RESUMEN
Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/IqE. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme.
Asunto(s)
Endorribonucleasas , Escherichia coli , Expresión Génica , Ribonucleasas , Endorribonucleasas/biosíntesis , Endorribonucleasas/química , Endorribonucleasas/genética , Endorribonucleasas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Ribonucleasas/biosíntesis , Ribonucleasas/química , Ribonucleasas/genética , Ribonucleasas/aislamiento & purificaciónRESUMEN
Antibiotic resistance is a serious problem which may be caused by bacterial dormancy. It has been suggested that bacterial toxin-antitoxin systems induce dormancy. We analyzed the genome-wide role of Staphylococcus aureus endoribonuclease toxin MazF using RNA-Seq, Ribo-Seq and quantitative proteomics. We characterized changes in transcriptome, translatome and proteome caused by MazF, and proposed that MazF decreases translation directly by cleaving mRNAs, and indirectly, by decreasing translation factors and by promoting ribosome hibernation. Important pathways affected during the early stage of MazF induction were identified: MazF increases cell wall thickness and decreases cell division; MazF activates SsrA-system which rescues stalled ribosomes, appearing as a result of MazF mRNA cleavage. These pathways may be promising targets for new antibacterial drugs that prevent bacteria dormancy. Finally, we described the overall impact of MazF on S. aureus cell physiology, and propose one of the mechanisms by which MazF might regulate cellular changes leading to dormancy.
Asunto(s)
Toxinas Bacterianas/metabolismo , Endorribonucleasas/fisiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Toxinas Bacterianas/biosíntesis , División Celular/genética , Pared Celular/genética , Pared Celular/metabolismo , Endorribonucleasas/biosíntesis , Endorribonucleasas/metabolismo , Biosíntesis de Proteínas , Proteoma , Staphylococcus aureus/enzimología , TranscriptomaRESUMEN
BACKGROUND: Spinal cord ischemia-reperfusion injury (SCIRI) often leads to neurological damage and mortality. In this regard, understanding the pathology of SCIRI and preventing its development are of great clinic value. METHODS: Herein, we analyzed the role of bone marrow mesenchymal stem cell (BMMSC)-derived exosomal microRNA (miR)-124-3p in SCIRI. A SCIRI rat model was established, and the expression of Ern1 and M2 macrophage polarization markers (Arg1, Ym1, and Fizz) was determined using immunohistochemistry, immunofluorescence assay, RT-qPCR, and western blot analysis. Targeting relationship between miR-124-3p and Ern1 was predicted using bioinformatic analysis and verified by dual-luciferase reporter assay. Macrophages were co-cultured with miR-124-3p-containing BMMSC-derived exosomes. M2 macrophages were identified using flow cytometry, and the expression of Arg1, Ym1, and Fizz was determined. In addition, SCIRI rats were injected with miR-124-3p-containing exosomes, spinal cord cell apoptosis was observed using TUNEL assay, and the pathological condition was evaluated with H&E staining. RESULTS: In SCIRI, Ern1 was highly expressed and M2 polarization markers were poorly expressed. Silencing Ern1 led to elevated expression of M2 polarization markers. MiR-124-3p targeted and negatively regulated Ern1. Exosomal miR-124-3p enhanced M2 polarization. Highly expressed exosomal miR-124-3p impeded cell apoptosis and attenuated SCIRI-induced tissue impairment and nerve injury. miR-124-3p from BMMSC-derived exosomes ameliorated SCIRI and its associated nerve injury through inhibiting Ern1 and promoting M2 polarization. CONCLUSION: In summary, exosomal miR-124-3p derived from BMMSCs attenuated nerve injury induced by SCIRI by regulating Ern1 and M2 macrophage polarization.
