RESUMEN
Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC.
Asunto(s)
Diferenciación Celular , Centro Germinal , Células Plasmáticas , Animales , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Ratones , Centro Germinal/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Ratones Endogámicos C57BL , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Ratones TransgénicosRESUMEN
Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Neutrófilos/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis/inmunología , Artritis Experimental/diagnóstico , Artritis Experimental/genética , Artritis Experimental/patología , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Quimiotaxis/genética , Quimiotaxis/inmunología , Colágeno/inmunología , Complemento C5a/inmunología , Complemento C5a/metabolismo , Citoplasma/inmunología , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Voluntarios Sanos , Humanos , Microscopía Intravital , Articulaciones/citología , Articulaciones/inmunología , Leucotrieno B4/inmunología , Leucotrieno B4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Polimorfismo de Nucleótido Simple , Proteómica , Índice de Severidad de la Enfermedad , Transducción de Señal/inmunología , Imagen de Lapso de TiempoRESUMEN
Histone lysine acylation, including acetylation and crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited to gene transcriptional activation. Here, we report that histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 in complex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits GAS41/SIN3A-HDAC1 complex to repress genes in chromatin, including cell-cycle inhibitor p21. GAS41 knockout or H3K27cr-binding depletion results in p21 de-repression, cell-cycle arrest, and tumor growth inhibition in mice, explaining a causal relationship between GAS41 and MYC gene amplification and p21 downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.
Asunto(s)
Cromatina , Histonas , Ratones , Animales , Cromatina/genética , Histonas/metabolismo , Lisina/metabolismo , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , AcetilaciónRESUMEN
Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.
Asunto(s)
COVID-19/metabolismo , COVID-19/virología , Interacciones Huésped-Patógeno , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , Sitios de Unión , COVID-19/genética , Línea Celular , Citocinas , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Lectinas Tipo C/química , Proteínas de la Membrana/química , Modelos Moleculares , Proteínas de Neoplasias/química , Unión Proteica , Conformación Proteica , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Relación Estructura-ActividadRESUMEN
Protein lipoylation, a crucial post-translational modification (PTM), plays a pivotal role in mitochondrial function and emerges as a key player in cell death through cuproptosis. This novel copper-driven cell death pathway is activated by excessive copper ions binding to lipoylated mitochondrial proteins, disrupting energy production and causing lethal protein aggregation and cell death. The intricate relationship among protein lipoylation, cellular energy metabolism, and cuproptosis offers a promising avenue for regulating essential cellular functions. This review focuses on the mechanisms of lipoylation and its significant impact on cell metabolism and cuproptosis, emphasizing the key genes involved and their implications for human diseases. It offers valuable insights into targeting dysregulated cellular metabolism for therapeutic purposes.
Asunto(s)
Cobre , Lipoilación , Mitocondrias , Humanos , Mitocondrias/metabolismo , Cobre/metabolismo , Animales , Proteínas Mitocondriales/metabolismo , Procesamiento Proteico-Postraduccional , Metabolismo EnergéticoRESUMEN
The regulation of protein-coding and noncoding RNAs is linked to nuclear processes, including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA-processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc-finger protein, Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Telómero/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , IntronesRESUMEN
BRD4 is a well-recognized transcriptional activator, but how it regulates gene transcriptional repression in a cell type-specific manner has remained elusive. In this study, we report that BRD4 works with Polycomb repressive complex 2 (PRC2) to repress transcriptional expression of the T-helper 2 (Th2)-negative regulators Foxp3 and E3-ubiqutin ligase Fbxw7 during lineage-specific differentiation of Th2 cells from mouse primary naïve CD4+ T cells. Brd4 binds to the lysine-acetylated-EED subunit of the PRC2 complex via its second bromodomain (BD2) to facilitate histone H3 lysine 27 trimethylation (H3K27me3) at target gene loci and thereby transcriptional repression. We found that Foxp3 represses transcription of Th2-specific transcription factor Gata3, while Fbxw7 promotes its ubiquitination-directed protein degradation. BRD4-mediated repression of Foxp3 and Fbxw7 in turn promotes BRD4- and Gata3-mediated transcriptional activation of Th2 cytokines including Il4, Il5, and Il13. Chemical inhibition of the BRD4 BD2 induces transcriptional de-repression of Foxp3 and Fbxw7, and thus transcriptional downregulation of Il4, Il5, and Il13, resulting in inhibition of Th2 cell lineage differentiation. Our study presents a previously unappreciated mechanism of BRD4's role in orchestrating a Th2-specific transcriptional program that coordinates gene repression and activation, and safeguards cell lineage differentiation.
