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BACKGROUND: The global outbreak of COVID-19 caused by the SARS-CoV-2 has led to millions of deaths. This unanticipated emergency has prompted virologists across the globe to delve deeper into the intricate dynamicity of the host-virus interface with an aim to identify antiviral targets and elucidate host and viral determinants of severe disease. AIM: The present study was undertaken to analyse the role of histone deacetylase 6 (HDAC6) in regulating SARS-CoV-2 infection. RESULTS: Gradual increase in HDAC6 expression was observed in different SARS-CoV-2-permissive cell lines following SARS-CoV-2 infection. The SARS-CoV-2 nucleocapsid protein (N protein) was identified as the primary viral factor responsible for upregulating HDAC6 expression. Downregulation of HDAC6 using shRNA or a specific inhibitor tubacin resulted in reduced viral replication suggesting proviral role of its deacetylase activity. Further investigations uncovered the interaction of HDAC6 with stress granule protein G3BP1 and N protein during infection. HDAC6-mediated deacetylation of SARS-CoV-2 N protein was found to be crucial for its association with G3BP1. CONCLUSION: This study provides valuable insights into the molecular mechanisms underlying the disruption of cytoplasmic stress granules during SARS-CoV-2 infection and highlights the significance of HDAC6 in the process.
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COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Histona Desacetilasa 6 , SARS-CoV-2 , Replicación Viral , Histona Desacetilasa 6/metabolismo , Histona Desacetilasa 6/genética , Humanos , SARS-CoV-2/fisiología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , COVID-19/virología , COVID-19/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Acetilación , Línea Celular , Chlorocebus aethiops , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células Vero , Animales , Interacciones Huésped-Patógeno , Proteínas de Unión a Poli-ADP-Ribosa , ADN Helicasas , ARN HelicasasRESUMEN
Profilin is an actin monomer-binding protein whose role in actin polymerization has been studied for nearly 50 years. While its principal biochemical features are now well understood, many questions remain about how profilin controls diverse processes within the cell. Dysregulation of profilin has been implicated in a broad range of human diseases, including neurodegeneration, inflammatory disorders, cardiac disease, and cancer. For example, mutations in the profilin 1 gene (PFN1) can cause amyotrophic lateral sclerosis (ALS), although the precise mechanisms that drive neurodegeneration remain unclear. While initial work suggested proteostasis and actin cytoskeleton defects as the main pathological pathways, multiple novel functions for PFN1 have since been discovered that may also contribute to ALS, including the regulation of nucleocytoplasmic transport, stress granules, mitochondria, and microtubules. Here, we will review these newly discovered roles for PFN1, speculate on their contribution to ALS, and discuss how defects in actin can contribute to these processes. By understanding profilin 1's involvement in ALS pathogenesis, we hope to gain insight into this functionally complex protein with significant influence over cellular physiology.
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induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1ß + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.
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Various stress conditions, such as heat stress (HS) and oxidative stress, can cause biomolecular condensates represented by stress granules (SGs) via liquid-liquid phase separation. We have previously shown that Hsp90 forms aggregates in response to HS and that Hsp90 aggregates transiently co-localize with SGs as visualized by Pabp. Here, we showed that arsenite, one of the well-described SG-inducing stimuli, induces Hsp90 aggregates distinct from conventional SGs in fission yeast. Arsenite induced Hsp90 granules in a dose-dependent manner, and these granules were significantly diminished by the co-treatment with a ROS scavenger N-acetyl cysteine (NAC), indicating that ROS are required for the formation of Hsp90 granules upon arsenite stress. Notably, Hsp90 granules induced by arsenite do not overlap with conventional SGs as represented by eIF4G or Pabp, while HS-induced Hsp90 granules co-localize with SGs. Nrd1, an RNA-binding protein known as a HS-induced SG component, was recruited into Hsp90 aggregates but not to the conventional SGs upon arsenite stress. The non-phosphorylatable eIF2α mutants significantly delayed the Hsp90 granule formation upon arsenite treatment. Importantly, inhibition of Hsp90 by geldanamycin impaired the Hsp90 granule formation and reduced the arsenite tolerance. Collectively, arsenite stimulates two types of distinct aggregates, namely conventional SGs and a novel type of aggregates containing Hsp90 and Nrd1, wherein Hsp90 plays a role as a center for aggregation, and stress-specific compartmentalization of biomolecular condensates.
