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1.
Cell ; 185(22): 4049-4066.e25, 2022 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-36208623

RESUMEN

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.


Asunto(s)
Antígeno B7-H1 , Neoplasias , Humanos , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos , Inhibidores de Puntos de Control Inmunológico , Microambiente Tumoral , Neoplasias/terapia , Neoplasias/patología , Ganglios Linfáticos/patología
2.
Nat Immunol ; 23(2): 303-317, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34949833

RESUMEN

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3ß kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2-AKT-GSK3ß axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Ferroptosis/inmunología , Memoria Inmunológica/inmunología , Longevidad/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Diana Mecanicista del Complejo 2 de la Rapamicina/inmunología , Animales , Glucógeno Sintasa Quinasa 3 beta/inmunología , Peroxidación de Lípido/inmunología , Activación de Linfocitos/inmunología , Recuento de Linfocitos/métodos , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/inmunología
3.
Mol Cell ; 81(1): 25-37.e4, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33238160

RESUMEN

Among the five KCNQ channels, also known as the Kv7 voltage-gated potassium (Kv) channels, KCNQ2-KCNQ5 control neuronal excitability. Dysfunctions of KCNQ2-KCNQ5 are associated with neurological disorders such as epilepsy, deafness, and neuropathic pain. Here, we report the cryoelectron microscopy (cryo-EM) structures of human KCNQ4 and its complexes with the opener retigabine or the blocker linopirdine at overall resolutions of 2.5, 3.1, and 3.3 Å, respectively. In all structures, a phosphatidylinositol 4,5-bisphosphate (PIP2) molecule inserts its head group into a cavity within each voltage-sensing domain (VSD), revealing an unobserved binding mode for PIP2. Retigabine nestles in each fenestration, inducing local shifts. Instead of staying within the central pore, linopirdine resides in a cytosolic cavity underneath the inner gate. Electrophysiological analyses of various mutants corroborated the structural observations. Our studies reveal the molecular basis for the modulatory mechanism of neuronal KCNQ channels and provide a framework for structure-facilitated drug discovery targeting these important channels.


Asunto(s)
Carbamatos/farmacología , Indoles/farmacología , Canales de Potasio KCNQ , Fenilendiaminas/farmacología , Piridinas/farmacología , Animales , Microscopía por Crioelectrón , Humanos , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Mutación , Fosfatidilinositol 4,5-Difosfato/metabolismo , Dominios Proteicos , Células Sf9 , Spodoptera
4.
Proc Natl Acad Sci U S A ; 121(30): e2401091121, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39024109

RESUMEN

Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor's orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.


Asunto(s)
Entropía , Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G , Ligandos , Animales , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Unión Proteica , Ratones , Microscopía por Crioelectrón , Receptores de Cannabinoides/metabolismo , Receptores de Cannabinoides/química , Sitios de Unión
5.
Blood ; 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38657191

RESUMEN

Hematopoietic differentiation is controlled by intrinsic regulators and the extrinsic hematopoietic niche. Activating transcription factor 4 (ATF4) plays a crucial role in the function of fetal and adult hematopoietic stem cell maintenance; however, the precise function of ATF4 in the bone marrow niche and the mechanism by which ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to achieve conditional knockout of Atf4 in Cdh5+ endothelial cells, Prx1+ bone marrow stromal cells, Osx+ osteo-progenitor cells, and Mx1+ hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. Intriguingly, depletion of Atf4 in niche cells does not affect hematopoiesis; however, Atf4-deficient hematopoietic cells exhibit erythroid differentiation defects, leading to hypoplastic anemia. Mechanistically, ATF4 mediates direct regulation of Rps19bp1 transcription, which is, in turn, involved in 40S ribosomal subunit assembly to coordinate ribosome biogenesis and promote erythropoiesis. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, our findings highlight the indispensable role of the ATF4-RPS19BP1 axis in the regulation of erythropoiesis.

