RESUMEN
Neonates are highly susceptible to inflammation and infection. Here, we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPCs) respond to inflammation, testing the hypothesis that deficits in the engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical adult-like EM transcriptional program. Moreover, we find that fetal HSPCs can respond to EM-inducing inflammatory stimuli in vitro but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that the loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of fetal demise. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection.
Asunto(s)
Inflamación , Interleucina-10 , Mielopoyesis , Animales , Ratones , Embarazo/inmunología , Feto , Hematopoyesis , Células Madre Hematopoyéticas/citología , Inflamación/inmunología , Interleucina-10/inmunología , Animales Recién Nacidos , FemeninoRESUMEN
Peripheral sensory neurons widely innervate various tissues to continuously monitor and respond to environmental stimuli. Whether peripheral sensory neurons innervate the spleen and modulate splenic immune response remains poorly defined. Here, we demonstrate that nociceptive sensory nerve fibers extensively innervate the spleen along blood vessels and reach B cell zones. The spleen-innervating nociceptors predominantly originate from left T8-T13 dorsal root ganglia (DRGs), promoting the splenic germinal center (GC) response and humoral immunity. Nociceptors can be activated by antigen-induced accumulation of splenic prostaglandin E2 (PGE2) and then release calcitonin gene-related peptide (CGRP), which further promotes the splenic GC response at the early stage. Mechanistically, CGRP directly acts on B cells through its receptor CALCRL-RAMP1 via the cyclic AMP (cAMP) signaling pathway. Activating nociceptors by ingesting capsaicin enhances the splenic GC response and anti-influenza immunity. Collectively, our study establishes a specific DRG-spleen sensory neural connection that promotes humoral immunity, suggesting a promising approach for improving host defense by targeting the nociceptive nervous system.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Centro Germinal , Inmunidad Humoral , Bazo , Animales , Masculino , Ratones , Linfocitos B/inmunología , Linfocitos B/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina/farmacología , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Ganglios Espinales/metabolismo , Centro Germinal/inmunología , Ratones Endogámicos C57BL , Nociceptores/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal , Bazo/inervación , Bazo/inmunología , FemeninoRESUMEN
The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.
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Limosilactobacillus reuteri , Melanoma , Microambiente Tumoral , Humanos , Dieta , Inhibidores de Puntos de Control Inmunológico , Limosilactobacillus reuteri/metabolismo , Melanoma/terapia , Triptófano/metabolismo , Linfocitos T CD8-positivos/inmunología , Receptores de Hidrocarburo de Aril/agonistasRESUMEN
Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.
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Embrión de Mamíferos , Células Madre Embrionarias , Animales , Técnicas de Cocultivo , Macaca fascicularis , Células Madre Embrionarias/metabolismo , Diferenciación Celular , Endodermo/metabolismo , Linaje de la CélulaRESUMEN
Neuroepithelial crosstalk is critical for gut physiology. However, the mechanisms by which sensory neurons communicate with epithelial cells to mediate gut barrier protection at homeostasis and during inflammation are not well understood. Here, we find that Nav1.8+CGRP+ nociceptor neurons are juxtaposed with and signal to intestinal goblet cells to drive mucus secretion and gut protection. Nociceptor ablation led to decreased mucus thickness and dysbiosis, while chemogenetic nociceptor activation or capsaicin treatment induced mucus growth. Mouse and human goblet cells expressed Ramp1, receptor for the neuropeptide CGRP. Nociceptors signal via the CGRP-Ramp1 pathway to induce rapid goblet cell emptying and mucus secretion. Notably, commensal microbes activated nociceptors to control homeostatic CGRP release. In the absence of nociceptors or epithelial Ramp1, mice showed increased epithelial stress and susceptibility to colitis. Conversely, CGRP administration protected nociceptor-ablated mice against colitis. Our findings demonstrate a neuron-goblet cell axis that orchestrates gut mucosal barrier protection.
Asunto(s)
Colitis , Células Caliciformes , Ratones , Humanos , Animales , Células Caliciformes/metabolismo , Nociceptores/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Colitis/metabolismo , Moco/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/metabolismoRESUMEN
Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.
Asunto(s)
Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Ratones , Animales , Microbioma Gastrointestinal/fisiología , Nociceptores/fisiología , Sustancia P , Disbiosis , InflamaciónRESUMEN
Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.