Asunto(s)
Endorribonucleasas/biosíntesis , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Complejos Multienzimáticos/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Daño por Reperfusión/patología , Traumatismos de la Médula Espinal/patología , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular/inmunología , Regulación hacia Abajo , Exosomas/metabolismo , Regulación de la Expresión Génica/fisiología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/metabolismoRESUMEN
Up to date, the mechanism of gastric cancer (GC) development is poorly understood. This study was to demonstrate the effects of LINC00339 on GC progression. Here, we found that LINC00339 was overexpressed expressed in GC tissues and predicted poor outcome. By CCK8, colony formation and Transwell assays, we showed LINC00339 knockdown suppressed GC cell proliferation, migration, and invasion in vitro. Flow cytometry analysis (FACS) indicated that LINC00339 knockdown induced tumor cell apoptosis. Besides, we utilized the xenograft assay and found that LINC00339 depletion led to decreased tumor growth in vivo. Mechanistically, miR-377-3p was found to be inhibited by LINC00339. And LINC00339 suppressed miR-377-3p to upregulate DCP1A, which consequently promoted GC progression. In conclusion, LINC00339 promotes gastric cancer progression by elevating DCP1A expression via inhibiting miR-377-3p.
Asunto(s)
Endorribonucleasas/biosíntesis , MicroARNs/genética , ARN Largo no Codificante/genética , Neoplasias Gástricas/patología , Transactivadores/biosíntesis , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Humanos , Invasividad Neoplásica/genética , Trasplante de Neoplasias , Oncogenes/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Neoplasias Gástricas/genética , Trasplante HeterólogoRESUMEN
The innate immune system is a broad collection of critical intra- and extra-cellular processes that limit the infectivity of diverse pathogens. The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes are important sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection by activating the latent ribonuclease (RNase L) to halt viral replication and establish an antiviral state. Attesting to the importance of the OAS/RNase L pathway, diverse viruses have developed numerous distinct strategies to evade the effects of OAS activation. How OAS proteins are regulated by viral or cellular RNAs is not fully understood but several recent studies have provided important new insights into the molecular mechanisms of OAS activation by dsRNA. Other studies have revealed unanticipated features of RNA sequence and structure that strongly enhance activation of at least one OAS family member. While these discoveries represent important advances, they also underscore the fact that much remains to be learned about RNA-mediated regulation of the OAS/RNase L pathway. In particular, defining the full complement of RNA molecular signatures that activate OAS is essential to our understanding of how these proteins maximize their protective role against pathogens while still accurately discriminating host molecules to avoid inadvertent activation by cellular RNAs. A more complete knowledge of OAS regulation may also serve as a foundation for the development of novel antiviral therapeutic strategies and lead the way to a deeper understanding of currently unappreciated cellular functions of the OAS/RNase L pathway in the absence of infection. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Activadores de Enzimas/metabolismo , Regulación de la Expresión Génica , Factores Inmunológicos/biosíntesis , ARN Bicatenario/metabolismo , Endorribonucleasas/biosíntesisRESUMEN
BACKGROUND: Ethanol (EtOH) exposure during pregnancy may result in fetal alcohol spectrum disorders (FASD). One of the most deleterious consequences of EtOH exposure is neuronal loss in the developing brain. Previously, we showed that EtOH exposure induced neuroapoptosis in the brain of postnatal day 4 (PD4) mice but not PD12 mice. This differential susceptibility may result from an insufficient cellular stress response system such as unfolded protein response (also known as endoplasmic reticulum [ER] stress) in PD4 mice. In this study, we compared the effect of EtOH on ER stress in PD4 and PD12 mice and determined whether the inhibition of ER stress could protect the developing brain against EtOH damage. METHODS: We used a third-trimester equivalent mouse model of FASD. PD4 and PD12 C57BL/6 mice were subcutaneously injected with saline (control), EtOH, EtOH plus 4-phenylbutyric acid (4-PBA), a chemical chaperone known as ER stress inhibitor, and 4-PBA alone. The expression of apoptosis marker, ER stress markers, and markers for glial cell activation was examined in the cerebral cortex. RESULTS: EtOH induced neuroapoptosis and increased the expression of ER stress markers, such as activating transcription factor 6, 78-kDa glucose-regulated protein, inositol-requiring enzyme 1α, mesencephalic astrocyte-derived neurotrophic factor, and caspase-12 in PD4 but not PD12 mice. EtOH exposure also activated microglia and astrocytes. Interestingly, treatment with 4-PBA attenuated EtOH-induced neuroapoptosis. Moreover, 4-PBA inhibited the expression of the aforementioned ER stress markers and EtOH-induced glial activation in PD4 mice. CONCLUSIONS: ER stress plays an important role in EtOH-induced damage to the developing brain. Inhibition of ER stress is neuroprotective and may provide a new therapeutic strategy for treating FASD.
