RESUMEN
AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. Here, we unexpectedly discover that fly and human AGO proteins, which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P2. The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, our study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation.
Asunto(s)
MicroARNs , Ubiquitina-Proteína Ligasas , Lípidos , MicroARNs/metabolismo , Péptidos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.
Asunto(s)
Proteínas de Drosophila/genética , Longevidad/genética , Factor 6 Asociado a Receptor de TNF/genética , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Enzimas Desubicuitinizantes , Drosophila/genética , Longevidad/fisiología , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Ubiquitina/genéticaRESUMEN
The cGAS-STING signaling pathway plays a pivotal role in sensing cytosolic DNA and initiating innate immune responses against various threats, with disruptions in this pathway being associated with numerous immune-related disorders. Therefore, precise regulation of the cGAS-STING signaling is crucial to ensure appropriate immune responses. Recent research, including ours, underscores the importance of protein condensation in driving the activation and maintenance of innate immune signaling within the cGAS-STING pathway. Consequently, targeting condensation processes in this pathway presents a promising approach for modulating the cGAS-STING signaling and potentially managing associated disorders. In this review, we provide an overview of recent studies elucidating the role and regulatory mechanism of protein condensation in the cGAS-STING signaling pathway while emphasizing its pathological implications. Additionally, we explore the potential of understanding and manipulating condensation dynamics to develop novel strategies for mitigating cGAS-STING-related disorders in the future.
Asunto(s)
Inmunidad Innata , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Humanos , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , ADN/metabolismoRESUMEN
In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células Cultivadas , Femenino , Células Germinativas/metabolismo , Humanos , Ovario/citología , Ovario/metabolismo , Fosforilación , Células Madre/metabolismo , Ubiquitinación , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
RIG-I plays important roles in pathogen sensing and activation of antiviral innate immune responses in response to RNA viruses. RIG-I-mediated signaling must be precisely controlled to maintain innate immune signaling homeostasis. Previous studies demonstrated that lysine 63 (K63)-linked polyubiquitination of RIG-I is vital for its activation, but the mechanisms through which RIG-I is deubiquitinated to control innate immune responses are not well understood. Here we identified USP27X as a negative regulator of antiviral signaling in response to RNA viruses through siRNA library screening. Further functional studies indicated that USP27X negatively modulated RIG-I-mediated antiviral signaling in a deubiquitinase-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I interferon signaling. Collectively, these studies uncover a novel negative regulatory role of USP27X in targeting RIG-I to balance innate immune responses.
Asunto(s)
Proteína 58 DEAD Box , Inmunidad Innata/genética , Transducción de Señal , Proteasas Ubiquitina-Específicas , Virus/inmunología , Animales , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Células HeLa , Células Hep G2 , Humanos , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Ratones , Células RAW 264.7 , Receptores Inmunológicos , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/inmunología , Ubiquitinación/genética , Ubiquitinación/inmunologíaRESUMEN
BACKGROUND: Through the comparison of the demographic, epidemiological, and clinical characteristics of hospital human influenza (influenza A (H1N1) pdm09, H3N2, and B)-related and hospitalized avian-origin influenza A (H7N9)-related viral pneumonia patients, find the different between them. METHODS: A retrospective study was conducted in hospitalized influenza-related viral pneumonia patients. RESULTS: Human influenza A-related patients in the 35-49-year-old group were more than those with B pneumonia patients (p = 0.027), and relatively less in the ≥ 65-year-old group than B pneumonia patients (p = 0.079). The proportion of comorbid condition to human influenza A pneumonia was 58%, lower than B pneumonia and H7N9 pneumonia patients (78% vs. 77.8%; p = 0.013). The proportion of invasive mechanical ventilation (IMV), lymphocytopenia, elevated lactate dehydrogenase to hospitalized human influenza A-related viral pneumonia patients was higher than B pneumonia patients (p < 0.05), but lower than H7N9 pneumonia patients (p < 0.05). In the multivariate analysis, pulmonary consolidation (odds ratio (OR): 13.67; 95% confidence interval (CI) 1.54-121.12; p = 0.019) and positive bacterial culture (sputum) (OR: 7.71; 95% CI 2.48-24.03; p < 0.001) were independently associated with IMV, while shock (OR: 13.16; 95% CI 2.06-84.07; p = 0.006), white