RESUMEN
The skin, the organ with the largest surface area in the body, is the most susceptible to chemical exposure from the external environment. In this study, we aimed to establish an in vitro skin toxicity monitoring system that utilizes the mechanism of stress granule (SG) formation induced by various cellular stresses. In HaCaT cells, a keratinocyte cell line that comprises the human skin, a green fluorescent protein (GFP) was knocked in at the C-terminal genomic locus of Ras GTPase-activating protein-binding protein 1 (G3BP1), a representative component of SGs. The G3BP1-GFP knock-in HaCaT cells and wild-type (WT) HaCaT cells formed SGs containing G3BP1-GFP upon exposure to arsenite and household chemicals, such as bisphenol A (BPA) and benzalkonium chloride (BAC), in real-time. In addition, the exposure of G3BP1-GFP knock-in HaCaT cells to BPA and BAC promoted the phosphorylation of eukaryotic initiation factor 2 alpha and protein kinase R-like endoplasmic reticulum kinase, which are cell signaling factors involved in SG formation, similar to WT HaCaT cells. In conclusion, this novel G3BP1-GFP knock-in human skin cell system can monitor SG formation in real-time and be utilized to assess skin toxicity to various substances.
Asunto(s)
Gránulos Citoplasmáticos , ADN Helicasas , Proteínas Fluorescentes Verdes , Queratinocitos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Humanos , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Arsenitos/toxicidad , Piel/efectos de los fármacos , Piel/metabolismo , Técnicas de Sustitución del Gen , Genes Reporteros/efectos de los fármacos , Fenoles/toxicidad , Células HaCaT , Fosforilación , Compuestos de Bencidrilo/toxicidad , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Pruebas de Toxicidad/métodosRESUMEN
Cathepsin X is a lysosomal peptidase that is involved in tumour progression and represents a potential target for therapeutic interventions. In addition, it regulates important functions of immune cells and is implicated in the modulation of tumour cell-immune cell crosstalk. Selective cathepsin X inhibitors have been proposed as prospective antitumour agents to prevent cancer progression; however, their impact on the antitumour immune response has been overlooked. Previous studies indicate that the migration and adhesion of T cells and dendritic cells are affected by diminished cathepsin X activity. Meanwhile, the influence of cathepsin X inhibition on natural killer (NK) cell function has not yet been explored. Here, we examined the localization patterns of cathepsin X and the role of its inhibitors on the cytotoxicity of cell line NK-92, which is used for adoptive cellular immunotherapy in cancer patients. NK-92 cells depend on lymphocyte function-associated antigen 1 (LFA-1) to form stable immunoconjugates with target cells, providing, in this way, optimal cytotoxicity. Since LFA-1 is a substrate for cathepsin X activity in other types of cells, we hypothesized that cathepsin X could disturb the formation of NK-92 immunoconjugates. Thus, we employed cathepsin X reversible and irreversible inhibitors and evaluated their effects on the NK-92 cell interactions with target cells and on the NK-92 cell cytotoxicity. We show that cathepsin X inhibition does not impair stable conjugate formation or the lytic activity of NK-92 cells. Similarly, the conjugate formation between Jurkat T cells and target cells was not affected by cathepsin X activity. Unlike in previous migration and adhesion studies on T cells, in NK-92 cells cathepsin X was not co-localized with LFA-1 at the plasma membrane but was, rather, redistributed to the cytotoxic granules and secreted during degranulation.
Asunto(s)
Catepsinas/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Sinapsis/efectos de los fármacos , Antineoplásicos/farmacología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Humanos , Inmunoterapia Adoptiva/métodos , Células Jurkat , Células K562 , Neoplasias/tratamiento farmacológico , Linfocitos T/efectos de los fármacosRESUMEN
We identified small-molecule enhancers of cellular stress granules by observing molecular crowding of proteins and RNAs in a time-dependent manner. Hit molecules sensitized the IRF3-mediated antiviral mechanism in the presence of poly(I:C) and inhibited the replication of SARS-CoV-2 by inducing stress granule formation. Thus, modulating multimolecular crowding can be a promising strategy against SARS-CoV-2.
