 gene expression, and plays a key role in epigenetic regulation of B cell development.
Asunto(s)
Linfocitos B/metabolismo , Factor de Transcripción PAX5/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismo , Animales , Línea Celular , PollosRESUMEN
The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression.
Asunto(s)
Apoptosis , Retículo Endoplásmico/metabolismo , Genes bcl-2 , Histona Acetiltransferasas/metabolismo , Regulación hacia Arriba , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Pollos , Retículo Endoplásmico/efectos de los fármacos , Histona Acetiltransferasas/genética , Tapsigargina/farmacologíaRESUMEN
The transcription factor paired box gene 5 (Pax5) is essential for B cell development. In this study, complementation analyses in Pax5-deficient DT40 cells showed that three Pax5 isoforms Pax5A, Pax5B and Pax5BΔEx8 (another spliced isoform of Pax5B lacking exon 8) exhibit distinct roles in transcriptional regulation of six B cell development-related genes (activation-induced cytidine deaminase, Aiolos, BTB and CNC homology 2, B cell lymphoma-6, early B cell factor 1, origin binding factor-1 genes), transcriptions of which are remarkably down-regulated by Pax5-deficiency. Moreover, ectopic expression study shows that these Pax5 isoforms may regulate themselves and each other at the transcriptional level.
Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción PAX5/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Animales , Línea Celular Transformada , Pollos , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Factor de Transcripción PAX5/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Transcripción GenéticaRESUMEN
The histone acetyltransferase p300/CBP-associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF-deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H-chain) synthesis. Remarkably, PCAF-deficiency causes an increase in the amount of secretory IgM H-chain mRNA, but not in that of IgM light chain and membrane-bound IgM H-chain mRNAs, resulting in dramatic up-regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H-chain synthesis.
Asunto(s)
Regulación hacia Abajo , Inmunoglobulina M/biosíntesis , Células Precursoras de Linfocitos B/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Animales , Línea Celular , Pollos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Células Precursoras de Linfocitos B/enzimología , Factores de Transcripción p300-CBP/genéticaRESUMEN
BACKGROUND: Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2(C96Y)) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins(C96Y)-induced cell death remains unclear. METHODS: We investigate the mechanisms of Ins2(C96Y)-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice. RESULTS: The expression of Ins(C96Y) activated the ASK1-p38 pathway. Deletion of ASK1 mitigated Ins(C96Y)-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins(C96Y)-induced MIN6 cell death. CONCLUSIONS: These findings suggest that ER stress-induced ASK1-p38 activation, which is triggered by the accumulation of Ins(C96Y), plays an important role in the pathogenesis of diabetes. GENERAL SIGNIFICANCE: Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1-p38 pathway may be an effective therapy for various types of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/enzimología , MAP Quinasa Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Sustitución de Aminoácidos , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Imidazoles/farmacología , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/patología , MAP Quinasa Quinasa Quinasa 5/genética , Ratones , Ratones Noqueados , Mutación Missense , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The extracellular signal-regulated kinase (ERK) pathway is an important signalling pathway that regulates a large number of cellular processes, including proliferation, differentiation and gene expression. Hyperosmotic stress activates the ERK pathway, whereas little is known about the regulatory mechanisms and physiological functions of ERK activation in hyperosmotic response. Here, we show that MAPK/ERK kinase kinase 2 (MEKK2), a member of the MAPKKK family, mediated the specific and transient activation of ERK, which was required for the induction of aquaporin 1 (AQP1) and AQP5 gene expression in response to hyperosmotic stress. Moreover, we identified the E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) as a binding partner of MEKK2. Depletion of CHIP by small-interference RNA or gene targeting attenuated the degradation of MEKK2 and prolonged the ERK activity. Interestingly, hyperosmolality-induced gene expression of AQP1 and AQP5 was suppressed by CHIP depletion and was reversed by inhibition of the prolonged phase of ERK activity. These findings show that transient activation of the ERK pathway, which depends not only on MEKK2 activation, but also on CHIP-dependent MEKK2 degradation, is crucial for proper gene expression in hyperosmotic stress response.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Acuaporinas/metabolismo , Células Cultivadas , Activación Enzimática , Humanos , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Noqueados , Presión Osmótica , Unión Proteica , Ratas , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Aberrant proteostasis is thought to be involved in the pathogenesis of neurodegenerative diseases. Some proteostasis abnormalities are ameliorated by chaperones. Chaperones are divided into three groups: molecular, pharmacological, and chemical. Chemical chaperones intended to alleviate stress in organelles, such as the endoplasmic reticulum (ER), are now being administered clinically. Of the chemical chaperones, 4-phenylbutyrate (4-PBA) has been used as a research reagent, and its mechanism of action includes chaperone effects and the inhibition of histone deacetylase. Moreover, it also binds to the B-site of SEC24 and regulates COPII-mediated transport from the ER. Although its therapeutic effect may not be strong, elucidating the mechanism of action of 4-PBA may contribute to the identification of novel therapeutic targets for neurodegenerative diseases.
