RESUMEN
Antisense oligonucleotide (ASO)-based therapy is one of the next-generation therapy, especially targeting neurological disorders. Many cases of ASO-dependent gene expression suppression have been reported. Recently, we developed a tocopherol conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO) as a new type of drug. Toc-HDO is more potent, stable, and efficiently taken up by the target tissues compared to the parental ASO. However, the detailed mechanisms of Toc-HDO, including its binding proteins, are unknown. Here, we developed native gel shift assays with fluorescence-labeled nucleic acids samples extracted from mice livers. These assays revealed two Toc-HDO binding proteins, annexin A5 (ANXA5) and carbonic anhydrase 8 (CA8). Later, we identified two more proteins, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and flap structure-specific endonuclease 1 (FEN1) by data mining. shRNA knockdown studies demonstrated that all four proteins regulated Toc-HDO activity in Hepa1-6, mouse hepatocellular cells. In vitro binding assays and fluorescence polarization assays with purified recombinant proteins characterized the identified proteins and pull-down assays with cell lysates demonstrated the protein binding to the Toc-HDO and ASO in a biological environment. Taken together, our findings provide a brand new molecular biological insight as well as future directions for HDO-based disease therapy.
Asunto(s)
Silenciador del Gen , Oligonucleótidos Antisentido/metabolismo , Animales , Anexina A5/metabolismo , Biomarcadores de Tumor/metabolismo , Anhidrasas Carbónicas/metabolismo , Línea Celular , Centrifugación por Gradiente de Densidad , ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Polarización de Fluorescencia , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Oligonucleótidos Antisentido/química , ARN , ARN Interferente Pequeño , alfa-TocoferolRESUMEN
Intramembrane cleavage of transmembrane proteins is a fundamental cellular process to produce important signals that elicit biological responses. These proteolytic events are known as regulated intramembrane proteolysis (RIP). ATF6 and BBF2H7 are transmembrane basic leucine zipper transcription factors and are subjected to RIP by site-1 protease (S1P) and site-2 protease (S2P) sequentially in response to endoplasmic reticulum (ER) stress. However, the detailed mechanisms responsible for RIP of the transcription factors, including the precise cutting sites, are still unknown. In this study, we demonstrated that S1P cleaves BBF2H7 just before the RXXL S1P recognition motif. Conversely, S2P cut at least three different sites in the membrane (next to Leu380, Met381, and Leu385), indicating that S2P cleaves the substrates at variable sites or via a multistep process. Interestingly, we found BBF2H7-derived small peptide (BSP) fragments located between the S1P and S2P cleavage sites in cells exposed to ER stress. Major type of BSP fragments was composed of 45 amino acid including partial transmembrane and luminal regions and easily aggregates like amyloid ß (Aß) protein. These results advance the understanding of poorly characterized ER stress-dependent RIP. Furthermore, the aggregable peptides produced by ER stress could link to the pathophysiology of neurodegenerative disorders.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/metabolismo , Proteolisis , Factor de Transcripción Activador 6/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Humanos , Fragmentos de Péptidos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transcripción Genética/fisiologíaRESUMEN
We previously reported that RNF183, a member of the RING finger (RNF) protein family, is specifically expressed in the renal collecting duct and that RNF183 mRNA is induced by the activity of nuclear factor of activated T cells 5 (NFAT5), which regulates the transcription of essential proteins for adaptation to hypertonic conditions. The renal medulla is the only tissue that is continuously hypertonic; therefore, RNF183 possibly plays an important role in adaptation to continuous hypertonic conditions. However, the mechanism of how cells adapt to long-term hypertonicity via RNF183 remains unclear. In this study, the Na, K-ATPase α1 subunit was identified as a candidate substrate of RNF183 by the BirA proximity-biotinylation technique. The Na, K-ATPase α1 subunit acts as an ion transporter along with the Na, K-ATPase ß1 subunit at the plasma membrane. We confirmed that RNF183 interacted with both α1 and ß1 subunits; however, we found that RNF183 ubiquitinated only the ß1 subunit, not the α1 subunit. Furthermore, RNF183 translocated both α1 and ß1 subunits from the plasma membrane to lysosomes. In addition, the expression levels of α1 and ß1 subunits in HEK293â¯cells stably expressing RNF183 were significantly decreased compared with mock control cells, and were restored by siRNA-mediated knockdown of RNF183. Moreover, in RNF183-expressing cells, chloroquine treatment increased the protein levels of the α1 and ß1 subunits. Therefore, our results suggest that Na, K-ATPase α1 and ß1 subunits are degraded in lysosomes by RNF183-mediated ubiquitination of ß1 subunit.