Asunto(s)
Antivirales/farmacología , Benzopiranos/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Pirazoles/farmacología , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Antivirales/química , Benzopiranos/química , Línea Celular Tumoral , Chlorocebus aethiops , Gránulos Citoplasmáticos/metabolismo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Humanos , Factor 3 Regulador del Interferón/metabolismo , Lopinavir/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Poli I-C/farmacología , Pirazoles/química , Relación Estructura-Actividad , Células VeroRESUMEN
Stress granules (SGs) are ribonucleoprotein functional condensates that form under stress conditions in all eukaryotic cells. Although their stress-survival function is far from clear, SGs have been implicated in the regulation of many vital cellular pathways. Consequently, SG dysfunction is thought to be a mechanistic point of origin for many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Additionally, SGs are thought to play a role in pathogenic pathways as diverse as viral infection and chemotherapy resistance. There is a growing consensus on the hypothesis that understanding the mechanistic regulation of SG physical properties is essential to understanding their function. Although the internal dynamics and condensation mechanisms of SGs have been broadly investigated, there have been fewer investigations into the timing of SG formation and clearance in live cells. Because the lifetime of SG persistence can be a key factor in their function and tendency toward pathological dysregulation, SG clearance mechanisms deserve particular attention. Here we show that resveratrol and its analogues piceatannol, pterostilbene, and 3,4,5,4'-tetramethoxystilbene induce G3BP-dependent SG formation with atypically rapid clearance kinetics. Resveratrol binds to G3BP, thereby reducing its protein-protein association valency. We suggest that altering G3BP valency is a pathway for the formation of uniquely transient SGs.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Resveratrol/farmacología , Gránulos de Estrés/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Cinética , Proteínas de Unión a Poli-ADP-Ribosa/efectos de los fármacos , ARN Helicasas/efectos de los fármacos , Proteínas con Motivos de Reconocimiento de ARN/efectos de los fármacos , Ribonucleoproteínas/metabolismo , Gránulos de Estrés/efectos de los fármacosRESUMEN
Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.
Asunto(s)
Gránulos Citoplasmáticos/genética , Retículo Endoplásmico/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Respuesta de Proteína Desplegada , Biomarcadores/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Citosol/efectos de los fármacos , Citosol/metabolismo , Dactinomicina/farmacología , Diterpenos/farmacología , Ditiotreitol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Compuestos Epoxi/farmacología , Expresión Génica , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Biogénesis de Organelos , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Fenantrenos/farmacología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Imagen Individual de Molécula , Estrés Fisiológico/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
Cellular senescence is recognized as a phenomenon wherein a proliferative cell undergoes a permanent growth arrest. The accumulation of senescent cells over time can become harmful and result in diseases and physiological decline. Plasminogen activator inhibitor (PAI-1) is considered as a critical marker and mediator of cellular senescence. The formation of stress granules (SGs) could prevent senescence through the sequestration of PAI-1, and we previously suggested that exogenous carbon monoxide (CO) could induce SG assembly via integrated stress response (ISR). Although CO is known to possess anti-inflammatory, antioxidative, and antiapoptotic properties, whether it exerts antisenescent effect is still not well defined. Here, to address whether CO-induced SGs could protect against cellular senescence, we first treated lung fibroblasts with bleomycin (BLM) to establish DNA damage-induced cellular senescence, and observed a significant increase of several hallmarks of senescence through SA-ß-gal staining, immunofluorescence, qRT-PCR, and Western blot assay. However, pre- and posttreatment of CO could remarkably attenuate these senescent phenotypes. According to our immunofluorescence results, CO-induced SGs could inhibit BLM-induced cellular senescence via sequestration of PAI-1, while it was abolished after the cotreatment of ISR inhibitor (ISRIB) due to the inhibition of SG assembly. Overall, our results proposed a novel role of CO in suppressing bleomycin-induced lung fibroblast senescence through the assembly of SGs.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Gasotransmisores/farmacología , Gránulos de Estrés/efectos de los fármacos , Apoptosis/efectos de los fármacos , Bleomicina/farmacología , Gránulos Citoplasmáticos/metabolismo , Fibroblastos/metabolismo , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismoRESUMEN
Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , ß1 , and ß4 expression suggested that integrin α2 ß1 , α5 ß1 , α6 ß1 , and α6 ß4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 ß1 , collagen interacting with integrin α2 ß1 , and laminin interacting with integrin α6 ß1 and α6 ß4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.
