RESUMO
Antiviral innate immunity is a complicated system initiated by the induction of type I interferon (IFN-I) and downstream interferon-stimulated genes (ISGs) and is finely regulated by numerous positive and negative factors at different signaling adaptors. During this process, posttranslational modifications, especially ubiquitination, are the most common regulatory strategy used by the host to switch the antiviral innate signaling pathway and are mainly controlled by E3 ubiquitin ligases from different protein families. A comprehensive understanding of the regulatory mechanisms and a novel discovery of regulatory factors involved in the IFN-I signaling pathway are important for researchers to identify novel therapeutic targets against viral infectious diseases based on innate immunotherapy. In this section, we use the E3 ubiquitin ligase as an example to guide the identification of a protein belonging to the RING Finger (RNF) family that regulates the RIG-I-mediated IFN-I pathway through ubiquitination.
Assuntos
Imunidade Inata , Interferon Tipo I , Transdução de Sinais , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Interferon Tipo I/metabolismo , Viroses/imunologia , Viroses/genética , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genética , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genéticaRESUMO
The innate immune system plays a pivotal role in pathogen recognition and the initiation of innate immune responses through its Pathogen Recognition Receptors (PRRs), which detect Pathogen-Associated Molecular Patterns (PAMPs). Nucleic acids, including RNA and DNA, are recognized as particularly significant PAMPs, especially in the context of viral pathogens. During RNA virus infections, specific sequences in the viral RNA mark it as non-self, enabling host recognition through interactions with RNA sensors, thereby triggering innate immunity. Given that some of the most lethal viruses are RNA viruses, they pose a severe threat to human and animal health. Therefore, understanding the immunobiology of RNA PRRs is crucial for controlling pathogen infections, particularly RNA virus infections. In this chapter, we will introduce a "pull-down" method for identifying RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensors.
Assuntos
Imunidade Inata , RNA Viral , Receptores de Reconhecimento de Padrão , Humanos , RNA Viral/genética , RNA Viral/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Animais , Receptores Toll-Like/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/genética , Vírus de RNA/imunologia , Vírus de RNA/genética , Interações Hospedeiro-Patógeno/imunologia , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologiaRESUMO
The incessant arms race between viruses and hosts has led to numerous evolutionary innovations that shape life's evolution. During this process, the interactions between viral receptors and viruses have garnered significant interest since viral receptors are cell surface proteins exploited by viruses to initiate infection. Our study sheds light on the arms race between the MDA5 receptor and 5'ppp-RNA virus in a lower vertebrate fish, Miichthys miiuy. Firstly, the frequent and independent loss events of RIG-I in vertebrates prompted us to search for alternative immune substitutes, with homology-dependent genetic compensation response (HDGCR) being the main pathway. Our further analysis suggested that MDA5 of M. miiuy and Gallus gallus, the homolog of RIG-I, can replace RIG-I in recognizing 5'ppp-RNA virus, which may lead to redundancy of RIG-I and loss from the species genome during evolution. Secondly, as an adversarial strategy, 5'ppp-RNA SCRV can utilize the m6A methylation mechanism to degrade MDA5 and weaken its antiviral immune ability, thus promoting its own replication and immune evasion. In summary, our study provides a snapshot into the interaction and coevolution between vertebrate and virus, offering valuable perspectives on the ecological and evolutionary factors that contribute to the diversity of the immune system.