blood cell count > 10,000/mm3 (OR: 7.22; 95% CI 1.47-35.58; p = 0.015) and positive bacterial culture(blood or sputum) (OR: 6.27; 95% CI 1.36-28.85; p = 0.018) were independently associated with death in the three types hospitalized influenza-related viral pneumonia patients. CONCLUSIONS: Hospital influenza B-related viral pneumonia mainly affects the elderly and people with underlying diseases, while human influenza A pneumonia mainly affects the young adults; however, the mortality was similar. The hospitalized human influenza A-related viral pneumonia patients was severer than B pneumonia patients, but milder than H7N9 pneumonia patients. Pulmonary consolidation and positive bacterial culture (sputum) were independently associated with IMV, while shock, white blood cell count > 10,000/mm3, and positive bacterial culture (blood or sputum) were independently associated with death to three types hospitalized influenza-related viral pneumonia patients.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Subtipo H7N9 del Virus de la Influenza A , Gripe Humana , Neumonía Viral , Adulto , Anciano , Demografía , Hospitales , Humanos , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana/complicaciones , Gripe Humana/epidemiología , Persona de Mediana Edad , Neumonía Viral/epidemiología , Estudios RetrospectivosRESUMEN
Drosophila germ-line stem cells (GSCs) provide an excellent model to study the regulatory mechanisms of stem cells in vivo. Bag of marbles (bam) has been demonstrated to be necessary and sufficient to promote GSC and cystoblast differentiation. Despite extensive investigation of its regulation and genetic functions, the biochemical nature of the Bam protein has been unknown. Here, we report that Bam is an ubiquitin-associated protein and controls the turnover of cyclin A (CycA). Mechanistically, we found that Bam associated with Otu to form a deubiquitinase complex that stabilized CycA by deubiquitination, thus providing a mechanism to explain how ectopic expression of Bam in GSCs promotes differentiation. Collectively, our findings not only identify a biochemical function of Bam, which contributes to GSC fate determination, but also emphasizes the critical role of proper expression of cyclin proteins mediated by both ubiquitination and deubiquitination pathways in balancing stem cell self-renewal and differentiation.
Asunto(s)
Ciclina A/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Proteínas de Drosophila/metabolismo , Células Germinativas/citología , Células Germinativas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/fisiología , Autorrenovación de las Células/fisiología , Ciclina A/química , Ciclina A/genética , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Ovario/citología , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Ubiquitina/metabolismoRESUMEN
TGF-ß/BMP (bone morphogenetic protein) signaling pathways play conserved roles in controlling embryonic development, tissue homeostasis, and stem cell regulation. Inhibitory Smads (I-Smads) have been shown to negatively regulate TGF-ß/BMP signaling by primarily targeting the type I receptors for ubiquitination and turnover. However, little is known about how I-Smads access the membrane to execute their functions. Here we show that Dad, the Drosophila I-Smad, associates with the cellular membrane via palmitoylation, thereby targeting the BMP type I receptor for ubiquitination. By performing systematic biochemistry assays, we characterized the specific cysteine (Cys556) essential for Dad palmitoylation and membrane association. Moreover, we demonstrate that dHIP14, a Drosophila palmitoyl acyl-transferase, catalyzes Dad palmitoylation, thereby inhibiting efficient BMP signaling. Thus, our findings uncover a modification of the inhibitory Smads that controls TGF-ß/BMP signaling activity.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Proteínas Smad/metabolismo , Aciltransferasas/metabolismo , Animales , Sitios de Unión , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Lipoilación , Unión Proteica , Transporte de Proteínas , Proteínas Smad/química , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chronic neuroinflammation is a characteristic of Parkinson's disease (PD). Previous investigations have shown that Parkin gene mutations are related to the early-onset recessive form of PD and isolated juvenile-onset PD. Further, Parkin plays important roles in mitochondrial quality control and cytokine-induced cell death. However, whether Parkin regulates other cellular events is still largely unknown. In this study, we performed overexpression and knockout experiments and found that Parkin negatively regulates antiviral immune responses against RNA and DNA viruses. Mechanistically, we show that Parkin interacts with tumor necrosis factor receptor-associated factor 3 (TRAF3) to regulate stability of TRAF3 protein by promoting Lys48-linked ubiquitination. Our findings suggest that Parkin plays a novel role in innate immune signaling by targeting TRAF3 for degradation and maintaining the balance of innate antiviral immunity.