Asunto(s)
Soluciones Hipertónicas/farmacología , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC-MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteoma/química , Sinapsis/química , Animales , Química Encefálica , Cromatografía Líquida de Alta Presión , Proteínas del Tejido Nervioso/aislamiento & purificación , Densidad Postsináptica/química , Proteoma/aislamiento & purificación , Proteómica , Ratas , Espectrometría de Masas en TándemRESUMEN
Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Animales , Atrofia , Conducta Animal/fisiología , Western Blotting , Cistatina C/metabolismo , Dependovirus , Modelos Animales de Enfermedad , Electromiografía , Fenómenos Electrofisiológicos , Vectores Genéticos , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/patología , Neuritas/patología , Ratas , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la Polimerasa , Especificidad de la Especie , Médula Espinal/metabolismo , Técnicas EstereotáxicasRESUMEN
Aggregation of the 43 kDa TAR DNA-binding protein (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA binding and TDP-43 N-terminal domain dimerisation has been suggested to ameliorate TDP-43 aggregation. However, the relationship between these factors and the solubility of TDP-43 is largely unknown. Therefore, we developed new oligonucleotides that can recruit two TDP-43 molecules and interfere with their intermolecular interactions via spatial separation. Using these oligonucleotides and TDP-43-preferable UG-repeats, we uncovered two distinct mechanisms for modulating TDP-43 solubility by RNA binding: One is N-terminal domain dimerisation, and the other is the spatial separation of two TDP-43 molecules. This study provides new molecular insights into the regulation of TDP-43 solubility.
Asunto(s)
Esclerosis Amiotrófica Lateral , Degeneración Lobar Frontotemporal , Humanos , Proteínas de Unión al ADN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Cuerpos de Inclusión/metabolismo , ARN/genética , ARN/metabolismoRESUMEN
We have recently shown that beta-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a beta-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-beta-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3zeta or - resulted in the disappearance of N-cadherin and beta-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3zeta/ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and beta-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and beta-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3zeta/ complex links the N-cadherin-beta-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.
Asunto(s)
Proteínas 14-3-3/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Dineínas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , beta Catenina/metabolismo , Proteínas 14-3-3/genética , Animales , Antígenos CD/genética , Células COS , Cadherinas/genética , Chlorocebus aethiops , Complejo Dinactina , Dineínas/genética , Retículo Endoplásmico/genética , Proteínas Activadoras de GTPasa/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Complejos Multiproteicos/genética , Transporte de Proteínas/fisiología , beta Catenina/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
DNA/RNA heteroduplex oligonucleotide (HDO), composed of DNA/locked nucleic acid (LNA) antisense oligonucleotide (ASO) and complementary RNA, is a next-generation antisense therapeutic agent. HDO is superior to the parental ASO in delivering to target tissues, and it exerts a more potent gene-silencing effect. In this study, we aimed to elucidate the intracellular trafficking mechanism of HDO-dependent gene silencing. HDO was more preferably transferred to the nucleus after transfection compared to the parental ASO. To determine when and where HDO is separated into the antisense strand (AS) and complementary strand (CS), we performed live-cell time-lapse imaging and fluorescence resonance energy transfer (FRET) assays. These assays demonstrated that HDO had a different intracellular trafficking mechanism than ASO. After endocytosis, HDO was separated in the early endosomes, and both AS and CS were released into the cytosol. AS was more efficiently transported to the nucleus than CS. Separation, endosomal release, and initiation of nuclear transport were a series of time-locked events occurring at a median of 30 s. CS cleavage was associated with efficient nuclear distribution and gene silencing in the nucleus. Understanding the unique intracellular silencing mechanisms of HDO will help us design more efficient drugs and might also provide insight into innate DNA/RNA cellular biology.