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Reno , Cicatrización de Heridas , Adulto , Animales , Humanos , Cicatriz/patología , Fibroblastos/patología , Trasplante de Piel , Piel/patología , Feto/patologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Asunto(s)
Neuronas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Biosíntesis de Proteínas , Serina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Animales , Axones/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Codón/genética , Femenino , Glicina/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/metabolismo , Tejido Nervioso/patología , Consumo de Oxígeno , Neoplasias Pancreáticas/patología , Pirazoles , Pirimidinas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , RatasRESUMEN
Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Código de Histonas/efectos de los fármacos , Código de Histonas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/fisiología , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mutación , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Activación Transcripcional , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Factores de Transcripción p300-CBP/fisiologíaRESUMEN
Aged skin heals wounds poorly, increasing susceptibility to infections. Restoring homeostasis after wounding requires the coordinated actions of epidermal and immune cells. Here we find that both intrinsic defects and communication with immune cells are impaired in aged keratinocytes, diminishing their efficiency in restoring the skin barrier after wounding. At the wound-edge, aged keratinocytes display reduced proliferation and migration. They also exhibit a dampened ability to transcriptionally activate epithelial-immune crosstalk regulators, including a failure to properly activate/maintain dendritic epithelial T cells (DETCs), which promote re-epithelialization following injury. Probing mechanism, we find that aged keratinocytes near the wound edge don't efficiently upregulate Skints or activate STAT3. Notably, when epidermal Stat3, Skints, or DETCs are silenced in young skin, re-epithelialization following wounding is perturbed. These findings underscore epithelial-immune crosstalk perturbations in general, and Skints in particular, as critical mediators in the age-related decline in wound-repair.
Asunto(s)
Envejecimiento/fisiología , Subgrupos Linfocitarios/citología , Transducción de Señal , Cicatrización de Heridas , Animales , Interleucina-6/administración & dosificación , Queratinocitos/metabolismo , Ratones , Piel/citología , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX. We show that OppX acts as an RNA sponge of MicF sRNA, a prototypical porin repressor. OppX originates from the 5' UTR of oppABCDF, encoding the major inner-membrane oligopeptide transporter, and sequesters MicF's seed region to derepress the synthesis of the porin OmpF. Intriguingly, OppX operates as a true sponge, storing MicF in an inactive complex without affecting its levels or stability. Conservation of the opp-OppX-MicF-ompF axis in related bacteria suggests that it serves an important mechanism, adjusting envelope porosity to specific transport capacity. These data also highlight the resource value of this Salmonella RNA interactome, which will aid in unraveling RNA-centric regulation in enteric pathogens.
Asunto(s)
Regiones no Traducidas 5' , Membrana Celular/genética , Proteínas de Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , ARN Bacteriano/genética , Salmonella enterica/genética , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/metabolismo , Interacciones Huésped-Patógeno , Permeabilidad , Porinas/genética , Porinas/metabolismo , ARN Bacteriano/metabolismo , RNA-Seq , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidadRESUMEN
In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.
Asunto(s)
ADP-Ribosilación , Núcleo Celular/enzimología , Mitocondrias/enzimología , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP-Ribosilación/efectos de los fármacos , Animales , Antimicina A/análogos & derivados , Antimicina A/farmacología , Línea Celular , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Cromatina/química , Cromatina/metabolismo , Transporte de Electrón/efectos de los fármacos , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Metacrilatos/farmacología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/enzimología , Oligomicinas/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Poli(ADP-Ribosa) Polimerasa-1/genética , Rotenona/farmacología , Tiazoles/farmacologíaRESUMEN
The cJun NH2-terminal kinase (JNK) signaling pathway is activated by metabolic stress and promotes the development of metabolic syndrome, including hyperglycemia, hyperlipidemia, and insulin resistance. This integrated physiological response involves cross-talk between different organs. Here we demonstrate that JNK signaling in adipocytes causes an increased circulating concentration of the hepatokine fibroblast growth factor 21 (FGF21) that regulates systemic metabolism. The mechanism of organ crosstalk is mediated by a feed-forward regulatory loop caused by JNK-regulated FGF21 autocrine signaling in adipocytes that promotes increased expression of the adipokine adiponectin and subsequent hepatic expression of the hormone FGF21. The mechanism of organ cross-talk places circulating adiponectin downstream of autocrine FGF21 expressed by adipocytes and upstream of endocrine FGF21 expressed by hepatocytes. This regulatory loop represents a novel signaling paradigm that connects autocrine and endocrine signaling modes of the same hormone in different tissues.
