RESUMO
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.
Assuntos
COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Desacetilase 6 de Histona , SARS-CoV-2 , Replicação Viral , Desacetilase 6 de Histona/metabolismo , Desacetilase 6 de Histona/genética , Humanos , SARS-CoV-2/fisiologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , COVID-19/virologia , COVID-19/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Acetilação , Linhagem Celular , Chlorocebus aethiops , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células Vero , Animais , Interações Hospedeiro-Patógeno , Proteínas de Ligação a Poli-ADP-Ribose , DNA Helicases , RNA HelicasesRESUMO
About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.
Assuntos
Neoplasias da Mama , Alvo Mecanístico do Complexo 1 de Rapamicina , Trastuzumab , Humanos , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptor ErbB-2/metabolismo , Animais , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA de Transferência/metabolismo , Camundongos , RNA Helicases/metabolismo , Camundongos Nus , Proteínas com Motivo de Reconhecimento de RNA/metabolismoRESUMO
Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role.
Assuntos
RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Humanos , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Corpos de Processamento/metabolismo , Corpos de Processamento/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Células HeLa , DNA Helicases/metabolismo , DNA Helicases/genética , Células HEK293 , Ligação Proteica , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas Proto-OncogênicasAssuntos
COVID-19 , DNA Helicases , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , SARS-CoV-2 , Replicação Viral , Humanos , Replicação Viral/fisiologia , SARS-CoV-2/fisiologia , COVID-19/metabolismo , COVID-19/virologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/fisiologia , RNA Helicases/metabolismo , RNA Helicases/fisiologia , DNA Helicases/metabolismo , DNA Helicases/fisiologia , Animais , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
Several biomolecular condensates assemble in mammalian cells in response to viral infection. The most studied of these are stress granules (SGs), which have been proposed to promote antiviral innate immune signaling pathways, including the RLR-MAVS, the protein kinase R (PKR), and the OAS-RNase L pathways. However, recent studies have demonstrated that SGs either negatively regulate or do not impact antiviral signaling. Instead, the SG-nucleating protein, G3BP1, may function to perturb viral RNA biology by condensing viral RNA into viral-aggregated RNA condensates, thus explaining why viruses often antagonize G3BP1 or hijack its RNA condensing function. However, a recently identified condensate, termed double-stranded RNA-induced foci, promotes the activation of the PKR and OAS-RNase L antiviral pathways. In addition, SG-like condensates known as an RNase L-induced bodies (RLBs) have been observed during many viral infections, including SARS-CoV-2 and several flaviviruses. RLBs may function in promoting decay of cellular and viral RNA, as well as promoting ribosome-associated signaling pathways. Herein, we review these recent advances in the field of antiviral biomolecular condensates, and we provide perspective on the role of canonical SGs and G3BP1 during the antiviral response.
Assuntos
RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA Viral , Grânulos de Estresse , Humanos , Animais , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Helicases/metabolismo , RNA Viral/metabolismo , Grânulos de Estresse/metabolismo , SARS-CoV-2/fisiologia , Imunidade Inata , Transdução de Sinais , Condensados Biomoleculares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Viroses/tratamento farmacológico , Viroses/metabolismo , DNA Helicases/metabolismo , eIF-2 Quinase/metabolismo , Endorribonucleases/metabolismo , COVID-19/virologia , COVID-19/imunologiaRESUMO
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
Assuntos
Infecções por Vírus de RNA , Grânulos de Estresse , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Grânulos Citoplasmáticos/metabolismo , Proteína DEAD-box 58/metabolismo , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Imunidade Inata , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores Imunológicos/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Grânulos de Estresse/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
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.
Assuntos
DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Replicação Viral , Animais , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Grânulos de Estresse/metabolismo , Bovinos , Fator de Iniciação 2 em Eucariotos/metabolismo , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Fosforilação , Linhagem Celular , Grânulos Citoplasmáticos/metabolismoRESUMO
In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.
Assuntos
Cloroplastos , Regulação da Expressão Gênica de Plantas , Oryza , Imunidade Vegetal , Proteínas de Plantas , Cloroplastos/metabolismo , Cloroplastos/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oryza/genética , Oryza/metabolismo , Oryza/imunologia , Proteínas de Repetições Ricas em Leucina , Sítios de Ligação , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Proteínas NLR/metabolismo , Proteínas NLR/genética , Edição de RNARESUMO
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.