Asunto(s)
Proteínas Nucleares , Complejo Represivo Polycomb 2 , Ratones , Animales , Complejo Represivo Polycomb 2/metabolismo , Proteínas Nucleares/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Interleucina-13/metabolismo , Interleucina-4/genética , Interleucina-5/metabolismo , Lisina , Diferenciación Celular/genética , Factores de Transcripción Forkhead/genéticaRESUMEN
Class II histone deacetylases (HDACs) are important in regulation of gene transcription during T cell development. However, our understanding of their cell-specific functions is limited. In this study, we reveal that class IIa Hdac4 and Hdac7 (Hdac4/7) are selectively induced in transcription, guiding the lineage-specific differentiation of mouse T-helper 17 (Th17) cells from naive CD4+ T cells. Importantly, Hdac4/7 are functionally dispensable in other Th subtypes. Mechanistically, Hdac4 interacts with the transcription factor (TF) JunB, facilitating the transcriptional activation of Th17 signature genes such as Il17a/f. Conversely, Hdac7 collaborates with the TF Aiolos and Smrt/Ncor1-Hdac3 corepressors to repress transcription of Th17 negative regulators, including Il2, in Th17 cell differentiation. Inhibiting Hdac4/7 through pharmacological or genetic methods effectively mitigates Th17 cell-mediated intestinal inflammation in a colitis mouse model. Our study uncovers molecular mechanisms where HDAC4 and HDAC7 function distinctively yet cooperatively in regulating ordered gene transcription during Th17 cell differentiation. These findings suggest a potential therapeutic strategy of targeting HDAC4/7 for treating Th17-related inflammatory diseases, such as ulcerative colitis.
Asunto(s)
Diferenciación Celular , Colitis , Histona Desacetilasas , Co-Represor 1 de Receptor Nuclear , Células Th17 , Animales , Células Th17/citología , Células Th17/metabolismo , Células Th17/inmunología , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Ratones , Colitis/genética , Colitis/metabolismo , Colitis/inmunología , Transcripción Genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , Co-Represor 2 de Receptor Nuclear/genética , Interleucina-17/metabolismo , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Humanos , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Interleucina-2/metabolismoRESUMEN
The tripartite motif (TRIM) protein family is the largest subfamily of E3 ubiquitin ligases, playing a crucial role in the antiviral process. In this study, we found that TRIM72, a member of the TRIM protein family, was increased in neuronal cells and mouse brains following rabies lyssavirus (RABV) infection. Over-expression of TRIM72 significantly reduced the viral titer of RABV in neuronal cells and mitigated the pathogenicity of RABV in mice. Furthermore, we found that TRIM72 over-expression effectively prevents the assembly and/or release of RABV. In terms of the mechanism, TRIM72 promotes the K48-linked ubiquitination of RABV Matrix protein (M), leading to the degradation of M through the proteasome pathway. TRIM72 directly interacts with M and the interaction sites were identified and confirmed through TRIM72-M interaction model construction and mutation analysis. Further investigation revealed that the degradation of M induced by TRIM72 was attributed to TRIM72's promotion of ubiquitination at site K195 in M. Importantly, the K195 site was found to be partially conserved among lyssavirus's M proteins, and TRIM72 over-expression induced the degradation of these lyssavirus M proteins. In summary, our study has uncovered a TRIM family protein, TRIM72, that can restrict lyssavirus replication by degrading M, and we have identified a novel ubiquitination site (K195) in lyssavirus M.
Asunto(s)
Rabia , Proteínas de Motivos Tripartitos , Animales , Ratones , Lyssavirus/metabolismo , Lyssavirus/patogenicidad , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Virus de la Rabia/metabolismo , Virus de la Rabia/patogenicidad , Rabia/genéticaRESUMEN
Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors.
Asunto(s)
Proteínas de Transporte de Catión , Hemocromatosis , Humanos , Hepcidinas/metabolismo , Hepcidinas/farmacología , Hierro/metabolismo , Proteínas de Transporte de Catión/metabolismoRESUMEN
Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.
Asunto(s)
Microscopía por Crioelectrón , Diacilglicerol O-Acetiltransferasa/química , Diacilglicerol O-Acetiltransferasa/metabolismo , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Sitios de Unión , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/ultraestructura , Diglicéridos/metabolismo , Humanos , Modelos Moleculares , Multimerización de Proteína , Relación Estructura-Actividad , Triglicéridos/metabolismoRESUMEN
Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.