Asunto(s)
Envejecimiento/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Etanol/antagonistas & inhibidores , Fenilbutiratos/farmacología , Factor de Transcripción Activador 6/biosíntesis , Animales , Astrocitos/metabolismo , Caspasa 12/biosíntesis , Corteza Cerebral/metabolismo , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/biosíntesis , Etanol/efectos adversos , Femenino , Proteínas de Choque Térmico/biosíntesis , Masculino , Ratones , Microglía/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Fármacos Neuroprotectores/farmacología , Proteínas Serina-Treonina Quinasas/biosíntesisRESUMEN
Expression of CRISPR-Cas systems is a prerequisite for their defensive role against invading genetic elements. Yet, much remains unknown about how this crucial step is regulated. We describe a new mechanism controlling CRISPR-cas expression, which requires an extracytoplasmic function (ECF) σ factor (DdvS), its membrane-bound anti-σ (DdvA) and a global regulatory complex (CarD-CarG). Transcriptomic analyses revealed that the DdvS/CarD/CarG-dependent regulon comprises a type III-B CRISPR-Cas system in Myxococcus xanthus. We mapped four DdvS-driven CarD/CarG-dependent promoters, with one lying immediately upstream of the cas cluster. Consistent with direct action, DdvS and CarD-CarG localize at these promoters in vivo. The cas genes are transcribed as a polycistronic mRNA that reads through the leader into the CRISPR array, a putative σA-dependent promoter in the leader having negligible activity in vivo. Consequently, expression of the entire CRISPR-Cas system and mature CRISPR-RNA (crRNA) production is DdvS/CarD/CarG-dependent. DdvA likely uses its large C-terminal domain to sense and transduce the extracytoplasmic signal triggering CRISPR-cas expression, which we show is not starvation-induced multicellular development. An ECF-σ/anti-σ pair and a global regulatory complex provide an effective mechanism to coordinate signal-sensing with production of precursor crRNA, its processing Cas6 endoribonuclease and other Cas proteins for mature crRNA biogenesis and interference.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endorribonucleasas/genética , Regulación Bacteriana de la Expresión Génica , Myxococcus xanthus/genética , Factor sigma/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Endorribonucleasas/biosíntesis , Endorribonucleasas/metabolismo , Myxococcus xanthus/metabolismo , Operón , Regiones Promotoras Genéticas , ARN Bacteriano/metabolismo , Regulón , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
Objectives: Labial salivary glands (LSGs) of SS patients show alterations related to endoplasmic reticulum stress. Glandular dysfunction could be partly the consequence of an altered inositol-requiring enzyme 1α (IRE1α)/X box-binding protein 1 (XBP-1) signalling pathway of the unfolded protein response, which then regulates genes involved in biogenesis of the secretory machinery. This study aimed to determine the expression, promoter methylation and localization of the IRE1α/XBP-1 pathway components in LSGs of SS patients and also their expression induced by IFN-γ in vitro. Methods: IRE1α, XBP-1 and glucose-regulated protein 78 (GRP78) mRNA and protein levels were measured by qPCR and western blot, respectively, in LSGs of SS patients (n = 47) and control subjects (n = 37). Methylation of promoters was evaluated by methylation-sensitive high resolution melting, localization was analysed by immunofluorescence and induction of the IRE1α/XBP-1 pathway components by IFN-γ was evaluated in 3D acini. Results: A significant decrease of IRE1α, XBP-1u, XBP-1s, total XBP-1 and GRP78 mRNAs was observed in LSGs of SS patients, which was correlated with increased methylation levels of their respective promoters, and consistently the protein levels for IRE1α, XBP-1s and GRP78 were observed to decrease. IFN-γ decreased the mRNA and protein levels of XBP-1s, IRE1α and GRP78, and increased methylation of their promoters. Significant correlations were also found between IRE1α/XBP-1 pathway components and clinical parameters. Conclusion: Decreased mRNA levels for IRE1α, XBP-1 and GRP78 can be partially explained by hypermethylation of their promoters and is consistent with chronic endoplasmic reticulum stress, which may explain the glandular dysfunction observed in LSGs of SS patients. Additionally, glandular stress signals, including IFN-γ, could modulate the expression of the IRE1α/XBP-1 pathway components.