Asunto(s)
Fibroblastos/inmunología , Inmunidad Innata , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/virología , Chlorocebus aethiops , Fibroblastos/citología , Fibroblastos/virología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Herpesvirus Humano 1/inmunología , Humanos , Ratones , Mitocondrias/inmunología , Mitocondrias/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Proteolisis , Virus Sendai/inmunología , Factor 3 Asociado a Receptor de TNF/inmunología , Transducción Genética , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitinación , Células Vero , Vesiculovirus/inmunologíaRESUMEN
Stimulator of interferon genes (STING, also known as MITA and ERIS) is critical in protecting the host against DNA pathogen invasion. However, the molecular mechanism underlying the regulation of STING remains unclear. Here, we show that PPM1A negatively regulates antiviral signaling by targeting STING in its phosphatase activity-dependent manner, and in a line with this, PPM1A catalytically dephosphorylates STING and TBK1 in vitro. Importantly, we provide evidence that whereas TBK1 promotes STING aggregation in a phosphorylation-dependent manner, PPM1A antagonizes STING aggregation by dephosphorylating both STING and TBK1, emphasizing that phosphorylation is crucial for the efficient activation of STING. Our findings demonstrate a novel regulatory circuit in which STING and TBK1 reciprocally regulate each other to enable efficient antiviral signaling activation, and PPM1A dephosphorylates STING and TBK1, thereby balancing this antiviral signal transduction.
Asunto(s)
Proteínas de la Membrana/inmunología , Fosfoproteínas Fosfatasas/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Transducción de Señal/inmunología , Animales , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Fosfoproteínas Fosfatasas/genética , Fosforilación/genética , Fosforilación/inmunología , Proteína Fosfatasa 2C , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Células VeroRESUMEN
N(6)-methyladenine (6mA) is one of the most abundant types of DNA methylation, and plays an important role in bacteria; however, its roles in higher eukaryotes, such as plants, insects, and mammals, have been considered less important. Recent studies highlight that 6mA does indeed occur, and that it plays an important role in eukaryotes, such as worm, fly, and green algae, and thus the regulation of 6mA has emerged as a novel epigenetic mechanism in higher eukaryotes. Despite this intriguing development, a number of important issues regarding its biological roles are yet to be addressed. In this review, we focus on the 5mC and 6mA modifications in terms of their production, distribution, and the erasure of 6mA in higher eukaryotes including mammals. We perform an analysis of the potential functions of 6mA, hence widening understanding of this new epigenetic mark in higher eukaryotes, and suggesting future studies in this field.
Asunto(s)
5-Metilcitosina/química , Adenina/análogos & derivados , Metilación de ADN/genética , Eucariontes/genética , Transducción de Señal/genética , Adenina/química , Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Marcadores Genéticos/genéticaRESUMEN
Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/enzimología , Proteínas Hedgehog/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Activación Enzimática , Estructura Terciaria de Proteína , Proteolisis , Transducción de Señal , Receptor Smoothened , Técnicas del Sistema de Dos Híbridos , Ubiquitinación , Alas de Animales/enzimología , Pez CebraRESUMEN
5-Hydroxymethylcytosine (5-hmC) may represent a new epigenetic modification of cytosine. While the dynamics of 5-hmC during neurodevelopment have recently been reported, little is known about its genomic distribution and function(s) in neurodegenerative diseases such as Huntington's disease (HD). We here observed a marked reduction of the 5-hmC signal in YAC128 (yeast artificial chromosome transgene with 128 CAG repeats) HD mouse brain tissues when compared with age-matched wild-type (WT) mice, suggesting a deficiency of 5-hmC reconstruction in HD brains during postnatal development. Genome-wide distribution analysis of 5-hmC further confirmed the diminishment of the 5-hmC signal in striatum and cortex in YAC128 HD mice. General genomic features of 5-hmC are highly conserved, not being affected by either disease or brain regions. Intriguingly, we have identiï¬ed disease-specific (YAC128 versus WT) differentially hydroxymethylated regions (DhMRs), and found that acquisition of DhmRs in gene body is a positive epigenetic regulator for gene expression. Ingenuity pathway analysis (IPA) of genotype-specific DhMR-annotated genes revealed that alternation of a number of canonical pathways involving neuronal development/differentiation (Wnt/ß-catenin/Sox pathway, axonal guidance signaling pathway) and neuronal function/survival (glutamate receptor/calcium/CREB, GABA receptor signaling, dopamine-DARPP32 feedback pathway, etc.) could be important for the onset of HD. Our results indicate that loss of the 5-hmC marker is a novel epigenetic feature in HD, and that this aberrant epigenetic regulation may impair the neurogenesis, neuronal function and survival in HD brain. Our study also opens a new avenue for HD treatment; re-establishing the native 5-hmC landscape may have the potential to slow/halt the progression of HD.