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Tejido Adiposo/fisiología , Comunicación Autocrina/genética , Factores de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica/genética , Transducción de Señal/genética , Adipocitos/metabolismo , Adiponectina/metabolismo , Tejido Adiposo/fisiopatología , Animales , Sistema Endocrino/metabolismo , Metabolismo Energético/genética , Retroalimentación Fisiológica/fisiología , Factores de Crecimiento de Fibroblastos/sangre , Hepatocitos/metabolismo , Resistencia a la Insulina/genética , Hígado/metabolismo , MAP Quinasa Quinasa 4/deficiencia , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , RatonesRESUMEN
The glucocorticoid receptor (GR) is a major nuclear receptor (NR) drug target for the treatment of inflammatory disorders and several cancers. Despite the effectiveness of GR ligands, their systemic action triggers a plethora of side effects, limiting long-term use. Here, we discuss new concepts of and insights into GR mechanisms of action to assist in the identification of routes toward enhanced therapeutic benefits. We zoom in on the communication between different GR domains and how this is influenced by different ligands. We detail findings on the interaction between GR and chromatin, and highlight how condensate formation and coregulator confinement can perturb GR transcriptional responses. Last, we discuss the potential of novel ligands and the therapeutic exploitation of crosstalk with other NRs.
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Receptores de Glucocorticoides , Transducción de Señal , Receptores de Glucocorticoides/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Animales , Cromatina/metabolismo , LigandosRESUMEN
Dysregulated macrophage responses and changes in tissue metabolism are hallmarks of chronic inflammation in the skin. However, the metabolic cues that direct and support macrophage functions in the skin are poorly understood. Here, we show that during sterile skin inflammation, the epidermis and macrophages uniquely depend on glycolysis and the TCA cycle, respectively. This compartmentalisation is initiated by ROS-induced HIF-1α stabilization leading to enhanced glycolysis in the epidermis. The end-product of glycolysis, lactate, is then exported by epithelial cells and utilized by the dermal macrophages to induce their M2-like fates through NF-κB pathway activation. In addition, we show that psoriatic skin disorder is also driven by such lactate metabolite-mediated crosstalk between the epidermis and macrophages. Notably, small-molecule inhibitors of lactate transport in this setting attenuate sterile inflammation and psoriasis disease burden, and suppress M2-like fate acquisition in dermal macrophages. Our study identifies an essential role for the metabolite lactate in regulating macrophage responses to inflammation, which may be effectively targeted to treat inflammatory skin disorders such as psoriasis.
Asunto(s)
Ácido Láctico , Psoriasis , Ratones , Animales , Ácido Láctico/metabolismo , Ácido Láctico/farmacología , Piel/metabolismo , Macrófagos/metabolismo , Inflamación/metabolismo , Psoriasis/metabolismoRESUMEN
The consumption of Western-type calorically rich diets combined with chronic overnutrition and a sedentary lifestyle in Western societies evokes a state of chronic metabolic inflammation, termed metaflammation. Metaflammation contributes to the development of many prevalent non-communicable diseases (NCDs), and these lifestyle-associated pathologies represent a rising public health problem with global epidemic dimensions. A better understanding of how modern lifestyle and Western diet (WD) activate immune cells is essential for the development of efficient preventive and therapeutic strategies for common NCDs. Here, we review the current mechanistic understanding of how the Western lifestyle can induce metaflammation, and we discuss how this knowledge can be translated to protect the public from the health burden associated with their selected lifestyle.
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Dieta Occidental , Sistema Inmunológico/fisiología , Animales , Dieta , Susceptibilidad a Enfermedades , Retroalimentación Fisiológica , Microbioma Gastrointestinal , Homeostasis , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Inflamación/etiología , Inflamación/metabolismo , Especificidad de ÓrganosRESUMEN
In the last two decades, the term synaptopathy has been largely used to underline the concept that impairments of synaptic structure and function are the major determinant of brain disorders, including neurodevelopmental disorders. This notion emerged from the progress made in understanding the genetic architecture of neurodevelopmental disorders, which highlighted the convergence of genetic risk factors onto molecular pathways specifically localized at the synapse. However, the multifactorial origin of these disorders also indicated the key contribution of environmental factors. It is well recognized that inflammation is a risk factor for neurodevelopmental disorders, and several immune molecules critically contribute to synaptic dysfunction. In the present review, we highlight this concept, which we define by the term "immune-synaptopathy," and we discuss recent evidence suggesting a bi-directional link between the genetic architecture of individuals and maternal activation of the immune system in modulating brain developmental trajectories in health and disease.
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Trastornos del Neurodesarrollo , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Sinapsis/metabolismo , FamiliaRESUMEN
The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that autoinhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By delineating the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.
Asunto(s)
Histona Metiltransferasas/ultraestructura , Histonas/ultraestructura , Cromatina/genética , Microscopía por Crioelectrón/métodos , Metilación de ADN/genética , Epigénesis Genética/genética , Epigenómica/métodos , Proteínas Fúngicas/química , Histona Metiltransferasas/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/genética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Metiltransferasas/metabolismo , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Nucleosomas/metabolismo , Subunidades de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Células 3T3 NIH , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.