Before the immune system can eliminate a bacterium, virus or other type of pathogen, it needs to be able to recognize these foreign elements. To achieve this, cells in the immune system have proteins called pattern recognition receptors (PRRs) which can identify distinct molecular features of certain pathogens. One specific group of PRRs is a family of retinoic acid-induced RIG-I-like receptors (RLRs), which help immune cells detect different types of viruses. Members of this family recognize distinct motifs on the genetic material of viruses known as RNA. For instance, RIG-I recognizes a marker known as 5'ppp on the end of single-stranded RNA molecules, whereas MDA5 recognizes long strands of double-stranded RNA. Many vertebrates including various mammals, birds, and fish lost the RIG-I receptor over the course of evolution. However, Geng et al. predicted that some animals lacking the RIG-I receptor may still be able to activate an immune response against viruses that contain the 5'ppp-RNA motif. To investigate this possibility, Geng et al. studied chickens and miiuy croakers (a type of ray-finned fish) which no longer have a RIG-I receptor. They found that both animals can still sense and eliminate two 5'ppp-RNA viruses called VSV and SCRV. Further experiments revealed that these two viruses are detected by a modified MDA5 receptor that had evolved to bind to 5'-ppp and activate the antiviral response. Viruses are also continuously evolving new ways to escape the immune system. This led Geng et al. to investigate whether SCRV, which causes serious harm to marine fish, has evolved a way to evade the MDA5 protection mechanism. Using miiuy croakers as a model, they found that SCRV causes the transcripts that produce the MDA5 protein to contain more molecules of m6a. This molecular tag degrades the transcript, leading to lower levels of MDA5, reducing the antiviral response against SCRV. The findings of Geng et al. offer valuable perspectives on how the immune system adapts over the course of evolution, and highlight the diversity of antiviral responses in vertebrates. Chickens and miiuy croakers are commonly farmed animals, and further work investigating how viruses invade these species could prevent illnesses from spreading and having a negative impact on the economy.
Assuntos
Proteína DEAD-box 58 , Helicase IFIH1 Induzida por Interferon , Animais , Helicase IFIH1 Induzida por Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Peixes/virologia , Peixes/genética , Peixes/imunologia , Vírus de RNA/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Evolução MolecularRESUMO
The aim of this study was to elucidate the effect of FAM13A on the differentiation of goat intramuscular precursor adipocytes and its mechanism of action. Here, we cloned the CDS region 2094 bp of the goat FAM13A gene, encoding a total of 697 amino acid residues. Functionally, overexpression of FAM13A inhibited the differentiation of goat intramuscular adipocytes with a concomitant reduction in lipid droplets, whereas interference with FAM13A expression promoted the differentiation of goat intramuscular adipocytes. To further investigate the mechanism of FAM13A inhibiting adipocyte differentiation, 104 differentially expressed genes were screened by RNA-seq, including 95 up-regulated genes and 9 down-regulated genes. KEGG analysis found that the RIG-I receptor signaling pathway, NOD receptor signaling pathway and toll-like receptor signaling pathway may affect adipogenesis. We selected the RIG-I receptor signaling pathway enriched with more differential genes as a potential adipocyte differentiation signaling pathway for verification. Convincingly, the RIG-I like receptor signaling pathway inhibitor (HY-P1934A) blocked this pathway to save the phenotype observed in intramuscular adipocyte with FAM13A overexpression. Finally, the upstream miRNA of FAM13A was predicted, and the targeted inhibition of miR-21-5p on the expression of FAM13A gene was confirmed. In this study, it was found that FAM13A inhibited the differentiation of goat intramuscular adipocytes through the RIG-I receptor signaling pathway, and the upstream miRNA of FAM13A (miR-21-5p) promoted the differentiation of goat intramuscular adipocytes. This work extends the genetic regulatory network of IMF deposits and provides theoretical support for improving human health and meat quality from the perspective of IMF deposits.
Assuntos
Adipócitos , Diferenciação Celular , Cabras , Transdução de Sinais , Animais , Cabras/genética , Cabras/metabolismo , Adipócitos/metabolismo , Adipócitos/citologia , Diferenciação Celular/genética , MicroRNAs/genética , Adipogenia/genética , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismoRESUMO
Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5' triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies.