Asunto(s)
Citoplasma/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Oocitos/citología , Sefarosa/farmacología , Adenosina Trifosfato/metabolismo , Animales , Blastocisto/efectos de los fármacos , Proteína Morfogenética Ósea 15 , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/metabolismo , Citoplasma/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Técnicas de Maduración In Vitro de los Oocitos , Integrinas/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Partenogénesis/efectos de los fármacos , Subunidades de Proteína/metabolismo , PorcinosRESUMEN
Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.
Asunto(s)
Apoptosis , Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Respuesta al Choque Térmico , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Receptores de Estrógenos/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Técnicas de Silenciamiento del Gen , Glicoles/farmacología , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Factores de Transcripción del Choque Térmico/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Modelos Biológicos , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismo , Temperatura , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin -which specifically cleavages VAMP1, VAMP2 or VAMP3- inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 -but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Exocitosis , Regulación de la Expresión Génica/efectos de los fármacos , Oocitos/fisiología , Proteínas SNARE/metabolismo , Toxina Tetánica/farmacología , Animales , Gránulos Citoplasmáticos/efectos de los fármacos , Femenino , Ratones , Neurotoxinas/farmacología , Oocitos/citología , Oocitos/efectos de los fármacos , Proteínas SNARE/genéticaRESUMEN
2,4-DCP (2,4-dichlorophenol) is an environmental estrogen that is ubiquitously distributed in the environment and widely used to produce herbicides and pharmaceutical intermediates. Although 2,4-DCP is suspected to have endocrine disruption, the reproductive toxicity of 2,4-DCP in mammals has not been adequately assessed. In the present study, we examined the effect of 2,4-DCP on the fertility of mouse eggs. The data showed that oral administration of 2,4-DCP (180 mg/kg/day for 7 days) compromises the fertilization rate of mouse oocytes. To further analyze the mechanism by which 2,4-DCP decreases fertilization, the key regulators and events during fertilization of mouse eggs were investigated. We found that the dynamics of cortical granules (CGs) were disrupted by showing the redistribution of CG free domain in 2,4-DCP-administered oocytes. This abnormality perturbed the sperm binding site in the zona pellucida (ZP) and dramatically reduced the number of sperm binding to the ZP of 2,4-DCP-administered oocytes. In addition, the abundance of Juno, a sperm receptor on the egg membrane, was also decreased and its distribution was mislocated in 2,4-DCP-administered oocytes. Finally, we validated that the defects of fertilization participants and events caused by 2,4-DCP might be mediated by the increased level of reactive oxygen species-induced apoptosis of oocytes. Therefore, we demonstrate that 2,4-DCP compromises the fertilization ability of mouse oocytes via inducing oxidative stress.