6.
Immunity ; 47(3): 538-551.e5, 2017 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-28930662

RESUMEN

Follicular regulatory T (Tfr) cells differentiate from conventional regulatory T (Treg) cells and suppress excessive germinal center (GC) responses by acting on both GC B cells and T follicular helper (Tfh) cells. Here, we examined the impact of mTOR, a serine/threonine protein kinase that senses and integrates diverse environmental cues, on the differentiation and functional competency of Tfr cells in response to protein immunization or viral infection. By genetically deleting Rptor or Rictor, essential components for mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), respectively, we found that mTORC1 but not mTORC2 is essential for Tfr differentiation. Mechanistically, mTORC1-mediated phosphorylation of the transcription factor STAT3 induced the expression of the transcription factor TCF-1 by promoting STAT3 binding to the Tcf7 5'-regulatory region. Subsequently, TCF-1 bound to the Bcl6 promoter to induce Bcl6 expression, which launched the Tfr cell differentiation program. Thus, mTORC1 initiates Tfr cell differentiation by activating the TCF-1-Bcl-6 axis during immunization or infection.


Asunto(s)
Inmunomodulación , Complejos Multiproteicos/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Biomarcadores , Diferenciación Celular/inmunología , Análisis por Conglomerados , Perfilación de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Inmunización , Inmunofenotipificación , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Transgénicos , Complejos Multiproteicos/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T Reguladores/citología , Serina-Treonina Quinasas TOR/genética
7.
Proc Natl Acad Sci U S A ; 120(44): e2306465120, 2023 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-37871214

RESUMEN

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.


Asunto(s)
Nanopartículas , Ácidos Nucleicos , Vacunas de ADN , Vacunas , Animales , Ratones , Vacunación Basada en Ácidos Nucleicos , Lisosomas , Sacarosa
8.
Proc Natl Acad Sci U S A ; 120(39): e2308435120, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37733739

RESUMEN

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.


Asunto(s)
Inhibición Psicológica , Neuralgia , Humanos , Microscopía por Crioelectrón , Unión Competitiva
9.
J Biol Chem ; 300(3): 105707, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38309505

RESUMEN

Liver cancer is notoriously refractory to conventional therapeutics. Tumor progression is governed by the interplay between tumor-promoting genes and tumor-suppressor genes. BRD4, an acetyl lysine-binding protein, is overexpressed in many cancer types, which promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains incompletely understood. In addition, understanding the regulatory mechanism of BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we investigated the potential relation between BRD4 protein level and P53, the most frequently dysregulated tumor suppressor. By analyzing the TCGA datasets, we first identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. Further investigation shows that P53 promotes BRD4 protein degradation. Mechanistically, P53 indirectly represses the transcription of USP1, a deubiquitinase, through the P21-RB1 axis. USP1 itself is also overexpressed in liver cancer and we show USP1 deubiquitinates BRD4 in vivo and in vitro, which increases BRD4 stability. With cell proliferation assays and xenograft model, we show the pro-tumor role of USP1 is partially mediated by BRD4. With functional transcriptomic analysis, we find the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB1-USP1-BRD4 axis in liver cancer.


Asunto(s)
Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular , Neoplasias Hepáticas , Proteínas Nucleares , Factores de Transcripción , Proteasas Ubiquitina-Específicas , Humanos , Proteínas que Contienen Bromodominio/genética , Proteínas que Contienen Bromodominio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Genes Supresores de Tumor , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión a Retinoblastoma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo
10.
Am J Hum Genet ; 109(4): 692-709, 2022 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-35271803