Asunto(s)
Endorribonucleasas/genética , Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/genética , Proteína 1 de Unión a la X-Box/genética , Adulto , Anciano , Western Blotting , Metilación de ADN , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Endorribonucleasas/biosíntesis , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/biosíntesis , Glándulas Salivales/metabolismo , Transducción de Señal , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Proteína 1 de Unión a la X-Box/biosíntesis , Adulto JovenRESUMEN
BACKGROUND: The endoplasmic reticulum (ER) is responsible for the control of correct protein folding and protein function which is crucial for cell survival. However, under pathological conditions, such as hypoxia-ischemia (HI), there is an accumulation of unfolded proteins thereby triggering the unfolded protein response (UPR) and causing ER stress which is associated with activation of several stress sensor signaling pathways, one of them being the inositol requiring enzyme-1 alpha (IRE1α) signaling pathway. The UPR is regarded as a potential contributor to neuronal cell death and inflammation after HI. In the present study, we sought to investigate whether microRNA-17 (miR-17), a potential IRE1α ribonuclease (RNase) substrate, arbitrates downregulation of thioredoxin-interacting protein (TXNIP) and consequent NLRP3 inflammasome activation in the immature brain after HI injury and whether inhibition of IRE1α may attenuate inflammation via miR-17/TXNIP regulation. METHODS: Postnatal day 10 rat pups (n = 287) were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O2). STF-083010, an IRE1α RNase inhibitor, was intranasally delivered at 1 h post-HI or followed by an additional one administration per day for 2 days. MiR-17-5p mimic or anti-miR-17-5p inhibitor was injected intracerebroventricularly at 48 h before HI. Infarct volume and body weight were used to evaluate the short-term effects while brain weight, gross and microscopic brain tissue morphologies, and neurobehavioral tests were conducted for the long-term evaluation. Western blots, immunofluorescence staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (Co-IP) were used for mechanism studies. RESULTS: Endogenous phosphorylated IRE1α expression was significantly increased after HI. Intranasal administration of STF-083010 alleviated brain injury and improved neurological behavior. MiR-17-5p expression was reduced after HI, and this decrease was attenuated by STF-083010 treatment. MiR-17-5p mimic administration ameliorated TXNIP expression, NLRP3 inflammasome activation, caspase-1 cleavage, and IL-1ß production, as well as brain infarct volume. Conversely, anti-miR-17-5p inhibitor reversed IRE1α inhibition-induced decrease in TXNIP expression and inflammasome activation, as well as exacerbated brain injury after HI. CONCLUSIONS: IRE1a-induced UPR pathway may contribute to inflammatory activation and brain injury following neonatal HI. IRE1a activation, through decay of miR-17-5p, elevated TXNIP expression to activate NLRP3 inflammasome and aggravated brain damage.