RESUMEN
Aggregation, fibril formation, and deposition of amyloid ß (Aß) protein are believed to be the central pathogeneses of Alzheimer's disease (AD). Numerous studies have shown that fibril formation is promoted by preformed seeds at the beginning of the aggregation process. Therefore, aggregated molecules that promote fibrillization of Aß protein as seeds could affect the pathology. We recently found that approximately 40 amino acid hydrophobic peptides, BBF2H7-derived small peptide (BSP) fragments, are generated via intramembranous cleavage under endoplasmic reticulum (ER) stress conditions. Interestingly, similar to Aß protein, the fragments exhibit a high aggregation propensity and form fibril structures. It has been noted that ER stress is involved in the pathogenesis of AD. In this study, we examined the effect of BSP fragments on aggregation and cytotoxicity of Aß1-40 protein, which is generated as a major species of Aß protein, but has a lower aggregative property than Aß1-42 protein. We demonstrated that BSP fragments promote aggregation of Aß1-40 protein. Aggregates of Aß1-40 protein mediated by BSP fragments also exhibited potent neurotoxicity. Our findings suggest the possibility that BSP fragments affect accumulation of Aß proteins and are involved in the pathogenesis of AD.
Asunto(s)
Amiloide/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Neuronas/metabolismo , Péptidos beta-Amiloides/metabolismo , Línea Celular , Humanos , Fragmentos de Péptidos/metabolismoRESUMEN
The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue.
RESUMEN
RNF183 is a ubiquitin ligase containing RING-finger and transmembrane domains, and its expression levels are increased in patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, and in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice. Here, we further demonstrate that RNF183 was induced to a greater degree in the dextran sulfate sodium (DSS)-treated IBD model at a very early stage than were inflammatory cytokines. In addition, fluorescence-activated cell sorting and polymerase chain reaction analysis revealed that RNF183 was specifically expressed in epithelial cells of DSS-treated mice, which suggested that increased levels of RNF183 do not result from the accumulation of immune cells. Furthermore, we identified death receptor 5 (DR5), a member of tumour necrosis factor (TNF)-receptor superfamily, as a substrate of RNF183. RNF183 mediated K63-linked ubiquitination and lysosomal degradation of DR5. DR5 promotes TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis signal through interaction with caspase-8. Inhibition of RNF183 expression was found to suppress TRAIL-induced activation of caspase-8 and caspase-3. Thus, RNF183 promoted not only DR5 transport to lysosomes but also TRAIL-induced caspase activation and apoptosis. Together, our results provide new insights into potential roles of RNF183 in DR5-mediated caspase activation in IBD pathogenesis.
Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Lisosomas/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Biomarcadores , Línea Celular , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Expresión Génica , Humanos , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Ratones , Membrana Mucosa/metabolismo , Membrana Mucosa/patología , Unión Proteica , Transporte de Proteínas , Proteolisis , ARN Mensajero , Índice de Severidad de la Enfermedad , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
GABAergic dysfunction underlies many neurodevelopmental and psychiatric disorders. GABAergic synapses exhibit several forms of plasticity at both pre- and postsynaptic levels. NMDA receptor (NMDAR)-dependent inhibitory long-term potentiation (iLTP) at GABAergic postsynapses requires an increase in surface GABAARs through promoted exocytosis; however, the regulatory mechanisms and the neuropathological significance remain unclear. Here we report that the autism-related protein PX-RICS is involved in GABAAR transport driven during NMDAR-dependent GABAergic iLTP. Chemically induced iLTP elicited a rapid increase in surface GABAARs in wild-type mouse hippocampal neurons, but not in PX-RICS/RICS-deficient neurons. This increase in surface GABAARs required the PX-RICS/GABARAP/14-3-3 complex, as revealed by gene knockdown and rescue studies. iLTP induced CaMKII-dependent phosphorylation of PX-RICS to promote PX-RICS-14-3-3 assembly. Notably, PX-RICS/RICS-deficient mice showed impaired amygdala-dependent fear learning, which was ameliorated by potentiating GABAergic activity with clonazepam. Our results suggest that PX-RICS-mediated GABAAR trafficking is a key target for GABAergic plasticity and its dysfunction leads to atypical emotional processing underlying autism.
Asunto(s)
Proteínas Activadoras de GTPasa/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Receptores de GABA-A/fisiología , Amígdala del Cerebelo/fisiología , Animales , Trastorno Autístico , Células Cultivadas , Miedo/fisiología , Hipocampo/citología , Aprendizaje/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients.
Asunto(s)
Trastorno del Espectro Autista/genética , Conducta Animal/fisiología , Proteínas Activadoras de GTPasa/genética , Síndrome de Deleción Distal 11q de Jacobsen/genética , Transporte de Proteínas/genética , Receptores de GABA-A/metabolismo , Conducta Social , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/psicología , Conducta Animal/efectos de los fármacos , Clonazepam/farmacología , Agonistas de Aminoácidos Excitadores/toxicidad , Moduladores del GABA/farmacología , Aseo Animal , Síndrome de Deleción Distal 11q de Jacobsen/metabolismo , Síndrome de Deleción Distal 11q de Jacobsen/psicología , Ácido Kaínico/toxicidad , Ratones , Ratones Noqueados , Percepción Olfatoria/efectos de los fármacos , Percepción Olfatoria/genética , Convulsiones/inducido químicamente , Convulsiones/genética , Conducta Estereotipada/efectos de los fármacos , Conducta Estereotipada/fisiología , Vocalización Animal/efectos de los fármacos , Vocalización Animal/fisiologíaRESUMEN
BACKGROUND: The mechanisms underlying stress-induced inflammation that contribute to major depressive disorder are unknown. We examine the role of the adenosine triphosphate (ATP)/purinergic type 2X7 receptor (P2X7R) pathway and the NLRP3 (nucleotide-binding, leucine-rich repeat, pyrin domain containing 3) inflammasome in interleukin (IL)-1ß and depressive behavioral responses to stress. METHODS: The influence of acute restraint stress on extracellular ATP, glutamate, IL-1ß, and tumor necrosis factor alpha in hippocampus was determined by microdialysis, and the influence of acute restraint stress on the NLRP3 inflammasome was determined by western blot analysis. The influence of P2X7R antagonist administration on IL-1ß and tumor necrosis factor alpha and on anxiety and depressive behaviors was determined in the chronic unpredictable stress rodent model. The role of the NLRP3 inflammasome was determined by analysis of Nlrp3 null mice. RESULTS: Acute restraint stress rapidly increased extracellular ATP, an endogenous agonist of P2X7R; the inflammatory cytokine IL-1ß; and the active form of the NLRP3 inflammasome in the hippocampus. Administration of a P2X7R antagonist completely blocked the release of IL-1ß and tumor necrosis factor alpha, another stress-induced cytokine, and activated NLRP3. Moreover, P2X7R antagonist administration reversed the anhedonic and anxiety behaviors caused by chronic unpredictable stress exposure, and deletion of the Nlrp3 gene rendered mice resistant to development of depressive behaviors caused by chronic unpredictable stress. CONCLUSIONS: These findings demonstrate that psychological "stress" is sensed by the innate immune system in the brain via the ATP/P2X7R-NLRP3 inflammasome cascade, and they identify novel therapeutic targets for the treatment of stress-related mood disorders and comorbid illnesses.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ansiedad/metabolismo , Conducta Animal/fisiología , Depresión/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Estrés Psicológico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anhedonia/fisiología , Animales , Ansiedad/etiología , Ansiedad/inmunología , Depresión/etiología , Depresión/inmunología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Noqueados , Agonistas del Receptor Purinérgico P2Y/metabolismo , Antagonistas del Receptor Purinérgico P2Y/metabolismo , Ratas , Ratas Sprague-Dawley , Estrés Psicológico/complicaciones , Estrés Psicológico/inmunologíaRESUMEN
Cadherins mediate Ca2+-dependent cell-cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with beta-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a beta-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/beta-catenin complex in the ER and ER exit site, resulting in a decrease in cell-cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with beta-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner gamma-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/beta-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling.
