RESUMEN
Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Metilación de ADN , Histonas , Nucleosomas , Ensamble y Desensamble de Cromatina , ADN , Metilasas de Modificación del ADN , Epigénesis Genética , Histonas/genética , Nucleosomas/genética , Semen , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Single-molecule magnetic tweezers deliver magnetic force and torque to single target molecules, permitting the study of dynamic changes in biomolecular structures and their interactions. Because the magnetic tweezer setups can generate magnetic fields that vary slowly over tens of millimeters-far larger than the nanometer scale of the single molecule events being observed-this technique can maintain essentially constant force levels during biochemical experiments while generating a biologically meaningful force on the order of 1-100 pN. When using bead-tether constructs to pull on single molecules, smaller magnetic beads and shorter submicrometer tethers improve dynamic response times and measurement precision. In addition, employing high-speed cameras, stronger light sources, and a graphics programming unit permits true high-resolution single-molecule magnetic tweezers that can track nanometer changes in target molecules on a millisecond or even submillisecond time scale. The unique force-clamping capacity of the magnetic tweezer technique provides a way to conduct measurements under near-equilibrium conditions and directly map the energy landscapes underlying various molecular phenomena. High-resolution single-molecule magnetic tweezerscan thus be used to monitor crucial conformational changes in single-protein molecules, including those involved in mechanotransduction and protein folding.
Asunto(s)
ADN , Mecanotransducción Celular , ADN/química , Fenómenos MagnéticosRESUMEN
The anatomic location and immunologic characteristics of brain tumors result in strong lymphocyte suppression. Consequently, conventional immunotherapies targeting CD8 T cells are ineffective against brain tumors. Tumor cells escape immunosurveillance by various mechanisms and tumor cell metabolism can affect the metabolic states and functions of tumor-infiltrating lymphocytes. Here, we discovered that brain tumor cells had a particularly high demand for oxygen, which affected γδ T cell-mediated antitumor immune responses but not those of conventional T cells. Specifically, tumor hypoxia activated the γδ T cell protein kinase A pathway at a transcriptional level, resulting in repression of the activatory receptor NKG2D. Alleviating tumor hypoxia reinvigorated NKG2D expression and the antitumor function of γδ T cells. These results reveal a hypoxia-mediated mechanism through which brain tumors and γδ T cells interact and emphasize the importance of γδ T cells for antitumor immunity against brain tumors.
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Neoplasias Encefálicas/inmunología , Citotoxicidad Inmunológica , Glioblastoma/inmunología , Linfocitos Intraepiteliales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Escape del Tumor , Microambiente Tumoral , Animales , Apoptosis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Antígenos CD8/genética , Antígenos CD8/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes Codificadores de la Cadena delta de los Receptores de Linfocito T , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Linfocitos Intraepiteliales/metabolismo , Linfocitos Intraepiteliales/patología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Fenotipo , Transducción de Señal , Hipoxia TumoralRESUMEN
Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Familia 4 del Citocromo P450/deficiencia , Familia 4 del Citocromo P450/genética , Descubrimiento de Drogas , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
During neurodegenerative disease, the toxic accumulation of aggregates and misfolded proteins is often accompanied with widespread changes in peripheral metabolism, even in cells in which the aggregating protein is not present. The mechanism by which the central nervous system elicits a distal reaction to proteotoxic stress remains unknown. We hypothesized that the endocrine communication of neuronal stress plays a causative role in the changes in mitochondrial homeostasis associated with proteotoxic disease states. We find that an aggregation-prone protein expressed in the neurons of C. elegans binds to mitochondria, eliciting a global induction of a mitochondrial-specific unfolded protein response (UPR(mt)), affecting whole-animal physiology. Importantly, dense core vesicle release and secretion of the neurotransmitter serotonin is required for the signal's propagation. Collectively, these data suggest the commandeering of a nutrient sensing network to allow for cell-to-cell communication between mitochondria in response to protein folding stress in the nervous system.