Asunto(s)
Proteínas Portadoras/metabolismo , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/biosíntesis , Hipoxia-Isquemia Encefálica/metabolismo , MicroARNs/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/biosíntesis , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Administración Intranasal , Animales , Animales Recién Nacidos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Ciclo Celular , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Inflamasomas/antagonistas & inhibidores , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sulfonamidas/administración & dosificación , Tiofenos/administración & dosificaciónRESUMEN
Pancreatic cancer is one of the most aggressive and difficult to treat cancers. Experimental and clinical evidence suggests that high basal state autophagy in pancreatic tumors could induce resistance to chemotherapy. Recently, we have demonstrated that penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis both in vitro and in vivo; however, the mechanism of autophagy induction by penfluridol was not clear. Several studies have established that endoplasmic reticulum stress could lead to autophagy and inhibit tumor progression. In this study, we demonstrated that penfluridol induced endoplasmic reticulum stress in BxPC-3, AsPC-1, and Panc-1 pancreatic cancer cell lines as indicated by upregulation of endoplasmic reticulum stress markers such as binding protein (BIP), C/EBP homologous protein (CHOP) and inositol requiring 1α (IRE1α) after treatment with penfluridol in a concentration-dependent manner. Inhibiting endoplasmic reticulum stress by pretreatment with pharmacological inhibitors such as sodium phenylbutyrate and mithramycin or by silencing CHOP using CHOP small interfering RNA, blocked penfluridol-induced autophagy. These results clearly indicate that penfluridol-induced endoplasmic reticulum stress lead to autophagy in our model. Western blot analysis of subcutaneously implanted AsPC-1 and BxPC-3 tumors as well as orthotopically implanted Panc-1 tumors demonstrated upregulation of BIP, CHOP, and IRE1α expression in the tumor lysates from penfluridol-treated mice as compared to tumors from control mice. Altogether, our study establishes that penfluridol-induced endoplasmic reticulum stress leads to autophagy resulting in reduced pancreatic tumor growth. Our study opens a new therapeutic target for advanced chemotherapies against pancreatic cancer.
Asunto(s)
Endorribonucleasas/biosíntesis , Proteínas de Choque Térmico/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Penfluridol/administración & dosificación , Proteínas Serina-Treonina Quinasas/biosíntesis , Factor de Transcripción CHOP/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Factor de Transcripción CHOP/antagonistas & inhibidores , Factor de Transcripción CHOP/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway.
Asunto(s)
Endorribonucleasas/genética , Ficusina/administración & dosificación , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa Quinasa 5/genética , Osteoporosis/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/genética , Anisomicina/administración & dosificación , Apoptosis/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Endorribonucleasas/biosíntesis , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 4/biosíntesis , MAP Quinasa Quinasa Quinasa 5/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Persona de Mediana Edad , Osteoblastos/efectos de los fármacos , Osteoporosis/genética , Osteoporosis/patología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Tunicamicina/administración & dosificación , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
PURPOSE: Proteasome inhibition with bortezomib eliminates multiple myeloma (MM) cells by partly disrupting unfolded protein response (UPR). However, the development of drug resistance limits its utility and resistance mechanism remains controversial. We aimed to investigate the role of IRE1α/Xbp-1 mediated branch of the UPR in bortezomib resistance. METHODS: The expression level of Xbp-1s was measured in 4 MM cell lines and correlated with sensitivity to bortezomib. LP1 and MY5 cells with different Xbp-1s level were treated with bortezomib; then pivotal UPR regulators were compared by immunoblotting. RPMI 8226 cells were transfected with plasmid pEX4-Xbp-1s and exposed to bortezomib; then apoptosis was determined by immunoblotting and flow cytometry. Bortezomib-resistant myeloma cells JJN3.BR were developed and the effect on UPR signaling pathway was determined. RESULTS: By analyzing 4 MM cell lines, we found little correlation between Xbp-1s basic level and bortezomib sensitivity. Bortezomib induced endoplasmic reticulum stress-initiated apoptosis via inhibiting IRE1α/Xbp-1 pathway regardless of Xbp-1s basic level. Exogenous Xbp-1s reduced cellular sensitivity to bortezomib, suggesting the change of Xbp-1s expression, not its basic level, is a potential marker of response to bortezomib in MM cells. Furthermore, sustained activation of IRE1α/Xbp-1 signaling pathway in JJN3.BR cells was identified. CONCLUSIONS: Our data indicate that reduced response of IRE1α/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells.