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The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
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Citoplasma , Polirribosomas , Ribonucleoproteínas , Polirribosomas/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulación de Dinámica Molecular , ARN Mensajero/metabolismo , ARN Mensajero/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Condensados Biomoleculares/metabolismo , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genéticaRESUMEN
AIM: Post-transcriptional modifications and their specific mechanisms are the focus of research on the regulation of myocardial damage. Stress granules (SGs) can inhibit the inflammatory response by inhibiting the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. This study investigated whether alkylation repair homologue protein 5 (ALKBH5) could affect myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion injury (IRI) through the cGAS-STING pathway via SGs. METHODS: A diabetes ischaemia-reperfusion rat model and a high glucose hypoxia/reoxygenation cell model were established. Adeno-associated virus (AAV) and lentivirus (LV) were used to overexpress ALKBH5, while the SG agonist arsenite (Ars) and the SG inhibitor anisomycin were used as interventions. Then, the levels of apoptosis and related indicators in the cell and rat models were measured. RESULTS: In the in vivo experiment, compared with the normal sham group, the degree of myocardial tissue damage, creatine kinase-MB and cardiac troponin I in serum, and myocardial apoptosis, the infarcted area of myocardium, and the level of B-cell lymphoma 2 associated X protein, cGAS-STING pathway and inflammatory factors in the diabetes ischaemia-reperfusion group were significantly increased. However, the expression of SGs and the levels of ALKBH5, rat sarcoma-GTPase-activating protein-binding protein 1, T-cell intracellular antigen-1 and Bcl2 were significantly decreased. After AAV-ALKBH5 intervention, the degree of myocardial tissue damage, degree of myocardial apoptosis, and extent of myocardial infarction in myocardial tissue were significantly decreased. In the in vitro experiment, compared with those in the normal control group, the levels of lactate dehydrogenase, inflammation and apoptosis were significantly greater, and cell viability and the levels of ALKBH5 and SGs were decreased in the high glucose and hypoxia/reoxygenation groups. In the high glucose hypoxia/reoxygenation cell model, the degree of cell damage, inflammation, and apoptosis was greater than those in the high glucose and hypoxia/reoxygenation models, and the levels of ALKBH5 and SGs were further decreased. LV-ALKBH5 and Ars alleviated the degree of cell damage and inhibited inflammation and cell apoptosis. The inhibition of SGs could partly reverse the protective effect of LV-ALKBH5. The cGAS agonist G140 antagonized the inhibitory effects of the SG agonist Ars on cardiomyocyte apoptosis, inflammation and the cGAS-STING pathway. CONCLUSION: Both ALKBH5 and SGs inhibited myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion. Mechanistically, ALKBH5 might inhibit the apoptosis of cardiomyocytes by promoting the expression of SGs through the cGAS-STING pathway.
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Apoptosis , Daño por Reperfusión Miocárdica , Transducción de Señal , Animales , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Ratas , Masculino , Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/patología , Ratas Sprague-Dawley , Diabetes Mellitus Experimental/metabolismoRESUMEN
Neurologic manifestations are an immediate consequence of SARS-CoV-2 infection, the etiologic agent of COVID-19, which, however, may also trigger long-term neurological effects. Notably, COVID-19 patients with neurological symptoms show elevated levels of biomarkers associated with brain injury, including Tau proteins linked to Alzheimer's pathology. Studies in brain organoids revealed that SARS-CoV-2 alters the phosphorylation and distribution of Tau in infected neurons, but the mechanisms are currently unknown. We hypothesize that these pathological changes are due to the recruitment of Tau into stress granules (SGs) operated by the nucleocapsid protein (NCAP) of SARS-CoV-2. To test this hypothesis, we investigated whether NCAP interacts with Tau and localizes to SGs in hippocampal neurons in vitro and in vivo. Mechanistically, we tested whether SUMOylation, a posttranslational modification of NCAP and Tau, modulates their distribution in SGs and their pathological interaction. We found that NCAP and Tau colocalize and physically interact. We also found that NCAP induces hyperphosphorylation of Tau and causes cognitive impairment in mice infected with NCAP in their hippocampus. Finally, we found that SUMOylation modulates NCAP SG formation in vitro and cognitive performance in infected mice. Our data demonstrate that NCAP induces Tau pathological changes both in vitro and in vivo. Moreover, we demonstrate that SUMO2 ameliorates NCAP-induced Tau pathology, highlighting the importance of the SUMOylation pathway as a target of intervention against neurotoxic insults, such as Tau oligomers and viral infection.