RESUMEN
By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to â¼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.
Asunto(s)
Fragmentación del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Factores de Transcripción p300-CBP/metabolismo , Acetilación/efectos de la radiación , Animales , Muerte Celular/genética , Muerte Celular/efectos de la radiación , Línea Celular , Pollos , Islas de CpG/genética , ADN/biosíntesis , ADN/genética , Reparación del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/genética , Histonas/genética , Histonas/metabolismo , Humanos , Unión Proteica/genética , Unión Proteica/efectos de la radiación , Transcripción Genética/genética , Transcripción Genética/efectos de la radiación , Factores de Transcripción p300-CBP/genéticaRESUMEN
Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress-induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress-activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1-dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.
Asunto(s)
Apoptosis , Inflamación/complicaciones , MAP Quinasa Quinasa Quinasa 5/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Neoplasias/enzimología , Animales , Línea Celular Tumoral , Femenino , Humanos , Inflamación/enzimología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias/etiología , Neoplasias/inmunología , Neoplasias Glandulares y Epiteliales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the selective loss of upper and lower motoneurons. Although >100 different Cu, Zn superoxide dismutase (SOD1) mutations have been identified in ALS patients, it remains controversial whether all of them are disease-causative mutations. Therefore, it is necessary to develop molecular mechanism-based diagnosis and treatment of ALS caused by SOD1 mutations. METHODS: We previously reported that 3 pathogenic mutations of SOD1 cause chronic endoplasmic reticulum (ER) stress by inducing the binding of SOD1 to Derlin-1, a component of the ER homeostatic machinery. Here, we systematically analyzed 132 SOD1 mutants and found that most have a constitutively exposed Derlin-1-binding region (DBR) that is occluded in the wild-type protein. To develop the novel molecular mechanism-based antibody that can specifically recognize the aberrant structure of toxic SOD1 mutants, we generated the monoclonal antibody against the DBR. RESULTS: MS785, a monoclonal antibody generated against the DBR, distinguished most ALS-causative SOD1 mutants from both wild-type and nontoxic mutants. Moreover, MS785 recognized endogenous SOD1 in B lymphocytes derived from 14 ALS patients carrying SOD1 mutations but not from 11 healthy controls. INTERPRETATION: This is the first study to address the common property of all ALS-causative SOD1 mutants. MS785 is the first molecular mechanism-based antibody that was shown to be able to distinguish ALS-linked toxic SOD1 mutants from both wild-type and nontoxic mutants. MS785 may thus become an innovative tool for the diagnosis of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/inmunología , Anticuerpos Monoclonales/metabolismo , Neuronas Motoras/fisiología , Superóxido Dismutasa/genética , Animales , Muerte Celular , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Neuronas Motoras/patología , Mutación/genética , Unión Proteica/genética , Conformación Proteica , Médula Espinal/patología , Superóxido Dismutasa-1 , TransfecciónRESUMEN
There are no available therapies targeting the underlying molecular mechanisms of neurodegenerative diseases. Although chaperone therapies that alleviate endoplasmic reticulum (ER) stress recently showed promise in the treatment of neurodegenerative diseases, the detailed mechanisms remain unclear. We previously reported that mice with central nervous system-specific deletion of Derlin-1, which encodes an essential component for ER quality control, are useful as models of neurodegenerative diseases such as spinocerebellar degeneration. Cholesterol biosynthesis is essential for brain development, and its disruption inhibits neurite outgrowth, causing brain atrophy. In this study, we report a novel mechanism by which chemical chaperones ameliorate brain atrophy and motor dysfunction. ER stress was induced in the cerebella of Derlin-1 deficiency mice, whereas the administration of a chemical chaperone did not alleviate ER stress. However, chemical chaperone treatment ameliorated cholesterol biosynthesis impairment through SREBP-2 activation and simultaneously relieved brain atrophy and motor dysfunction. Altogether, these findings demonstrate that ER stress may not be the target of action of chaperone therapies and that chemical chaperone-mediated improvement of brain cholesterol biosynthesis is a promising novel therapeutic strategy for neurodegenerative diseases.
Asunto(s)
Proteínas de la Membrana , Enfermedades Neurodegenerativas , Animales , Ratones , Atrofia/tratamiento farmacológico , Colesterol , Estrés del Retículo Endoplásmico , Proteínas de la Membrana/química , Chaperonas Moleculares , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genéticaRESUMEN
Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function.