Asunto(s)
Clorofenoles/toxicidad , Gránulos Citoplasmáticos/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Oocitos/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Femenino , Fertilización In Vitro , Ratones Endogámicos ICR , Oocitos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Ensuring that oocytes are fertilized by a single sperm during broadcast spawning is crucial for the fertilization success of many marine invertebrates. Although the adverse impacts of ocean acidification (OA) on various marine species have been revealed in recent years, its impact on polyspermy and the underlying mechanisms involved remain largely unknown. Therefore, in the present study, the effect of OA on polyspermy risk was assessed in a broadcast spawning bivalve, Tegillarca granosa. In addition, the impacts of OA on the two polyspermy blocking processes, the fast block (membrane depolarization) and the permanent block (cortical reaction), were investigated. The results show that the exposure of oocytes to two future OA scenarios (pH 7.8 and pH 7.4) leads to significant increases in polyspermy risk, about 1.70 and 2.38 times higher than the control, respectively. The maximum change in the membrane potential during oocyte membrane depolarization markedly decreased to 15.79 % (pH 7.8) and 34.06 % (pH 7.4) of the control value. Moreover, the duration of oocyte membrane depolarization was significantly reduced to approximately 63.38 % (pH 7.8) and 21.91 % (pH 7.4) of the control. In addition, cortical granule exocytosis, as well as microfilament migration, were significantly arrested by OA treatment. Exposure to future OA scenarios also led to significant reductions in the ATP and Ca2+ content of the oocytes, which may explain the hampered polyspermy blocking. Overall, the present study suggests that OA may significantly increase polyspermy risk in T. granosa by inhibiting membrane depolarization and arresting cortical granule exocytosis.
Asunto(s)
Ácidos/química , Bivalvos/fisiología , Gránulos Citoplasmáticos/metabolismo , Exocitosis , Potenciales de la Membrana/fisiología , Océanos y Mares , Espermatozoides/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Exocitosis/efectos de los fármacos , Femenino , Fertilización/efectos de los fármacos , Concentración de Iones de Hidrógeno , Masculino , Potenciales de la Membrana/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Helicasas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Esclerosis Tuberosa/metabolismo , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/química , Evolución Molecular , Femenino , Humanos , Insulina/farmacología , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa/química , ARN Helicasas/química , Proteínas con Motivos de Reconocimiento de ARN/química , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Pez Cebra/metabolismoRESUMEN
BACKGROUND: Ras-GTPase activating protein SH3-domain-binding proteins (G3BP) are a small family of RNA-binding proteins implicated in regulating gene expression. Changes in expression of G3BPs are correlated to several cancers including thyroid, colon, pancreatic and breast cancer. G3BPs are important regulators of stress granule (SG) formation and function. SG are ribonucleoprotein (RNP) particles that respond to cellular stresses to triage mRNA resulting in transcripts being selectively degraded, stored or translated resulting in a change of gene expression which confers a survival response to the cell. These changes in gene expression contribute to the development of drug resistance. Many RNA viruses, including Chikungunya (and potentially Coronavirus), dismantle SG so that the cell cannot respond to the viral infection. Non-structural protein 3 (nsP3), from the Chikungunya virus, has been shown to translocate G3BP away from SG. Interestingly in cancer cells, the formation of SG is correlated to drug-resistance and blocking SG formation has been shown to reestablish the efficacy of the anticancer drug bortezomib. METHODS: Chikungunya nsP3 was transfected into breast cancer cell lines T47D and MCF7 to disrupt SG formation. Changes in the cytotoxicity of bortezomib were measured. RESULTS: Bortezomib cytotoxicity in breast cancer cell lines changed with a 22 fold decrease in its IC50 for T47D and a 7 fold decrease for MCF7 cells. CONCLUSIONS: Chikungunya nsP3 disrupts SG formation. As a result, it increases the cytotoxicity of the FDA approved drug, bortezomib. In addition, the increased cytotoxicity appears to correlate to improved bortezomib selectivity when compared to control cell lines.
Asunto(s)
Bortezomib/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/genética , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Fiebre Chikungunya/metabolismo , Fiebre Chikungunya/patología , Virus Chikungunya/metabolismo , Chlorocebus aethiops , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/patología , ADN Helicasas/genética , Regulación hacia Abajo , Resistencia a Antineoplásicos , Femenino , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Transfección , Células Vero , Proteínas no Estructurales Virales/administración & dosificación , Proteínas no Estructurales Virales/genéticaRESUMEN
Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.