Asunto(s)
Bortezomib/administración & dosificación , Endorribonucleasas/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína 1 de Unión a la X-Box/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/biosíntesis , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mieloma Múltiple/patología , Proteínas Serina-Treonina Quinasas/biosíntesis , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box/biosíntesisRESUMEN
In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.
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Candida albicans/genética , Proteínas de Ciclo Celular/genética , Endorribonucleasas/genética , Manosiltransferasas/genética , Biosíntesis de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Candida albicans/crecimiento & desarrollo , Proteínas de Ciclo Celular/biosíntesis , Endorribonucleasas/biosíntesis , Glicosilación , Humanos , Oligonucleótidos/genética , Fenotipo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Ribosomas/genética , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesisRESUMEN
Previous studies have shown that infection of Prochlorococcus MED4 by the cyanophage P-SSP7 leads to increased transcript levels of host endoribonuclease (RNase) E. However, it has remained enigmatic whether this is part of a host defense mechanism to degrade phage messenger RNA (mRNA) or whether this single-strand RNA-specific RNase is utilized by the phage. Here we describe a hitherto unknown means through which this cyanophage increases expression of RNase E during phage infection and concomitantly protects its own RNA from degradation. We identified two functionally different RNase E mRNA variants, one of which is significantly induced during phage infection. This transcript lacks the 5' UTR, is considerably more stable than the other transcript, and is likely responsible for increased RNase E protein levels during infection. Furthermore, selective enrichment and in vivo analysis of double-stranded RNA (dsRNA) during infection revealed that phage antisense RNAs (asRNAs) sequester complementary mRNAs to form dsRNAs, such that the phage protein-coding transcriptome is nearly completely covered by asRNAs. In contrast, the host protein-coding transcriptome is only partially covered by asRNAs. These data suggest that P-SSP7 orchestrates degradation of host RNA by increasing RNase E expression while masking its own transcriptome from RNase E degradation in dsRNA complexes. We propose that this combination of strategies contributes significantly to phage progeny production.
Asunto(s)
Bacteriófagos/genética , Endorribonucleasas/genética , Prochlorococcus/genética , Transcriptoma/genética , Bacteriófagos/patogenicidad , Endorribonucleasas/biosíntesis , Genoma Viral , Interacciones Huésped-Patógeno/genética , Prochlorococcus/virología , Estabilidad del ARN/genética , ARN sin Sentido/biosíntesis , ARN sin Sentido/genética , ARN Bicatenario/genética , ARN Mensajero/genéticaRESUMEN
UNLABELLED: The oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2',5'-oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2',5'-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2(H126R)) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2(H126R) replicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient (Ifnar1(-/-)) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions of Oas gene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2(H126R) activated RNase L in Ifih1(-/-) BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2(H126R) failed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as in Ifnar1(-/-) BMM, both expressing low basal levels of Oas genes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basal Oas gene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels. IMPORTANCE: The oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basal Oas gene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Endorribonucleasas/biosíntesis , Expresión Génica , Interacciones Huésped-Patógeno , Virus de la Hepatitis Murina/fisiología , Células Mieloides/enzimología , Células Mieloides/virología , Animales , Células Cultivadas , Ratones , Ratones NoqueadosRESUMEN
Tob1 is a member of the BTG/TOB family of proteins with established antiproliferative function. In Danio rerio and Xenopus laevis, the Tob1 gene is expressed from the one-cell stage through to early gastrula stages, followed in later development by discrete expression in many tissues including the notochord and somites. In both mouse and human, Tob1 is expressed in many adult tissues including the testis and ovary; however, the specific cell types are unknown. We examine Tob1 gene expression in mouse in developing germ cells and in sorted male germ cells (gonocytes, spermatogonia, pachytene spermatocytes and round spermatids) by reverse transcription and droplet digital polymerase chain reaction (RT-ddPCR) and in adult ovary and testis by immunofluorescence with anti-Tob1 protein staining. By RT-ddPCR, Tob1 expression was low in developing male germ cells but was highly expressed in round spermatids. In developing female germ cells undergoing entry into meiosis, it increased 10-fold. Tob1 was also highly expressed in round spermatids and in oocytes in all stages of folliculogenesis. Notably, a marker for P-bodies, Dcp-2, was also highly expressed in round spermatids and all oocyte stages examined. The cytoplasmic presence of Tob1 protein in round spermatids and oocytes and the association of Tob1 protein with Dcp2 in both cell types suggest that Tob1 protein plays a role in post-transcriptional mechanisms.