Asunto(s)
Cadherinas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Aparato de Golgi/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
In our previous study, we identified RICS, a novel beta-catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits.
Asunto(s)
Empalme Alternativo/genética , Proteínas Activadoras de GTPasa/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Cadherinas/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario , Homólogo 4 de la Proteína Discs Large , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Aparato de Golgi/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Neuritas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de N-Metil-D-Aspartato/metabolismo , beta Catenina/metabolismoRESUMEN
Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral nerve disorder. The causative gene for axonal type CMT2E has been identified as neurofilament light (NF-L) chain. Using cultured cells and in vitro assays, we analyzed the filament formation ability of Pro22 CMT mutant proteins of NF-L, P22S and P22T. NF-L Pro22 mutant proteins formed large aggregates in SW13- cells and cortical neurons and assembled into short twisty threads thinner than 10 nm filaments in vitro. Those threads associated with each other at their ends and entangled into large aggregates, also abnormalities, were detected at steps in oligomer formation. Pro22 mutations abolished Thr21 phosphorylation by cyclin-dependent kinase 5 and external signal regulated kinase, which suppressed filament assembly, but phosphorylation by protein kinase A (PKA) inhibited aggregate formation in vitro and alleviated aggregates in cortical neurons. These results indicate that the Pro22 CMT mutation induces abnormal filament aggregates by disrupting proper oligomer formation and the aggregates are mitigated by phosphorylation with PKA, which makes it a viable target for the development for therapeutics.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Prolina/genética , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Enfermedad de Charcot-Marie-Tooth/patología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Humanos , Proteínas de Neurofilamentos/química , Fosforilación , Estructura Terciaria de Proteína/genética , Ratas , Treonina/genética , Treonina/metabolismoRESUMEN
One of the major pathological hallmarks of Alzheimer disease is neurofibrillary tangles. Neurofibrillary tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is one of the tau protein kinases that increase paired helical filament epitopes in tau by phosphorylation. Recently, various mutations of tau have been identified in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we investigated the phosphorylation of FTDP-17 mutant tau proteins, K257T, P301L, P301S, and R406W, by Cdk5 complexed with p35, p25, or p39 in vitro and in cultured cells. The extent of phosphorylation by all Cdk5 species was slightly lower in mutant tau than in wild-type tau. Major phosphorylation sites, including Ser202, Ser235, and Ser404, were the same among the wild-type, K257T, P301L, and P301S tau proteins phosphorylated by any Cdk5. On the other hand, R406W tau was less phosphorylated at Ser404 than were the other variants. This was not due to the simple replacement of amino acid Arg406 with Trp close to the phosphorylation site, because Ser404 in a R406W peptide was equally phosphorylated in a wild-type peptide. The decreased phosphorylation of mutant tau by Cdk5s was canceled when tau protein bound to microtubules was phosphorylated. These results indicate that FTDP-17 mutations do not affect the phosphorylatability of tau by Cdk5 complexed with p35, p25, or p39 and may explain part of the discrepancy reported previously between in vivo and in vitro phosphorylation of FTDP-17 tau mutants.