Assuntos
Proteína DEAD-box 58 , Vírus da Hepatite E , Interferência de RNA , RNA Interferente Pequeno , Replicação Viral , Humanos , Vírus da Hepatite E/fisiologia , Vírus da Hepatite E/genética , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , RNA Interferente Pequeno/genética , Células A549 , Receptores Imunológicos , Hepatite E/virologia , Hepatite E/imunologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Antivirais/farmacologia , Transdução de SinaisRESUMO
Cytomegalovirus (CMV) is a pathogen that is common worldwide and is often present in individuals infected with human immunodeficiency virus (HIV). Pattern recognition receptors (PRRs) are host sensors that activate the immune response against infectious agents. However, it is unclear whether PRR single-nucleotide polymorphisms (SNPs) are associated with the occurrence of CMV DNAemia in subjects coinfected with HIV and CMV. HIV/CMV-coinfected patients with and without CMV DNAemia were recruited for this study. The DDX58 rs10813831 and IFIH1 (rs3747517 and rs1990760) polymorphisms were genotyped using the TaqMan Allelic Discrimination Assay, whereas the DDX58 rs12006123 and TLR3 (rs3775291 and rs3775296) SNPs were analyzed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. A mutation present in at least one allele of the DDX58 rs12006123 SNP occurred at least two times more frequently in HIV/CMV-coinfected patients with CMV DNAemia than in coinfected subjects without CMV DNAemia (OR, 2.50; 95% CI, 1.33-4.68; p = 0.004, in the dominant model). A higher level of CMV DNAemia was observed in subjects who had the heterozygous (GA) or homozygous recessive (AA) genotype for the DDX58 rs12006123 SNP compared with those who had the wild-type (GG) genotype (p = 0.0003). Moreover, in subjects with a mutation detected in at least one allele of the DDX58 rs12006123 SNP, a lower serum IFN-ß concentration was found compared with those who had a wild-type (GG) genotype for this polymorphism (p = 0.024). The DDX58 rs12006123 SNP is associated with CMV DNAemia in HIV/CMV-coinfected patients.
Assuntos
Coinfecção , Infecções por Citomegalovirus , Citomegalovirus , Infecções por HIV , Polimorfismo de Nucleotídeo Único , Receptor 3 Toll-Like , Humanos , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/complicações , Infecções por HIV/virologia , Infecções por HIV/complicações , Infecções por HIV/genética , Coinfecção/virologia , Coinfecção/genética , Feminino , Masculino , Adulto , Citomegalovirus/genética , Receptor 3 Toll-Like/genética , Pessoa de Meia-Idade , Proteína DEAD-box 58/genética , DNA Viral/genética , DNA Viral/sangue , Genótipo , Helicase IFIH1 Induzida por Interferon/genética , Receptores ImunológicosRESUMO
Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.
Assuntos
Proteínas do Capsídeo , Carpas , Doenças dos Peixes , Proteínas de Peixes , Imunidade Inata , Infecções por Reoviridae , Reoviridae , Transdução de Sinais , Proteínas com Motivo Tripartido , Animais , Reoviridae/fisiologia , Reoviridae/imunologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , Carpas/imunologia , Infecções por Reoviridae/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Replicação Viral , Ligação Proteica , Simulação de Acoplamento Molecular , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genéticaRESUMO
The escape of mitochondrial double-stranded dsRNA (mt-dsRNA) into the cytosol has been recently linked to a number of inflammatory diseases. Here, we report that the release of mt-dsRNA into the cytosol is a general feature of senescent cells and a critical driver of their inflammatory secretome, known as senescence-associated secretory phenotype (SASP). Inhibition of the mitochondrial RNA polymerase, the dsRNA sensors RIGI and MDA5, or the master inflammatory signaling protein MAVS, all result in reduced expression of the SASP, while broadly preserving other hallmarks of senescence. Moreover, senescent cells are hypersensitized to mt-dsRNA-driven inflammation due to their reduced levels of PNPT1 and ADAR1, two proteins critical for mitigating the accumulation of mt-dsRNA and the inflammatory potency of dsRNA, respectively. We find that mitofusin MFN1, but not MFN2, is important for the activation of the mt-dsRNA/MAVS/SASP axis and, accordingly, genetic or pharmacologic MFN1 inhibition attenuates the SASP. Finally, we report that senescent cells within fibrotic and aged tissues present dsRNA foci, and inhibition of mitochondrial RNA polymerase reduces systemic inflammation associated to senescence. In conclusion, we uncover the mt-dsRNA/MAVS/MFN1 axis as a key driver of the SASP and we identify novel therapeutic strategies for senescence-associated diseases.