Assuntos
Grânulos Citoplasmáticos , DNA Helicases , Proteínas de Fluorescência Verde , Queratinócitos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Humanos , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Arsenitos/toxicidade , Pele/efeitos dos fármacos , Pele/metabolismo , Técnicas de Introdução de Genes , Genes Reporter/efeitos dos fármacos , Fenóis/toxicidade , Células HaCaT , Fosforilação , Compostos Benzidrílicos/toxicidade , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Testes de Toxicidade/métodosRESUMO
Acute stress caused by short-term exposure to deleterious chemicals can induce the aggregation of RNA-binding proteins (RBPs) in the cytosol and the formation of stress granules (SGs). The cytoplasmic RBP, Ras GTPase-activating protein-binding protein 1 (G3BP1) is a critical organizer of SG, and its aggregation is considered a hallmark of cellular stress. However, assembly of SG is a highly dynamic process that involves RBPs; hence, existing methods based on fixation processes or overexpression of RBPs exhibit limited efficacy in detecting the assembly of SG under stress conditions. In this study, we established a G3BP1- Green fluorescent protein (GFP) reporter protein in a human neuroblastoma cell line to overcome these limitations. GFP was introduced into the G3BP1 genomic sequence via homologous recombination to generate a G3BP1-GFP fusion protein and further analyze the aggregation processes. We validated the assembly of SG under stress conditions using the G3BP1-GFP reporter system. Additionally, this system supported the evaluation of bisphenol A-induced SG response in the established human neuroblastoma cell line. In conclusion, the established G3BP1-GFP reporter system enables us to monitor the assembly of the SG complex in a human neuroblastoma cell line in real time and can serve as an efficient tool for assessing potential neurotoxicity associated with short-term exposure to chemicals.
Assuntos
DNA Helicases , Proteínas de Fluorescência Verde , Neuroblastoma , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Humanos , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Linhagem Celular Tumoral , RNA Helicases/genética , RNA Helicases/metabolismo , Neuroblastoma/patologia , DNA Helicases/metabolismo , Grânulos de Estresse , Estresse Fisiológico , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.
Assuntos
Herpesvirus Humano 1 , Proteínas Virais , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ribonucleases , DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Herpesvirus Humano 1/genética , Endorribonucleases/metabolismo , Estabilidade de RNA , Vírion/genética , Vírion/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Aberrant expression of forkhead box transcription factor 1 (FOXM1) plays critical roles in a variety of human malignancies and predicts poor prognosis. However, little is known about the crosstalk between FOXM1 and long noncoding RNAs (lncRNAs) in tumorigenesis. The present study identifies a previously uncharacterized lncRNA XLOC_008672 in gastric cancer (GC), which is regulated by FOXM1 and possesses multiple copies of tandem repetitive sequences. LncRNA microarrays are used to screen differentially expressed lncRNAs in FOXM1 knockdown GC cells, and then the highest fold downregulation lncRNA XLOC_008672 is screened out. Sequence analysis reveals that the new lncRNA contains 62 copies of 37-bp tandem repeats. It is transcriptionally activated by FOXM1 and functions as a downstream effector of FOXM1 in GC cells through in vitro and in vivo functional assays. Elevated expression of XLOC_008672 is found in GC tissues and indicates worse prognosis. Mechanistically, XLOC_008672 can bind to small nuclear ribonucleoprotein polypeptide A (SNRPA), thereby enhancing mRNA stability of Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and, consequently, facilitating GC cell proliferation and migration. Our study discovers a new uncharacterized lncRNA XLOC_008672 involved in GC carcinogenesis and progression. Targeting FOXM1/XLOC_008672/SNRPA/G3BP1 signaling axis might be a promising therapeutic strategy for GC.
Assuntos
Carcinogênese , Proliferação de Células , Proteína Forkhead Box M1 , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante , Neoplasias Gástricas , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA Helicases , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Camundongos Nus , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Prognóstico , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Sequências de Repetição em Tandem/genéticaRESUMO
Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.