Asunto(s)
Encéfalo/metabolismo , Cuerpo Estriado/metabolismo , Citosina/análogos & derivados , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , 5-Metilcitosina/análogos & derivados , Animales , Encéfalo/fisiopatología , Cuerpo Estriado/fisiopatología , Citosina/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Epigenómica , Humanos , Enfermedad de Huntington/fisiopatología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.
Asunto(s)
Señalización del Calcio , Daño del ADN/genética , ADN Mitocondrial/metabolismo , Genoma Mitocondrial/genética , Enfermedad de Huntington/patología , Mitocondrias/metabolismo , Superóxidos/metabolismo , Animales , Bradiquinina/farmacología , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Embrión de Mamíferos/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/patología , Neostriado/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Resorcinoles/farmacologíaRESUMEN
Innate antiviral immunity is the first line of the host defense system that rapidly detects invading viruses. Mitochondria function as platforms for innate antiviral signal transduction in mammals through the adaptor protein, MAVS. Excessive activation of MAVS-mediated antiviral signaling leads to dysfunction of mitochondria and cell apoptosis that likely causes the pathogenesis of autoimmunity. However, the mechanism of how MAVS is regulated at mitochondria remains unknown. Here we show that the Cytochrome c Oxidase (CcO) complex subunit COX5B physically interacts with MAVS and negatively regulates the MAVS-mediated antiviral pathway. Mechanistically, we find that while activation of MAVS leads to increased ROS production and COX5B expression, COX5B down-regulated MAVS signaling by repressing ROS production. Importantly, our study reveals that COX5B coordinates with the autophagy pathway to control MAVS aggregation, thereby balancing the antiviral signaling activity. Thus, our study provides novel insights into the link between mitochondrial electron transport system and the autophagy pathway in regulating innate antiviral immunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Complejo IV de Transporte de Electrones/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Proteína 5 Relacionada con la Autofagia , Western Blotting , Proliferación Celular , Células Cultivadas , Complejo IV de Transporte de Electrones/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunidad Innata , Inmunoprecipitación , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Unión Proteica , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Estomatitis Vesicular/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/genética , Virión/metabolismoRESUMEN
Aberrant activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway causes autoimmunity in humans and mice; however, the exact mechanism by which the cGAS-STING pathway initiates adaptive immunity and tissue pathology is still not fully understood. Here, we used a cGAS knockin (KI) mouse model that develops systemic autoimmunity. In the lungs of cGAS-KI mice, blood vessels were enclosed by organized lymphoid tissues that resemble tertiary lymphoid structures (TLSs). Cell-intrinsic cGAS induction promoted up-regulation of CCR5 in CD8+ T cells and led to CCL5 production in vascular endothelial cells. Peripheral CD8+ T cells were recruited to the lungs and produced CXCL13 and interferon-γ. The latter triggered endothelial cell death, potentiated CCL5 production, and was essential for TLS establishment. Blocking CCL5 or CCR5, or depleting CD8+ T cells, impaired TLS formation. cGAS-mediated TLS formation also enhanced humoral and antitumor responses. These data demonstrate that cGAS signaling drives a specialized lymphoid structure that underlies autoimmune tissue pathology.
Asunto(s)
Linfocitos T CD8-positivos , Células Endoteliales , Nucleotidiltransferasas , Estructuras Linfoides Terciarias , Animales , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ratones , Células Endoteliales/inmunología , Estructuras Linfoides Terciarias/inmunología , Linfocitos T CD8-positivos/inmunología , Quimiocina CCL5/inmunología , Quimiocina CCL5/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal/inmunología , Receptores CCR5/inmunología , Receptores CCR5/genética , Receptores CCR5/metabolismo , Autoinmunidad/inmunologíaRESUMEN
Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here, we employ the zebrafish model to investigate mechanisms of CFM pathogenesis. In early embryos, tet2 and tet3 are essential for pharyngeal cartilage development. Single-cell RNA sequencing reveals that loss of Tet2/3 impairs chondrocyte differentiation due to insufficient BMP signaling. Moreover, biochemical and genetic evidence reveals that the sequence-specific 5mC/5hmC-binding protein, Sall4, binds the promoter of bmp4 to activate bmp4 expression and control pharyngeal cartilage development. Mechanistically, Sall4 directs co-phase separation of Tet2/3 with Sall4 to form condensates that mediate 5mC oxidation on the bmp4 promoter, thereby promoting bmp4 expression and enabling sufficient BMP signaling. These findings suggest the TET-BMP-Sall4 regulatory axis is critical for pharyngeal cartilage development. Collectively, our study provides insights into understanding craniofacial development and CFM pathogenesis.