Asunto(s)
Ácido Ascórbico , Purinas , Animales , Transporte Biológico , Purinas/metabolismo , Mutación , Sitios de Unión , Ácido Ascórbico/metabolismo , Mamíferos/metabolismoRESUMEN
Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.
Asunto(s)
Ácido Araquidónico , Ferroptosis , Gotas Lipídicas , Replicación Viral , Ferroptosis/efectos de los fármacos , Gotas Lipídicas/metabolismo , Gotas Lipídicas/efectos de los fármacos , Animales , Replicación Viral/efectos de los fármacos , Ratones , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Virus de la Encefalomiocarditis/efectos de los fármacos , Diacilglicerol O-Acetiltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Peroxidación de Lípido/efectos de los fármacos , Coenzima A Ligasas/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
DNA methylation plays a crucial role in the regulation of plant growth and the biosynthesis of secondary metabolites. Danshen (Salvia miltiorrhiza) is a valuable Chinese herbal medicine commonly used to treat cardiovascular diseases; its active ingredients are tanshinones and phenolic acids, which primarily accumulate in roots. Here, we conducted a targeted metabolic analysis of S. miltiorrhiza roots at 3 distinct growth stages: 40 d old (r40), 60 d old (r60), and 90 d old (r90). The contents of tanshinones (cryptotanshinone, tanshinone I, tanshinone IIA, and rosmariquinone) and phenolic acids (rosmarinic acid and salvianolic acid B) gradually increased during plant development. Whole-genome bisulfite sequencing and transcriptome sequencing of roots at the 3 growth stages revealed an increased level of DNA methylation in the CHH context (H represents A, T, or C) context at r90 compared with r40 and r60. Increased DNA methylation levels were associated with elevated expression of various genes linked to epigenetic regulations, including CHROMOMETHYLASE2 (SmCMT2), Decrease in DNA Methylation 1 (SmDDM1), Argonaute 4 (SmAGO4), and DOMAINS REARRANGED METHYLTRANSFERASE 1 (SmDRM1). Moreover, expression levels of many genes involved in tanshinone and salvianolic acid biosynthesis, such as copalyldiphosphate synthase 5 (SmCPS5), cytochrome P450-related enzyme (SmCYP71D464), geranylgeranyl diphosphate synthase (SmGGPPS1), geranyl diphosphate synthase (SmGPPS), hydroxyphenylpyruvate reductase (SmHPPR), and hydroxyphenylpyruvate dioxygenase (SmHPPD), were altered owing to hyper-methylation, indicating that DNA methylation plays an important role in regulating tanshinone and phenolic acid accumulation. Our data shed light on the epigenetic regulation of root growth and the biosynthesis of active ingredients in S. miltiorrhiza, providing crucial clues for further improvement of active compound production via molecular breeding in S. miltiorrhiza.
Asunto(s)
Abietanos , Hidroxibenzoatos , Salvia miltiorrhiza , Salvia miltiorrhiza/genética , Salvia miltiorrhiza/metabolismo , Metilación de ADN , Epigénesis Genética , Raíces de Plantas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The onset and progression of atherosclerosis are closely linked to the involvement of macrophages. While the contribution of NLRP3 inflammasome activation to the creation of a local highly inflammatory microenvironment is well recognized, the precise triggers remain unclear. In this study, we aimed to investigate the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia-induced glycolysis involving PFKFB3 in the development of atherosclerosis. To develop an atherosclerosis model, we selected ApoE knockout mice treated with a high-fat western diet. We then quantified the expression of HIF-1α, PFKFB3, and NLRP3. In addition, we administered the PFKFB3 inhibitor PFK158 during atherosclerosis modeling. The glycolytic activity was subsequently determined through 18F-FDG micro-PET/CT, ex vivo glucose uptake, and ECAR analysis. Furthermore, we employed lipopolysaccharide (LPS) and TNF-α to induce the differentiation of bone marrow-derived macrophages (BMDMs) into M1-like phenotypes under both hypoxic and normoxic conditions. Our histological analyses revealed the accumulation of PFKFB3 in human atherosclerotic plaques, demonstrating colocalization with NLRP3 expression and macrophages. Treatment with PFK158 reduced glycolytic activity and NLRP3 inflammasome activation, thereby mitigating the occurrence of atherosclerosis. Mechanistically, hypoxia promoted glycolytic reprogramming and NLRP3 inflammasome activation in BMDMs. Subsequent blocking of either HIF-1α or PFKFB3 downregulated the NLRP3/Caspase-1/IL-1ß pathway in hypoxic BMDMs. Our study demonstrated that the HIF-1α/PFKFB3/NLRP3 axis serves as a crucial mechanism for macrophage inflammation activation in the emergence of atherosclerosis. The therapeutic potential of PFKFB3 inhibition may represent a promising strategy for atheroprotection.