Asunto(s)
Proteínas Portadoras/biosíntesis , Células Germinales Embrionarias/metabolismo , Endorribonucleasas/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , Espermatogonias/metabolismo , Animales , Biomarcadores/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oogénesis/fisiología , Ovario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermatogénesis/fisiología , Testículo/metabolismoRESUMEN
Aberrant deposition of fat including free fatty acids in the liver often causes damage to hepatocytes, namely lipotoxicity, which is a key pathogenic event in the development and progression of fatty liver diseases. This study demonstrates a pivotal role of sphingosine kinase 1 (SphK1) in protecting hepatocytes from lipotoxicity. Exposure of primary murine hepatocytes to palmitate resulted in dose-dependent cell death, which was enhanced significantly in Sphk1-deficient cells. In keeping with this, expression of dominant-negative mutant SphK1 also markedly promoted palmitate-induced cell death. In contrast, overexpression of wild-type SphK1 profoundly protected hepatocytes from lipotoxicity. Mechanistically, the protective effect of SphK1 is attributable to suppression of ER stress-mediated pro-apoptotic pathways, as reflected in the inhibition of IRE1α activation, XBP1 splicing, JNK phosphorylation, and CHOP induction. Of note, SphK1 inhibited the IRE1α pathway by reducing IRE1α expression at the transcriptional level. Moreover, S1P mimicked the effect of SphK1, suppressing IRE1α expression in a receptor-dependent manner. Furthermore, enforced overexpression of IRE1α significantly blocked the protective effect of SphK1 against lipotoxicity. Therefore, this study provides new insights into the role of SphK1 in hepatocyte survival and uncovers a novel mechanism for protection against ER stress-mediated cell death.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Endorribonucleasas/biosíntesis , Inhibidores Enzimáticos/efectos adversos , Hepatocitos/metabolismo , Ácido Palmítico/efectos adversos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/genética , Inhibidores Enzimáticos/farmacología , Hepatocitos/patología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Ácido Palmítico/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Serina-Treonina Quinasas/genética , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Proteína 1 de Unión a la X-BoxRESUMEN
We evaluated the effects of down-regulated heme oxygenase (HO)-1 expression on the proliferation of the acute myelocytic leukemia Kasumi-1 cell line by using the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) in combination with daunorubicin (DNR), and evaluated the mechanism. The proliferation rates of cells treated with 10 mg/mL DNR and 10 mM ZnPPIX individually or in combination for different time periods were detected using the MTT assay. The apoptotic outcomes of the blank control, ZnPPIX, DNR, and ZnPPIX groups in combination with the DNR group were detected by flow cytometry. The expression of HO-1, activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α mRNA and proteins were detected by fluorescent quantitative real-time polymerase chain reaction and western blotting, respectively. Combined administration inhibited the cells most potently and time-dependently, decreased the expression of HO-1, and significantly increased the expression of activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α expression levels. The cell apoptotic rates in the blank control, DNR, ZnPPIX, and combined administration groups were 8.32 ± 0.53, 39.16 ± 1.46, 10.46 ± 0.88, and 56.26 ± 2.24%, respectively. Inhibiting HO-1 expression can enhance the damaging effects of DNR on Kasumi-1 cells, providing experimental evidence for the improvement of therapeutic effects on acute myelocytic leukemia in clinical practice.