Assuntos
Senescência Celular , Citosol , Inflamação , Mitocôndrias , RNA de Cadeia Dupla , RNA de Cadeia Dupla/metabolismo , Humanos , Citosol/metabolismo , Mitocôndrias/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Animais , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Fenótipo Secretor Associado à Senescência , Helicase IFIH1 Induzida por Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Camundongos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , Exorribonucleases/metabolismo , Exorribonucleases/genética , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Transdução de SinaisRESUMO
Insights into mechanisms driving either activation or inhibition of immune response are crucial in understanding the pathology of various diseases. The differentiation of viral from endogenous RNA in the cytoplasm by pattern-recognition receptors, such as retinoic acid-inducible gene I (RIG-I), is one of the essential paths for timely activation of an antiviral immune response through induction of type I interferons (IFN). In this mini-review, we describe the most recent developments centered around RIG-I's structure and mechanism of action. We summarize the paradigm-changing work over the past few years that helped us better understand RIG-I's monomeric and oligomerization states and their role in conveying immune response. We also discuss potential applications of the modulation of the RIG-I pathway in preventing autoimmune diseases or induction of immunity against viral infections. Overall, our review aims to summarize innovative research published in the past few years to help clarify questions that have long persisted around RIG-I.
Assuntos
Proteína DEAD-box 58 , Receptores Imunológicos , Humanos , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/química , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Animais , Viroses/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Transdução de Sinais , Multimerização Proteica , Imunidade InataRESUMO
BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.
Assuntos
Metilação de DNA , Imunidade Inata , Vacinas contra Influenza , Influenza Humana , Humanos , Metilação de DNA/genética , Metilação de DNA/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Imunidade Inata/genética , Feminino , Masculino , Influenza Humana/prevenção & controle , Influenza Humana/imunologia , Influenza Humana/genética , Pessoa de Meia-Idade , Adulto , Transdução de Sinais , Linfócitos T/imunologia , Estudos Longitudinais , Epigênese Genética , Vacinação , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismoRESUMO
Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.
Assuntos
Caspase 8 , Proteína DEAD-box 58 , Enzima Desubiquitinante CYLD , Imunidade Inata , Vírus da Influenza A , Influenza Humana , MAP Quinase Quinase Quinases , Ubiquitinação , Humanos , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , Caspase 8/metabolismo , Caspase 8/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Vírus da Influenza A/imunologia , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Células A549 , Animais , Transdução de Sinais/imunologia , Receptores ImunológicosRESUMO
Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.