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Homeostasis , Transducción de Señal , Respuesta de Proteína Desplegada , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/metabolismo , Comunicación Celular , Mitocondrias/metabolismo , Células Neuroendocrinas/metabolismo , Neuronas/metabolismo , Neuronas/patología , Péptidos/metabolismo , Pliegue de Proteína , Serotonina/metabolismoRESUMEN
Defects in mitochondrial metabolism have been increasingly linked with age-onset protein-misfolding diseases such as Alzheimer's, Parkinson's, and Huntington's. In response to protein-folding stress, compartment-specific unfolded protein responses (UPRs) within the ER, mitochondria, and cytosol work in parallel to ensure cellular protein homeostasis. While perturbation of individual compartments can make other compartments more susceptible to protein stress, the cellular conditions that trigger cross-communication between the individual UPRs remain poorly understood. We have uncovered a conserved, robust mechanism linking mitochondrial protein homeostasis and the cytosolic folding environment through changes in lipid homeostasis. Metabolic restructuring caused by mitochondrial stress or small-molecule activators trigger changes in gene expression coordinated uniquely by both the mitochondrial and cytosolic UPRs, protecting the cell from disease-associated proteins. Our data suggest an intricate and unique system of communication between UPRs in response to metabolic changes that could unveil new targets for diseases of protein misfolding.
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Citosol/fisiología , Respuesta al Choque Térmico/fisiología , Lípidos/biosíntesis , Mitocondrias/fisiología , Respuesta de Proteína Desplegada/fisiología , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/genética , Homeostasis , Humanos , Metabolismo de los Lípidos/genética , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/genética , Pliegue de ProteínaRESUMEN
The upper airway is an important site of infection, but immune memory in the human upper airway is poorly understood, with implications for COVID-19 and many other human diseases1-4. Here we demonstrate that nasal and nasopharyngeal swabs can be used to obtain insights into these challenging problems, and define distinct immune cell populations, including antigen-specific memory B cells and T cells, in two adjacent anatomical sites in the upper airway. Upper airway immune cell populations seemed stable over time in healthy adults undergoing monthly swabs for more than 1 year, and prominent tissue resident memory T (TRM) cell and B (BRM) cell populations were defined. Unexpectedly, germinal centre cells were identified consistently in many nasopharyngeal swabs. In subjects with SARS-CoV-2 breakthrough infections, local virus-specific BRM cells, plasma cells and germinal centre B cells were identified, with evidence of local priming and an enrichment of IgA+ memory B cells in upper airway compartments compared with blood. Local plasma cell populations were identified with transcriptional profiles of longevity. Local virus-specific memory CD4+ TRM cells and CD8+ TRM cells were identified, with diverse additional virus-specific T cells. Age-dependent upper airway immunological shifts were observed. These findings provide new understanding of immune memory at a principal mucosal barrier tissue in humans.
Asunto(s)
Memoria Inmunológica , Células B de Memoria , Células T de Memoria , Mucosa Nasal , Nasofaringe , SARS-CoV-2 , Adulto , Humanos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/citología , COVID-19/inmunología , COVID-19/virología , Centro Germinal/inmunología , Centro Germinal/citología , Inmunoglobulina A/inmunología , Memoria Inmunológica/inmunología , Células B de Memoria/inmunología , Células T de Memoria/inmunología , Mucosa Nasal/inmunología , Mucosa Nasal/virología , Nasofaringe/virología , Nasofaringe/inmunología , Células Plasmáticas/inmunología , Células Plasmáticas/citología , SARS-CoV-2/inmunologíaRESUMEN
Spin nematic is a magnetic analogue of classical liquid crystals, a fourth state of matter exhibiting characteristics of both liquid and solid1,2. Particularly intriguing is a valence-bond spin nematic3-5, in which spins are quantum entangled to form a multipolar order without breaking time-reversal symmetry, but its unambiguous experimental realization remains elusive. Here we establish a spin nematic phase in the square-lattice iridate Sr2IrO4, which approximately realizes a pseudospin one-half Heisenberg antiferromagnet in the strong spin-orbit coupling limit6-9. Upon cooling, the transition into the spin nematic phase at TC ≈ 263 K is marked by a divergence in the static spin quadrupole susceptibility extracted from our Raman spectra and concomitant emergence of a collective mode associated with the spontaneous breaking of rotational symmetries. The quadrupolar order persists in the antiferromagnetic phase below TN ≈ 230 K and becomes directly observable through its interference with the antiferromagnetic order in resonant X-ray diffraction, which allows us to uniquely determine its spatial structure. Further, we find using resonant inelastic X-ray scattering a complete breakdown of coherent magnon excitations at short-wavelength scales, suggesting a many-body quantum entanglement in the antiferromagnetic state10,11. Taken together, our results reveal a quantum order underlying the Néel antiferromagnet that is widely believed to be intimately connected to the mechanism of high-temperature superconductivity12,13.