RESUMEN

Recent works have shown that SNP heritability-which is dominated by low-effect common variants-may not be the most relevant quantity for localizing high-effect/critical disease genes. Here, we introduce methods to estimate the proportion of phenotypic variance explained by a given assignment of SNPs to a single gene ("gene-level heritability"). We partition gene-level heritability by minor allele frequency (MAF) to find genes whose gene-level heritability is explained exclusively by "low-frequency/rare" variants (0.5% ≤ MAF < 1%). Applying our method to ∼16K protein-coding genes and 25 quantitative traits in the UK Biobank (N = 290K "White British"), we find that, on average across traits, ∼2.5% of nonzero-heritability genes have a rare-variant component and only ∼0.8% (327 gene-trait pairs) have heritability exclusively from rare variants. Of these 327 gene-trait pairs, 114 (35%) were not detected by existing gene-level association testing methods. The additional genes we identify are significantly enriched for known disease genes, and we find several examples of genes that have been previously implicated in phenotypically related Mendelian disorders. Notably, the rare-variant component of gene-level heritability exhibits trends different from those of common-variant gene-level heritability. For example, while total gene-level heritability increases with gene length, the rare-variant component is significantly larger among shorter genes; the cumulative distributions of gene-level heritability also vary across traits and reveal differences in the relative contributions of rare/common variants to overall gene-level polygenicity. While nonzero gene-level heritability does not imply causality, if interpreted in the correct context, gene-level heritability can reveal useful insights into complex-trait genetic architecture.


Asunto(s)
Estudio de Asociación del Genoma Completo , Herencia Multifactorial , Frecuencia de los Genes/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Herencia Multifactorial/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética
11.
Gastroenterology ; 167(2): 281-297, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38492894

RESUMEN

BACKGROUND & AIMS: Because pancreatic cancer responds poorly to chemotherapy and immunotherapy, it is necessary to identify novel targets and compounds to overcome resistance to treatment. METHODS: This study analyzed genomic single nucleotide polymorphism sequencing, single-cell RNA sequencing, and spatial transcriptomics. Ehf-knockout mice, KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+ and Pdx1-Cre) mice, CD45.1+ BALB/C nude mice, and CD34+ humanized mice were also used as subjects. Multiplexed immunohistochemistry and flow cytometry were performed to investigate the proportion of tumor-infiltrated C-X-C motif chemokine receptor 2 (CXCR2)+ neutrophils. In addition, multiplexed cytokines assays and chromatin immunoprecipitation assays were used to examine the mechanism. RESULTS: The TP53 mutation-mediated loss of tumoral EHF increased the recruitment of CXCR2+ neutrophils, modulated their spatial distribution, and further induced chemo- and immunotherapy resistance in clinical cohorts and preclinical syngeneic mice models. Mechanistically, EHF deficiency induced C-X-C motif chemokine ligand 1 (CXCL1) transcription to enhance in vitro and in vivo CXCR2+ neutrophils migration. Moreover, CXCL1 or CXCR2 blockade completely abolished the effect, indicating that EHF regulated CXCR2+ neutrophils migration in a CXCL1-CXCR2-dependent manner. The depletion of CXCR2+ neutrophils also blocked the in vivo effects of EHF deficiency on chemotherapy and immunotherapy resistance. The single-cell RNA-sequencing results of PDAC treated with Nifurtimox highlighted the therapeutic significance of Nifurtimox by elevating the expression of tumoral EHF and decreasing the weightage of CXCL1-CXCR2 pathway within the microenvironment. Importantly, by simultaneously inhibiting the JAK1/STAT1 pathway, it could significantly suppress the recruitment and function of CXCR2+ neutrophils, further sensitizing PDAC to chemotherapy and immunotherapies. CONCLUSIONS: The study demonstrated the role of EHF in the recruitment of CXCR2+ neutrophils and the promising role of Nifurtimox in sensitizing pancreatic cancer to chemotherapy and immunotherapy.


Asunto(s)
Quimiocina CXCL1 , Resistencia a Antineoplásicos , Infiltración Neutrófila , Neutrófilos , Neoplasias Pancreáticas , Receptores de Interleucina-8B , Animales , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Receptores de Interleucina-8B/antagonistas & inhibidores , Humanos , Infiltración Neutrófila/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Ratones , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/genética , Línea Celular Tumoral , Ratones Noqueados , Microambiente Tumoral , Inmunoterapia/métodos , Ratones Desnudos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Ratones Endogámicos BALB C , Antineoplásicos/farmacología , Transducción de Señal , Mutación , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología
12.
Brief Bioinform ; 24(4)2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37328639