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COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Hipocampo , Neuronas , SARS-CoV-2 , Sumoilación , Proteínas tau , Proteínas tau/metabolismo , Animales , Ratones , Humanos , Hipocampo/metabolismo , Hipocampo/patología , COVID-19/metabolismo , COVID-19/patología , COVID-19/virología , SARS-CoV-2/patogenicidad , SARS-CoV-2/metabolismo , Fosforilación , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Gránulos de Estrés/metabolismo , Ratones Endogámicos C57BL , Fosfoproteínas/metabolismo , Masculino , Proteínas de la Nucleocápside/metabolismo , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Disfunción Cognitiva/virologíaRESUMEN
Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.
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Cuerpos de Inclusión Viral , Antígeno Intracelular 1 de las Células T , Replicación Viral , eIF-2 Quinasa , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Animales , Antígeno Intracelular 1 de las Células T/metabolismo , Antígeno Intracelular 1 de las Células T/genética , Chlorocebus aethiops , Células Vero , Cuerpos de Inclusión Viral/metabolismo , Humanos , Gránulos de Estrés/metabolismo , Cuerpos de Inclusión/metabolismo , Interacciones Huésped-Patógeno , Gránulos Citoplasmáticos/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Separación de FasesRESUMEN
Small extracellular vesicles (sEVs) contain abundant circular RNAs (circRNAs) and are involved in cellular processes, particularly hypoxia. However, the process that packaging of circRNAs into neuronal sEVs under hypoxia is unclear. This study revealed the spatial mechanism of the Fused in Sarcoma protein (FUS) that facilitates the loading of functional circRNAs into sEVs in hypoxia neurons. It is found that FUS translocated from the nucleus to the cytoplasm and is more enriched in hypoxic neuronal sEVs than in normal sEVs. Cytoplasmic FUS formed aggregates with the sEVs marker protein CD63 in cytoplasmic stress granules (SGs) under hypoxic stress. Meanwhile, cytoplasmic FUS recruited of functional cytoplasmic circRNAs to SGs. Upon relief of hypoxic stress and degradation of SGs, cytoplasmic FUS is transported with those circRNAs from SGs to sEVs. Validation of FUS knockout dramatically reduced the recruitment of circRNAs from SGs and led to low circRNA loading in sEVs, which is also confirmed by the accumulation of circRNAs in the cytoplasm. Furthermore, it is showed that the FUS Zf_RanBP domain regulates the transport of circRNAs to sEVs by interacting with hypoxic circRNAs in SGs. Overall, these findings have revealed a FUS-mediated transport mechanism of hypoxia-related cytoplasmic circRNAs loaded into sEVs under hypoxic conditions.
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Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.
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Proteína 1 de la Respuesta de Crecimiento Precoz , Lomustina , ARN Mensajero , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Lomustina/farmacología , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Apoptosis/efectos de los fármacos , Antineoplásicos Alquilantes/farmacologíaRESUMEN
Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts.
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Arabidopsis , Gránulos Citoplasmáticos , Estrés Fisiológico , Arabidopsis/metabolismo , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/química , Proteínas de Arabidopsis/metabolismo , Protoplastos/metabolismo , Nicotiana/metabolismo , Inmunoprecipitación/métodos , Espectrometría de Masas/métodosRESUMEN
The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.
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Citoplasma , Homeostasis , ARN Mensajero , Gránulos de Estrés , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Gránulos de Estrés/metabolismo , Citoplasma/metabolismo , Caperuzas de ARN/metabolismo , Arsenitos/farmacología , Estrés Oxidativo , Transporte Activo de Núcleo Celular , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/genética , Compuestos de Sodio/farmacología , Proteína Exportina 1 , Carioferinas/metabolismo , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Gránulos Citoplasmáticos/metabolismo , Estabilidad del ARN , Núcleo Celular/metabolismo , Línea Celular Tumoral , NucleotidiltransferasasRESUMEN
The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.