Assuntos
DNA Helicases , Inflamação , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Animais , Suínos , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/metabolismo , RNA Helicases/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Replicação Viral , Coronavirus/imunologia , Coronavirus/fisiologia , Linhagem Celular , Doenças dos Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/genética , Imunidade InataRESUMO
Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (ß) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor ß. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.
Assuntos
COVID-19 , Diabetes Mellitus , Adulto , Animais , Humanos , Idoso , SARS-CoV-2 , Receptor de Insulina , Peptídeo C , DNA Helicases , Estudos Retrospectivos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , GlucoseRESUMO
Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, ß-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, ß-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.
Assuntos
Barreira Hematotesticular , DNA Helicases , Masculino , Animais , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , beta Catenina , RNA , Claudinas , MamíferosRESUMO
G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , DNA Helicases/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Virulência , RNA Guia de Sistemas CRISPR-Cas , Proteínas do Nucleocapsídeo , Replicação Viral , RNA Viral/genéticaRESUMO
Although the composition and assembly of stress granules (SGs) are well understood, the molecular mechanisms underlying SG disassembly remain unclear. Here, we identify that heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is associated with SGs and that its absence specifically enhances the disassembly of arsenite-induced SGs depending on the ubiquitination-proteasome system but not the autophagy pathway. hnRNPA2B1 interacts with many core SG proteins, including G3BP1, G3BP2, USP10, and Caprin-1; USP10 can deubiquitinate G3BP1; and hnRNPA2B1 depletion attenuates the G3BP1-USP10/Caprin-1 interaction but elevates the G3BP1 ubiquitination level under arsenite treatment. Moreover, the disease-causing mutation FUSR521C also disassembles faster from SGs in HNRNPA2B1 mutant cells. Furthermore, knockout of hnRNPA2B1 in mice leads to Sertoli cell-only syndrome (SCOS), causing complete male infertility. Consistent with this, arsenite-induced SGs disassemble faster in Hnrnpa2b1 knockout (KO) mouse Sertoli cells as well. These findings reveal the essential roles of hnRNPA2B1 in regulating SG disassembly and male mouse fertility.
Assuntos
Arsenitos , Masculino , Animais , Camundongos , Arsenitos/toxicidade , DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , FertilidadeRESUMO
Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Progressão da Doença , Escherichia coli , NF-kappa B , Grânulos de Estresse , Animais , Humanos , Camundongos , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Modelos Animais de Doenças , DNA Helicases , Escherichia coli/genética , Escherichia coli/metabolismo , Gencitabina , NF-kappa B/metabolismo , NF-kappa B/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Transdução de Sinais/genética , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genéticaRESUMO
Proper regulation of cellular response to environmental stress is crucial for maintaining biological homeostasis and is achieved by the balance between cell death processes, such as the formation of the pyroptosis-inducing NLRP3 inflammasome, and pro-survival processes, such as stress granule (SG) assembly. However, the functional interplay between these two stress-responsive organelles remains elusive. Here, we identified DHX33, a viral RNA sensor for the NLRP3 inflammasome, as a SG component, and the SG-nucleating protein G3BP as an NLRP3 inflammasome component. We also found that a decrease in intracellular potassium (K+) concentration, a key 'common' step in NLRP3 inflammasome activation, markedly inhibited SG assembly. Therefore, when macrophages are exposed to stress stimuli with the potential to induce both SGs and the NLRP3 inflammasome, such as cytoplasmic poly(I:C) stimulation, they preferentially form the NLRP3 inflammasome but avoid SG assembly by sequestering G3BP into the inflammasome and by inducing a reduction in intracellular K+ levels. Thus, under such conditions, DHX33 is primarily utilized as a viral RNA sensor for the inflammasome. Our data reveal the functional crosstalk between NLRP3 inflammasome-mediated pyroptosis and SG-mediated cell survival pathways and delineate a molecular mechanism that regulates cell-fate decisions and anti-viral innate immunity under stress.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Grânulos de Estresse , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Humanos , Grânulos de Estresse/metabolismo , Camundongos , Animais , Potássio/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Piroptose , RNA Helicases/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Poli I-C/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , DNA HelicasesRESUMO
Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.