Asunto(s)
Factor de Transcripción Activador 4/biosíntesis , Endorribonucleasas/biosíntesis , Hemo-Oxigenasa 1/biosíntesis , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Factor de Transcripción CHOP/biosíntesis , Factor de Transcripción Activador 4/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Endorribonucleasas/genética , Inhibidores Enzimáticos/administración & dosificación , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/genéticaRESUMEN
Radiation therapy is the most widely used treatment for patients with cervical cancer. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis and sensitizes tumor cells to radiotherapy, which reportedly induces ER stress in cells. Classical key tumor suppressor p53 is involved in the response to a variety of cellular stresses, including those incurred by ionizing irradiation. A recent study demonstrated that small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) increased the radiosensitivity of tumor cells expressing mutant p53 (mtp53). In the present study, we explored the effects and the underlying mechanisms of RITA in regards to the radiosensitivity and ER stress in mtp53-expressing human cervix cancer cells. Treatment with 1 µM of RITA for 24 h before irradiation markedly decreased survival and increased apoptosis in C-33A and HT-3 cells; the effects were not significantly altered by knockdown of p53. In the irradiated C-33A and HT-3 cells, RITA significantly increased the expression of IRE1α, the spliced XBP1 mRNA level, as well as apoptosis; the effects were abolished by knockdown of IRE1α. Transcriptional pulse-chase assays revealed that RITA significantly increased the stability of IRE1α mRNA in the irradiated C-33A and HT-3 cells. In contrast, the same RITA treatment did not show any significant effect on sham-irradiated cells. In conclusion, the present study provides initial evidence that RITA upregulates the expression level of IRE1α by increasing the stability of IRE1α mRNA in irradiated mtp53-expressing cervical cancer cells; the effect leads to enhanced IRE1α/XBP1 ER stress signaling and increased apoptosis in the cells. The present study offers novel insight into the pharmacological potential of RITA in the radiotherapy for cervical cancer.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Endorribonucleasas/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Factores de Transcripción/biosíntesis , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Mutación , Proteínas de Neoplasias/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Radiación , Tolerancia a Radiación , Factores de Transcripción del Factor Regulador X , Transducción de Señal/efectos de la radiación , Factores de Transcripción/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/radioterapia , Proteína 1 de Unión a la X-BoxRESUMEN
Sorafenib is one of the preferred drugs for the treatment of advanced primary hepatocellular carcinoma (HCC). However, its side-effects and acquired resistance limit its use. The unfolded protein response (UPR) induced by chemotherapeutics has been demonstrated to be required for tumor cells to maintain malignancy and therapy resistance. Activation of the IRE1/XBP1 pathway during the UPR is important for tumor survival under pathophysiological conditions. In the present study, we found that the UPR was activated and RACK1 was overexpressed in three human HCC cell lines and in HCC samples. Activation of the IRE1/XBP1 signaling pathway plays a protective role when HCC cells encounter endoplasmic reticulum (ER) stress due to in vitro sorafenib treatment. We then found that the interaction between IRE1 and RACK1 was essential for the activation of IRE1 signaling in sorafenib-treated cells. Exogenous overexpression of RACK1 enhanced the phosphorylation level of IRE1 and increased XBP1 mRNA splicing activity, which protected the HCC cells from sorafenib-induced apoptosis. However, the re-expression of RACK1 led HCC cells to regain susceptibility to sorafenib-induced apoptosis. Taken together, the present study suggests that the RACK1/IRE1 complex may contribute to activation of the UPR in HCC cells. Targeting RACK1 in combination with sorafenib administration is a potential strategy for clinical trials of advanced HCC treatment.