RESUMEN
Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , ADN/genética , ADN/metabolismo , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/genéticaRESUMEN
The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/-) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Resistencia a la Enfermedad/inmunología , Interacciones Huésped-Patógeno/inmunología , Virosis/inmunología , Virosis/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Animales , Antivirales/farmacología , Modelos Animales de Enfermedad , Inmunidad Innata , Ratones , Ratones Noqueados , Péptidos/farmacología , Fosforilación , Unión Proteica , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/metabolismo , Infecciones por Virus ARN/virología , Virus ARN/efectos de los fármacos , Virus ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Ubiquitinación , Virosis/virología , Replicación ViralRESUMEN
Genome-wide association studies (GWASs) have unraveled a large number of cancer risk alleles. Understanding how these allelic variants predispose to disease is a major bottleneck confronting translational application. In this issue, Li and colleagues combine GWASs with The Cancer Genome Atlas (TCGA) to disambiguate the contributions of germline and somatic variants to tumorigenic gene expression programs. They find that close to half of the known risk alleles for estrogen receptor (ER)-positive breast cancer are expression quantitative trait loci (eQTLs) acting upon major determinants of gene expression in tumors.
RESUMEN
Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Indoles/farmacología , Neoplasias Pulmonares/metabolismo , Triazinas/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteína Coatómero/metabolismo , Femenino , Genes ras , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Lisosomas/metabolismo , Ratones , Terapia Molecular Dirigida , Proteína con Dominio Pirina 3 de la Familia NLR , Trasplante de Neoplasias , Fosforilación OxidativaRESUMEN
The Notch signaling pathway is a highly conserved, fundamental process to embryogenesis and neurogenesis. While force-induced conformational change is known to activate Notch receptors, Smyrlaki et al. recently used DNA origami to reveal an additional, force-independent mode of Notch activation via soluble presentation of spatially controlled ligand nanopatterns.
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Receptores Notch , Transducción de Señal , Receptores Notch/genética , Receptores Notch/metabolismo , Desarrollo Embrionario , Neurogénesis , ADN/genéticaRESUMEN
Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.
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Trastorno del Espectro Autista , Cilios , Epéndimo , Ratones Noqueados , Fenotipo , Animales , Masculino , Ratones , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/fisiopatología , Conducta Animal , Cilios/metabolismo , Modelos Animales de Enfermedad , Epéndimo/metabolismo , Hipocampo/metabolismo , Hidrocefalia/genética , Hidrocefalia/metabolismo , Hidrocefalia/patología , Hidrocefalia/fisiopatología , Katanina/metabolismo , Katanina/genética , Ratones Endogámicos C57BL , Neuronas/metabolismo , Sinapsis/metabolismo , Transcriptoma/genéticaRESUMEN
Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mitofagia , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Dinaminas , Metabolismo Energético , GTP Fosfohidrolasas/genética , Células HEK293 , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Mutación , Neuronas/metabolismo , Neuronas/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Tiempo , Zinc/metabolismoRESUMEN
Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral/metabolismo , Metaboloma/fisiología , Biomarcadores de Tumor/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Regulación Neoplásica de la Expresión Génica/fisiología , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Redes y Vías Metabólicas/genética , Metabolómica/métodos , Neoplasias/metabolismoRESUMEN
The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs.