RESUMEN

Precise targeting of transcription factor binding sites (TFBSs) is essential to comprehending transcriptional regulatory processes and investigating cellular function. Although several deep learning algorithms have been created to predict TFBSs, the models' intrinsic mechanisms and prediction results are difficult to explain. There is still room for improvement in prediction performance. We present DeepSTF, a unique deep-learning architecture for predicting TFBSs by integrating DNA sequence and shape profiles. We use the improved transformer encoder structure for the first time in the TFBSs prediction approach. DeepSTF extracts DNA higher-order sequence features using stacked convolutional neural networks (CNNs), whereas rich DNA shape profiles are extracted by combining improved transformer encoder structure and bidirectional long short-term memory (Bi-LSTM), and, finally, the derived higher-order sequence features and representative shape profiles are integrated into the channel dimension to achieve accurate TFBSs prediction. Experiments on 165 ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) datasets show that DeepSTF considerably outperforms several state-of-the-art algorithms in predicting TFBSs, and we explain the usefulness of the transformer encoder structure and the combined strategy using sequence features and shape profiles in capturing multiple dependencies and learning essential features. In addition, this paper examines the significance of DNA shape features predicting TFBSs. The source code of DeepSTF is available at https://github.com/YuBinLab-QUST/DeepSTF/.


Asunto(s)
ADN , Redes Neurales de la Computación , Sitios de Unión , Unión Proteica , ADN/genética , ADN/química , Factores de Transcripción/genética , Factores de Transcripción/química
13.
Plant Cell ; 34(11): 4274-4292, 2022 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-35929087

RESUMEN

Lipid droplets (LDs) are evolutionarily conserved organelles that serve as hubs of cellular lipid and energy metabolism in virtually all organisms. Mobilization of LDs is important in light-induced stomatal opening. However, whether and how LDs are involved in stomatal development remains unknown. We show here that Arabidopsis thaliana LIPID DROPLETS AND STOMATA 1 (LDS1)/RABC1 (At1g43890) encodes a member of the Rab GTPase family that is involved in regulating LD dynamics and stomatal morphogenesis. The expression of RABC1 is coordinated with the different phases of stomatal development. RABC1 targets to the surface of LDs in response to oleic acid application in a RABC1GEF1-dependent manner. RABC1 physically interacts with SEIPIN2/3, two orthologues of mammalian seipin, which function in the formation of LDs. Disruption of RABC1, RABC1GEF1, or SEIPIN2/3 resulted in aberrantly large LDs, severe defects in guard cell vacuole morphology, and stomatal function. In conclusion, these findings reveal an aspect of LD function and uncover a role for lipid metabolism in stomatal development in plants.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Animales , Arabidopsis/metabolismo , Gotas Lipídicas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo de los Lípidos/genética , Mamíferos/metabolismo
14.
FASEB J ; 38(13): e23701, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38941193

RESUMEN

Zearalenone (ZEN) is a mycotoxin known for its estrogen-like effects, which can disrupt the normal physiological function of endometrial cells and potentially lead to abortion in female animals. However, the precise mechanism by which ZEN regulates endometrial function remains unclear. In this study, we found that the binding receptor estrogen receptors for ZEN is extensively expressed across various segments of the uterus and within endometrial cells, and a certain concentration of ZEN treatment reduced the proliferation capacity of goat endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Meanwhile, cell cycle analysis revealed that ZEN treatment leaded to cell cycle arrest in goat EECs and ESCs. To explore the underlying mechanism, we investigated the mitochondrial quality control systems and observed that ZEN triggered excessive mitochondrial fission and disturbed the balance of mitochondrial fusion-fission dynamics, impaired mitochondrial biogenesis, increased mitochondrial unfolded protein response and mitophagy in goat EECs and ESCs. Additionally, ZEN treatment reduced the activities of mitochondrial respiratory chain complexes, heightened the production of hydrogen peroxide and reactive oxygen species, and caused cellular oxidative stress and mitochondrial dysfunction. These results suggest that ZEN has adverse effects on goat endometrium cells by disrupting the mitochondrial quality control system and affecting cell cycle and proliferation. Understanding the underlying molecular pathways involved in ZEN-induced mitochondrial dysfunction and its consequences on cell function will provide critical insights into the reproductive toxicity of ZEN and contribute to safeguarding the health and wellbeing of animals and humans exposed to this mycotoxin.