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Exposure to toxic molecules from food or oral medications induces toxicity in colon cells that cause various human diseases; however, in vitro monitoring systems for colon cell toxicity are not well established. Stress granules are nonmembranous foci that form in cells exposed to cellular stress. When cells sense toxic environments, they acutely and systemically promote stress granule formation, with Ras GTPase-activating protein-binding protein 1 (G3BP1) acting as a core component to protect their mRNA from abnormal degradation. Here, we knocked in green fluorescent protein (GFP)-coding sequences into the C-terminal region of the G3BP1 gene in a human colon cell line through CRISPR-Cas9-mediated homologous recombination and confirmed the formation of stress granules with the G3BP1-GFP protein in these cells under cellular stress exposure. We demonstrated the formation and dissociation of stress granules in G3BP1-GFP expressing colon cells through real-time monitoring using a fluorescence microscope. Furthermore, we validated the toxicity monitoring system in the established colon cell line by observing stress granule formation following exposure to dihydrocapsaicin, bisphenol A, and sorbitol. Taken together, we established a stress granule reporter system in a colon cell line, providing a novel assessment for the real-time monitoring of colon toxicity in response to various chemicals.
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In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.
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Fenotipo , Proteína que Contiene Valosina , Proteína que Contiene Valosina/metabolismo , Proteína que Contiene Valosina/genética , Humanos , Animales , Mutación , Autofagia/genética , Reparación del ADNRESUMEN
(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.
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Supervivencia Celular , Transducción de Señal , Gránulos de Estrés , Humanos , Supervivencia Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células HeLa , Gránulos de Estrés/metabolismo , Fosforilación/efectos de los fármacos , Tolueno/análogos & derivados , Tolueno/farmacología , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.
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ADN Helicasas , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Replicación Viral , Animales , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , Gránulos de Estrés/metabolismo , Bovinos , Factor 2 Eucariótico de Iniciación/metabolismo , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/metabolismo , Interacciones Huésped-Patógeno/inmunología , Fosforilación , Línea Celular , Gránulos Citoplasmáticos/metabolismoRESUMEN
Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.
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Antineoplásicos , Sorafenib , Gránulos de Estrés , Humanos , Sorafenib/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Gránulos de Estrés/metabolismo , Células HeLa , Resistencia a Antineoplásicos/efectos de los fármacos , Péptidos/farmacología , Péptidos/química , Supervivencia Celular/efectos de los fármacos , Ubiquitina Tiolesterasa/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Línea Celular Tumoral , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/químicaRESUMEN
Tandem CCCH zinc finger (TZF) proteins play diverse roles in plant growth and stress response. Although as many as 11 TZF proteins have been identified in Arabidopsis, little is known about the mechanism by which TZF proteins select and regulate the target mRNAs. Here, we report that Arabidopsis TZF1 is a bona-fide stress granule protein. Ectopic expression of TZF1 (TZF1 OE), but not an mRNA binding-defective mutant (TZF1H186Y OE), enhances salt stress tolerance in Arabidopsis. RNA-seq analyses of NaCl-treated plants revealed that the down-regulated genes in TZF1 OE plants are enriched for functions in salt and oxidative stress responses. Because many of these down-regulated mRNAs contain AU- and/or U-rich elements (AREs and/or UREs) in their 3'-UTRs, we hypothesized that TZF1-ARE/URE interaction might contribute to the observed gene expression changes. Results from RNA immunoprecipitation-quantitative PCR analysis, gel-shift, and mRNA half-life assays indicate that TZF1 binds and triggers degradation of the autoinhibited Ca2+-ATPase 11 (ACA11) mRNA, which encodes a tonoplast-localized calcium pump that extrudes calcium and dampens signal transduction pathways necessary for salt stress tolerance. Furthermore, this salt stress-tolerance phenotype was recapitulated in aca11 null mutants. Collectively, our findings demonstrate that TZF1 binds and initiates degradation of specific mRNAs to enhance salt stress tolerance.