Asunto(s)
Aterosclerosis , Glucólisis , Inflamasomas , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Fosfofructoquinasa-2 , Animales , Fosfofructoquinasa-2/metabolismo , Fosfofructoquinasa-2/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Ratones , Macrófagos/metabolismo , Inflamasomas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Masculino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/metabolismo , Ratones NoqueadosRESUMEN
BET proteins commonly activate cellular gene expression, yet inhibiting their recruitment paradoxically reactivates latent HIV-1 transcription. Here we identify the short isoform of BET family member BRD4 (BRD4S) as a corepressor of HIV-1 transcription. We found that BRD4S was enriched in chromatin fractions of latently infected T cells, and it was more rapidly displaced from chromatin upon BET inhibition than the long isoform. BET inhibition induced marked nucleosome remodeling at the latent HIV-1 promoter, which was dependent on the activity of BRG1-associated factors (BAF), an SWI/SNF chromatin-remodeling complex with known repressive functions in HIV-1 transcription. BRD4S directly bound BRG1, a catalytic subunit of BAF, via its bromodomain and extraterminal (ET) domain, and this isoform was necessary for BRG1 recruitment to latent HIV-1 chromatin. Using chromatin immunoprecipitation sequencing (ChIP-seq) combined with assay for transposase-accessible chromatin coupled to high-throughput sequencing (ATAC-seq) data, we found that the latent HIV-1 promoter phenotypically resembles endogenous long terminal repeat (LTR) sequences, pointing to a select role of BRD4S-BRG1 complexes in genomic silencing of invasive retroelements.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN Viral/metabolismo , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Latencia del Virus , Azepinas/farmacología , Proteínas de Ciclo Celular , Cromatina/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/efectos de los fármacos , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Viral/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Regulación Viral de la Expresión Génica , Células HEK293 , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas , Interferencia de ARN , Retroelementos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/virología , Factores de Tiempo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Transfección , Triazoles/farmacología , Latencia del Virus/efectos de los fármacosRESUMEN
Heterochromatin can be epigenetically inherited in cis, leading to stable gene silencing. However, the mechanisms underlying heterochromatin inheritance remain unclear. Here, we identify Fft3, a fission yeast homolog of the mammalian SMARCAD1 SNF2 chromatin remodeler, as a factor uniquely required for heterochromatin inheritance, rather than for de novo assembly. Importantly, we find that Fft3 suppresses turnover of histones at heterochromatic loci to facilitate epigenetic transmission of heterochromatin in cycling cells. Moreover, Fft3 also precludes nucleosome turnover at several euchromatic loci to prevent R-loop formation, ensuring proper replication progression. Our analyses show that overexpression of Clr4/Suv39h, which is also required for efficient replication through these loci, suppresses phenotypes associated with the loss of Fft3. This work uncovers a conserved factor critical for epigenetic inheritance of heterochromatin and describes a mechanism in which suppression of nucleosome turnover prevents formation of structural barriers that impede replication at fragile regions in the genome.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , ADN de Hongos/biosíntesis , Epigénesis Genética , Herencia , Heterocromatina/metabolismo , Nucleosomas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN de Hongos/genética , Genotipo , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación , Nucleosomas/genética , Fenotipo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Factores de TiempoRESUMEN
The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.
Asunto(s)
Inmunidad Adaptativa , Diferenciación Celular , Cromatina/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , Células Th17/enzimología , Factores de Transcripción/metabolismo , Transcripción Genética , Acetilación , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Ciclina T/genética , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Nucleares/genética , Fenotipo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Células Th17/inmunología , Factores de Transcripción/genética , Transfección , CohesinasRESUMEN
Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.
Asunto(s)
Miembro 4 de la Familia de Transportadores de Soluto 12 , Simportadores , Cationes/metabolismo , Cloruros/metabolismo , Microscopía por Crioelectrón , Humanos , Transporte Iónico , Conformación Proteica en Hélice alfa , Miembro 4 de la Familia de Transportadores de Soluto 12/química , Simportadores/antagonistas & inhibidores , Simportadores/química , Cotransportadores de K ClRESUMEN
SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.