Assuntos
Proteína DEAD-box 58 , Histona Desacetilases , Imunidade Inata , Receptores Imunológicos , Transdução de Sinais , Vesiculovirus , Replicação Viral , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Humanos , Histona Desacetilases/metabolismo , Vesiculovirus/imunologia , Receptores Imunológicos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Acetilação , Células HEK293 , Interferons/metabolismo , Interferons/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno/imunologia , Animais , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Hippocampal neural stem/progenitor cells (NSPCs) are highly vulnerable to different stress stimuli, resulting in adult neurogenesis decline and eventual cognitive defects. Our previous study demonstrated that NOD-like receptor family pyrin domain-containing 6 (Nlrp6) highly expressed in NSPCs played a critical role in sustaining hippocampal neurogenesis to resist stress-induced depression, but the underlying mechnistms are still unclear. Here, we found that Nlrp6 depletion led to cognitive defects and hippocampal NSPC loss in mice. RNA-sequencing analysis of the primary NSPCs revealed that Nlrp6 deficiency altered gene expression profiles of mitochondrial energy generation and ferroptotic process. Upon siNlrp6 transfection, as well as corticosterone (CORT) exposure, downregulation of Nlrp6 suppressed retinoic acid-inducible gene I (RIG-1)/mitochondrial antiviral signaling proteins (MAVS)-mediated autophagy, but drove NSPC ferroptotic death. More interesting, short chain fatty acids (SCFAs) upregulated Nlrp6 expression and promoted RIG-1/MAVS-mediated mitophagy, preventing CORT-induced NSPC ferroptosis. Our study further demonstrates that Nlrp6 should be a sensor for RIG-1/MAVS-mediated mitophagy and play a critical role in maintain mitochondrial homeostasis of hippocampal NSPCs. These results suggests that Nlrp6 should be a potential drug target to combat neurodegenerative diseases relative with chronic stress.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Corticosterona , Proteína DEAD-box 58 , Ferroptose , Mitofagia , Células-Tronco Neurais , Animais , Camundongos , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Corticosterona/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Células-Tronco Neurais/metabolismo , Hipocampo/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Receptores de Superfície CelularRESUMO
Mammalian orthoreovirus (MRV) outer capsid protein σ3 is a multifunctional protein containing a double-stranded RNA-binding domain, which facilitates viral entry and assembly. We reasoned that σ3 has an innate immune evasion function. Here, we show that σ3 protein localizes in the mitochondria and interacts with mitochondrial antiviral signaling protein (MAVS) to activate the intrinsic mitochondria-mediated apoptotic pathway. Consequently, σ3 protein promotes the degradation of MAVS through the intrinsic caspase-9/caspase-3 apoptotic pathway. Moreover, σ3 protein can also inhibit the expression of the components of the RNA-sensing retinoic acid-inducible gene (RIG)-like receptor (RLR) signaling pathway to block antiviral type I interferon responses. Mechanistically, σ3 inhibits RIG-I and melanoma differentiation-associated gene 5 expression is independent of its inhibitory effect on MAVS. Overall, we demonstrate that the MRV σ3 protein plays a vital role in negatively regulating the RLR signaling pathway to inhibit antiviral responses. This enables MRV to evade host defenses to facilitate its own replication providing a target for the development of effective antiviral drugs against MRV. IMPORTANCE: Mammalian orthoreovirus (MRV) is an important zoonotic pathogen, but the regulatory role of its viral proteins in retinoic acid-inducible gene-like receptor (RLR)-mediated antiviral responses is still poorly understood. Herein, we show that MRV σ3 protein co-localizes with mitochondrial antiviral signaling protein (MAVS) in the mitochondria and promotes the mitochondria-mediated intrinsic apoptotic pathway to cleave and consequently degrade MAVS. Furthermore, tryptophan at position 133 of σ3 protein plays a key role in the degradation of MAVS. Importantly, we show that MRV outer capsid protein σ3 is a key factor in antagonizing RLR-mediated antiviral responses, providing evidence to better unravel the infection and transmission mechanisms of MRV.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Capsídeo , Orthoreovirus de Mamíferos , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Humanos , Orthoreovirus de Mamíferos/genética , Animais , Apoptose , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Mitocôndrias/metabolismo , Imunidade Inata , Camundongos , Evasão da Resposta Imune , Células HEK293 , Receptores Imunológicos/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Linhagem Celular , Interações Hospedeiro-PatógenoRESUMO
Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , RNA de Cadeia Dupla , Ribonuclease III , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Ribonuclease III/química , Ribonuclease III/genética , Microscopia Crioeletrônica , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/química , Ligação Proteica , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/químicaRESUMO
Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.