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Células Madre Pluripotentes Inducidas , Síndrome de QT Prolongado , Humanos , Miocitos Cardíacos/metabolismo , Mutación del Sistema de Lectura , Células Madre Pluripotentes Inducidas/metabolismo , Canales de Potasio Éter-A-Go-Go/genética , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/metabolismo , Heterocigoto , Mutación , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismoRESUMEN
BACKGROUND: Atherosclerosis is a chronic inflammatory disease causing a fatal plaque rupture, and its key aspect is a failure to resolve inflammation. We hypothesize that macrophage-targeted near-infrared fluorescence emitting photoactivation could simultaneously assess macrophage/lipid-rich plaques in vivo and facilitate inflammation resolution. METHODS: We fabricated a Dectin-1-targeted photoactivatable theranostic agent through the chemical conjugation of the near-infrared fluorescence-emitting photosensitizer chlorin e6 and the Dectin-1 ligand laminarin (laminarin-chlorin e6 [LAM-Ce6]). Intravascular photoactivation by a customized fiber-based diffuser after administration of LAM-Ce6 effectively reduced inflammation in the targeted plaques of atherosclerotic rabbits in vivo as serially assessed by dual-modal optical coherence tomography-near-infrared fluorescence structural-molecular catheter imaging after 4 weeks. RESULTS: The number of apoptotic macrophages peaked at 1 day after laser irradiation and then resolved until 4 weeks. Autophagy was strongly augmented 1 hour after the light therapy, with the formation of autophagolysosomes. LAM-Ce6 photoactivation increased the terminal deoxynucleotidyl transferase dUTP (deoxyuridine triphosphate) nick end labeling/RAM11 (rabbit monocyte/macrophage antibody)- and MerTK (c-Mer tyrosine kinase)-positive cells in the plaques, suggesting enhanced efferocytosis. In line with inflammation resolution, photoactivation reduced the plaque burden through fibrotic replacement via the TGF (transforming growth factor)-ß/CTGF (connective tissue growth factor) pathway. CONCLUSIONS: Optical coherence tomography-near-infrared fluorescence imaging-guided macrophage Dectin-1-targetable photoactivation could induce the transition of macrophage/lipid-rich plaques into collagen-rich lesions through autophagy-mediated inflammation resolution and TGF-ß-dependent fibrotic replacement. This novel strategy offers a new opportunity for the catheter-based theranostic strategy.
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Clorofilidas , Imagen Multimodal , Fármacos Fotosensibilizantes , Placa Aterosclerótica , Porfirinas , Tomografía de Coherencia Óptica , Animales , Placa Aterosclerótica/diagnóstico por imagen , Conejos , Imagen Multimodal/métodos , Tomografía de Coherencia Óptica/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Macrófagos/metabolismo , Nanomedicina Teranóstica/métodos , Ratones , Masculino , Autofagia , Tirosina Quinasa c-Mer/metabolismo , ApoptosisRESUMEN
Mechanosensory feedback from the digestive tract to the brain is critical for limiting excessive food and water intake, but the underlying gut-brain communication pathways and mechanisms remain poorly understood1-12. Here we show that, in mice, neurons in the parabrachial nucleus that express the prodynorphin gene (hereafter, PBPdyn neurons) monitor the intake of both fluids and solids, using mechanosensory signals that arise from the upper digestive tract. Most individual PBPdyn neurons are activated by ingestion as well as the stimulation of the mouth and stomach, which indicates the representation of integrated sensory signals across distinct parts of the digestive tract. PBPdyn neurons are anatomically connected to the digestive periphery via cranial and spinal pathways; we show that, among these pathways, the vagus nerve conveys stomach-distension signals to PBPdyn neurons. Upon receipt of these signals, these neurons produce aversive and sustained appetite-suppressing signals, which discourages the initiation of feeding and drinking (fully recapitulating the symptoms of gastric distension) in part via signalling to the paraventricular hypothalamus. By contrast, inhibiting the same population of PBPdyn neurons induces overconsumption only if a drive for ingestion exists, which confirms that these neurons mediate negative feedback signalling. Our findings reveal a neural mechanism that underlies the mechanosensory monitoring of ingestion and negative feedback control of intake behaviours upon distension of the digestive tract.
Asunto(s)
Ingestión de Alimentos , Retroalimentación , Neuronas/fisiología , Animales , Encefalinas/genética , Encefalinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Tracto Gastrointestinal Superior/fisiologíaRESUMEN
During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.