Asunto(s)
Adenosina/análogos & derivados , Gránulos Citoplasmáticos/metabolismo , Citoprotección , Estrés Fisiológico , Adenosina/metabolismo , Arsenitos/toxicidad , Gránulos Citoplasmáticos/efectos de los fármacos , Citoprotección/efectos de los fármacos , Células HeLa , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Metilación/efectos de los fármacos , Modelos Biológicos , Conformación Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Estrés Fisiológico/efectos de los fármacos , ARNt Metiltransferasas/metabolismoRESUMEN
Septins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbß3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbß3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.
Asunto(s)
Plaquetas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Activación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Septinas/deficiencia , Animales , Plaquetas/efectos de los fármacos , Venenos de Crotálidos/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Exocitosis , Femenino , Fibrinógeno/metabolismo , Genotipo , Lectinas Tipo C , Lisosomas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria , Septinas/sangre , Septinas/genética , Trombina/metabolismoRESUMEN
A comprehensive overview of the interplay between glucocorticoids (GCs) and adult hippocampal neurogenesis (AHN) is presented, particularly, in the context of a diseased brain. The effectors of GCs in the dentate gyrus neurogenic niche of the hippocampal are reviewed, and the consequences of the GC signaling on the generation and integration of new neurons are discussed. Recent findings demonstrating how GC signaling mediates impairments of the AHN in various brain pathologies are overviewed. GC-mediated effects on the generation and integration of adult-born neurons in the hippocampal dentate gyrus depend on the nature, severity, and duration of the acting stress factor. GCs realize their effects on the AHN primarily via specific glucocorticoid and mineralocorticoid receptors. Disruption of the reciprocal regulation between the hypothalamic-pituitary-adrenal (HPA) axis and the generation of the adult-born granular neurons is currently considered to be a key mechanism implicating the AHN into the pathogenesis of numerous brain diseases, including those without a direct hippocampal damage. These alterations vary from reduced proliferation of stem and progenitor cells to increased cell death and abnormalities in morphology, connectivity, and localization of young neurons. Although the involvement of the mutual regulation between the HPA axis and the AHN in the pathogenesis of cognitive deficits and mood impairments is evident, several unresolved critical issues are stated. Understanding the details of GC-mediated mechanisms involved in the alterations in AHN could enable the identification of molecular targets for ameliorating pathology-induced imbalance in the HPA axis/AHN mutual regulation to conquer cognitive and psychiatric disturbances.
Asunto(s)
Encefalopatías/fisiopatología , Glucocorticoides/farmacología , Hipocampo/fisiopatología , Neurogénesis/efectos de los fármacos , Animales , Gránulos Citoplasmáticos/efectos de los fármacos , Giro Dentado/fisiopatología , Hipocampo/crecimiento & desarrollo , Humanos , Sistema Hipotálamo-Hipofisario/fisiopatología , Sistema Hipófiso-Suprarrenal/fisiopatologíaRESUMEN
Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. However, dysregulated LLPS can also facilitate aberrant phase transitions and lead to protein aggregation and disease. Accordingly, there is great interest in identifying small molecules that modulate LLPS. Here, we demonstrate that 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and similar compounds are potent biphasic modulators of protein LLPS. Depending on context, bis-ANS can both induce LLPS de novo as well as prevent formation of homotypic liquid droplets. Our study also reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. These findings may provide a foundation for the rational design of small molecule modulators of LLPS with therapeutic value.