Assuntos
Gastroenterite Suína Transmissível , Glicólise , Inflamação , Organoides , Vírus da Gastroenterite Transmissível , Animais , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Gastroenterite Suína Transmissível/virologia , Gastroenterite Suína Transmissível/metabolismo , Gastroenterite Suína Transmissível/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/metabolismo , Inflamação/virologia , Intestinos/virologia , Intestinos/patologia , NF-kappa B/metabolismo , Organoides/virologia , Organoides/metabolismo , Organoides/patologia , Transdução de Sinais , Suínos , Vírus da Gastroenterite Transmissível/fisiologiaRESUMO
Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.
Assuntos
Autofagia , Proteína DEAD-box 58 , Glaucoma , Pressão Intraocular , Malha Trabecular , Animais , Feminino , Humanos , Masculino , Camundongos , Doenças da Aorta , Autofagia/efeitos dos fármacos , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Hipoplasia do Esmalte Dentário , Glaucoma/patologia , Glaucoma/metabolismo , Glaucoma/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/metabolismo , Interferon beta/metabolismo , Pressão Intraocular/genética , Metacarpo/anormalidades , Camundongos Endogâmicos C57BL , Doenças Musculares , Mutação , Odontodisplasia , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Atrofia Óptica/patologia , Osteoporose , Linhagem , Receptores Imunológicos , Malha Trabecular/metabolismo , Malha Trabecular/efeitos dos fármacos , Calcificação VascularRESUMO
PEDV, a single-stranded RNA virus, causes significant economic losses in the pig industry. Sin3-associated protein 18 (SAP18) is known for its role in transcriptional inhibition and RNA splicing. However, research on SAP18's involvement in PEDV infection is limited. Here, we identified an interaction between SAP18 and PEDV nonstructural protein 10 (Nsp10) using immunoprecipitation-mass spectrometry (IP-MS) and confirmed it through immunoprecipitation and laser confocal microscopy. Additionally, PEDV Nsp10 reduced SAP18 protein levels and induced its cytoplasmic accumulation. Overexpressing SAP18 suppressed PEDV replication, meanwhile its knockdown via short interfering RNA (siRNA) enhanced replication. SAP18 overexpression boosted IRF3 and NF-κB P65 phosphorylation, nuclear translocation, and IFN-ß antiviral response. Furthermore, SAP18 upregulated RIG-I expression and facilitated its dephosphorylation, while SAP18 knockdown had the opposite effect. Finally, SAP18 interacted with phosphatase 1 (PP1) catalytic subunit alpha (PPP1CA), promoting PPP1CA-RIG-I interaction during PEDV infection. These findings highlight SAP18's role in activating the type I interferon pathway and inhibiting viral replication by promoting RIG-I dephosphorylation through its interaction with PPP1CA.
Assuntos
Vírus da Diarreia Epidêmica Suína , Proteínas não Estruturais Virais , Replicação Viral , Animais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Vírus da Diarreia Epidêmica Suína/genética , Fosforilação , Suínos , Linhagem Celular , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Chlorocebus aethiopsRESUMO
Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis.
Assuntos
Células Epiteliais , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Transdução de Sinais , Animais , Células Epiteliais/microbiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Suínos , Haemophilus parasuis/patogenicidade , Haemophilus parasuis/genética , Traqueia/microbiologia , Traqueia/citologia , Doenças dos Suínos/microbiologia , Sorogrupo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Inflamassomos/metabolismo , Inflamassomos/genética , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Infecções por Haemophilus/veterinária , Infecções por Haemophilus/microbiologiaRESUMO
BACKGROUND: Current cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5'-C; antisense strand, 3'-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy. METHODS: IsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5'-C; antisense strand, 3'-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-ß and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection. RESULTS: IsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-ß and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors. CONCLUSIONS: This study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5'-C; antisense strand, 3'-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.