Asunto(s)
Cartílago , Pez Cebra , Animales , Pez Cebra/metabolismo , Cartílago/metabolismo , Diferenciación Celular/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Condrogénesis/genéticaRESUMEN
Microglia are versatile regulators in brain development and disorders. Emerging evidence links microRNA (miRNA)-mediated regulation to microglial function; however, the exact underlying mechanism remains largely unknown. Here, we uncover the enrichment of miR-137, a neuropsychiatric-disorder-associated miRNA, in the microglial nucleus, and reveal its unexpected nuclear functions in maintaining the microglial global transcriptomic state, phagocytosis, and inflammatory response. Mechanistically, microglial Mir137 deletion increases chromatin accessibility, which contains binding motifs for the microglial master transcription factor Pu.1. Through biochemical and bioinformatics analyses, we propose that miR-137 modulates Pu.1-mediated gene expression by suppressing Pu.1 binding to chromatin. Importantly, we find that increased Pu.1 binding upregulates the target gene Jdp2 (Jun dimerization protein 2) and that knockdown of Jdp2 significantly suppresses the impaired phagocytosis and pro-inflammatory response in Mir137 knockout microglia. Collectively, our study provides evidence supporting the notion that nuclear miR-137 acts as a transcriptional modulator and that this microglia-specific function is essential for maintaining normal adult brain function.
Asunto(s)
MicroARNs , Microglía , Microglía/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Encéfalo/metabolismo , Homeostasis , Cromatina/metabolismoRESUMEN
Ribosome-associated protein quality control (RQC) is a protein surveillance mechanism that eliminates defective nascent polypeptides. The E3 ubiquitin ligase, Ltn1, is a key regulator of RQC that targets substrates for ubiquitination. Argonaute proteins (AGOs) are central players in miRNA-mediated gene silencing and have recently been shown to also regulate RQC by facilitating Ltn1. Therefore, AGOs directly coordinate post-transcriptional gene silencing and RQC, ensuring efficient gene silencing. We summarize the principles of RQC and the functions of AGOs in miRNA-mediated gene silencing, and discuss how AGOs associate with the endoplasmic reticulum (ER) to assist Ltn1 in controlling RQC. We highlight that RQC not only eliminates defective nascent polypeptides but also removes unwanted protein products when AGOs participate.
Asunto(s)
MicroARNs , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Péptidos/genética , MicroARNs/genética , MicroARNs/metabolismo , Biosíntesis de ProteínasRESUMEN
BACKGROUND: Oncolytic viral therapy is a promising method for tumor treatment. Currently, several oncolytic viruses (OVs) have been used as tumor therapy at different phases of research and clinical trials. OVs not only directly lyse tumor cells due to viral replication but also initiate host antitumor immune responses. Previous studies have primarily focused on how OVs activate adaptive immune responses in immune cells. However, the role of innate immune responses in tumors induced by OVs remains unclear. METHODS: To determine the innate immune responses induced by vesicular stomatitis virus (VSV), the mutant VSVΔM51 strain was used for the infection and quantitative polymerase chain reaction (qPCR) was employed to measure the transcriptional levels of antiviral genes. The knockdown efficiency of RIG-I was examined by qPCR. Viral titers were measured by plaque assays. Tumor models were established by intradermally implanting RIG-I-knockdown and control LLC cells into the flank of wild type C57BL/6J mice. When the tumors reached approximately 50mm3 , they were infected with VSVΔM51 via intratumoral injections to examine its therapeutic effect. RESULTS: Infection with VSVΔM51 triggered remarkable innate immune responses in several tumor cell lines through the cytoplasmic RIG-I sensing pathway. Moreover, we found that intratumoral injection of VSVΔM51 effectively reduced tumor growth in murine LCC lung cancer model. Importantly, VSVΔM51 -induced antitumor therapy was more effective in murine LLC tumor model established using Rig-I-knockdown cells compared with the tumor model established using control cells. CONCLUSION: RIG-I-mediated innate immune signaling in tumor cells plays a negative role in regulating antitumor therapy with VSVΔM51 virus.