Asunto(s)
Naftalenosulfonatos de Anilina/química , Naftalenosulfonatos de Anilina/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Proteínas de Unión al ADN/química , Transición de Fase , Naftalenosulfonatos de Anilina/toxicidad , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Citosol/metabolismo , Células HCT116 , Heparina/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Poli A/química , Dominios Proteicos/genéticaRESUMEN
Stress granules (SGs) are non-membranous cytosolic protein-RNA aggregates that process mRNAs through stalled translation initiation in response to cellular stressors and in disease. DEAD-Box RNA helicase 3 (DDX3) is an active target of drug development for the treatment of viral infections, cancers, and neurodegenerative diseases. DDX3 plays a critical role in RNA metabolism, including SGs, but the role of DDX3 enzymatic activity in SG dynamics is not well understood. Here, we address this question by determining the effects of DDX3 inhibition on the dynamics of SG assembly and disassembly. We use two small molecule inhibitors of DDX3, RK33 and 16D, with distinct inhibitory mechanisms that target DDX3's ATPase activity and RNA helicase site, respectively. We find that both DDX3 inhibitors reduce the assembly of SGs, with a more pronounced reduction from RK-33. In contrast, both compounds only marginally affect the disassembly of SGs. RNA-mediated knockdown of DDX3 caused a similar reduction in SG assembly and minimal effect on SG disassembly. Collectively, these results reveal that the enzymatic activity of DDX3 is required for the assembly of SGs and pharmacological inhibition of DDX3 could be relevant for the treatment of SG-dependent pathologies.
Asunto(s)
Azepinas/farmacología , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/metabolismo , Imidazoles/farmacología , Línea Celular Tumoral , Gránulos Citoplasmáticos/efectos de los fármacos , Humanos , ARN Interferente Pequeño/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismoRESUMEN
creativecommons.org/licenses/by-nc-sa/3.0/. Cells assemble stress granules (SGs) to protect their RNAs from exposure to harmful chemical reactions induced by environmental stress. These SGs release RNAs, which resume translation once the stress is relieved. During stem cell differentiation, gene expression is altered to allow cells to adopt various functional and morphological features necessary to differentiate. This process induces stress within a cell, and cells that cannot overcome this stress die. Here, we investigated the role of SGs in the progression of stem cell differentiation. SGs aggregated during the neuronal differentiation of human bone marrow-mesenchymal stem cells, and not in cell lines that could not undergo differentiation. SGs were observed between one and three hours post-induction; RNA translation was restrained at the same time. Immediately after disassembly of SGs, the expression of the neuronal marker neurofilament-M (NFM) gradually increased. Assembled SGs that persisted in cells were exposed to salubrinal, which inhibited the dephosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), and in eIF2α/S51D mutant cells. When eIF2α/S51A mutant cells differentiated, SGs were not assembled. In all experiments, the disruption of SGs was accompanied by delayed NF-M expression and the number of neuronally differentiated cells was decreased. Decreased differentiation was accompanied by decreased cell viability, indicating the necessity of SGs for preventing cell death during neuronal differentiation. Collectively, these results demonstrate the essential role of SGs during the neuronal differentiation of stem cells.
Asunto(s)
Cinamatos/farmacología , Gránulos Citoplasmáticos/metabolismo , Células Madre Mesenquimatosas/citología , Neuronas/citología , Tiourea/análogos & derivados , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Gránulos Citoplasmáticos/efectos de los fármacos , ADN Helicasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Antígeno Intracelular 1 de las Células T/metabolismo , Tiourea/farmacologíaRESUMEN
Stress granules (SGs) are assemblies of mRNA and proteins that form from mRNAs stalled in translation initiation in response to stress. Chronic stress might even induce formation of cytotoxic pathological SGs. SGs participate in various biological functions including response to apoptosis, inflammation, immune modulation, and signalling pathways; moreover, SGs are involved in pathogenesis of neurodegenerative diseases, viral infection, aging, cancers and many other diseases. Emerging evidence has shown that small molecules can affect SG dynamics, including assembly, disassembly, maintenance and clearance. Thus, targeting SGs is a potential therapeutic strategy for the treatment of human diseases and the promotion of health. The established methods for detecting SGs provided ready tools for large-scale screening of agents that alter the dynamics of SGs. Here, we describe the effects of small molecules on SG assembly, disassembly, and their roles in the disease. Moreover, we provide perspective for the possible application of small molecules targeting SGs in the treatment of human diseases.