Asunto(s)
Proliferación Celular , Endometrio , Cabras , Mitocondrias , Zearalenona , Animales , Femenino , Endometrio/citología , Endometrio/metabolismo , Endometrio/efectos de los fármacos , Zearalenona/toxicidad , Zearalenona/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Cultivadas , Dinámicas Mitocondriales/efectos de los fármacos , Mitofagia/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/citología
15.
J Cell Physiol ; : e31368, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38982727

RESUMEN

Neuromedin S (NMS) plays key roles in reproductive regulation, while its function and mechanism in follicular development remain unclear. The current study aims to investigate the specific role and mechanisms of NMS and its receptors in regulating the proliferation and steroidogenesis of ovarian granulosa cells (GCs). Phenotypically, a certain concentration of NMS addition promoted the proliferation and estrogen production of goat GCs, accompanied by an increase in the G1/S cell population and upregulation of the expression levels of cyclin D1, cyclin dependent kinase 6, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450, family 11, subfamily A, polypeptide 1, while the effects of NMS treatment were effectively hindered by knockdown of neuromedin U receptor type 2 (NMUR2). Mechanistically, activation of NMUR2 with NMS maintained endoplasmic reticulum (ER) calcium (Ca2+) homeostasis by triggering the PLCG1-IP3R pathway, which helped preserve ER morphology, sustained an appropriate level of endoplasmic reticulum unfolded protein response (UPRer), and suppressed the nuclear translocation of activating transcription factor 4. Moreover, NMS maintained intracellular Ca2+ homeostasis to activate the calmodulin 1-large tumor suppressor kinase 1 pathway, ultimately orchestrating the regulation of goat GC proliferation and estrogen production through the Yes1 associated transcriptional regulator-ATF4-c-Jun pathway. Crucially, the effects of NMS were mitigated by concurrent knockdown of the NMUR2 gene. Collectively, these data suggest that activation of NMUR2 by NMS enhances cell proliferation and estrogen production in goat GCs through modulating the ER and intracellular Ca2+ homeostasis, leading to activation of the YAP1-ATF4-c-Jun pathway. These findings offer valuable insights into the regulatory mechanisms involved in follicular growth and development, providing a novel perspective for future research.

16.
Neuroimage ; 297: 120740, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39047590

RESUMEN

Modular dynamic graph theory metrics effectively capture the patterns of dynamic information interaction during human brain development. While existing research has employed modular algorithms to examine the overall impact of dynamic changes in community structure throughout development, there is a notable gap in understanding the cross-community dynamic changes within different functional networks during early childhood and their potential contributions to the efficiency of brain information transmission. This study seeks to address this gap by tracing the trajectories of cross-community structural changes within early childhood functional networks and modeling their contributions to information transmission efficiency. We analyzed 194 functional imaging scans from 83 children aged 2 to 8 years, who participated in passive viewing functional magnetic resonance imaging sessions. Utilizing sliding windows and modular algorithms, we evaluated three spatiotemporal metrics-temporal flexibility, spatiotemporal diversity, and within-community spatiotemporal diversity-and four centrality metrics: within-community degree centrality, eigenvector centrality, between-community degree centrality, and between-community eigenvector centrality. Mixed-effects linear models revealed significant age-related increases in the temporal flexibility of the default mode network (DMN), executive control network (ECN), and salience network (SN), indicating frequent adjustments in community structure within these networks during early childhood. Additionally, the spatiotemporal diversity of the SN also displayed significant age-related increases, highlighting its broad pattern of cross-community dynamic interactions. Conversely, within-community spatiotemporal diversity in the language network exhibited significant age-related decreases, reflecting the network's gradual functional specialization. Furthermore, our findings indicated significant age-related increases in between-community degree centrality across the DMN, ECN, SN, language network, and dorsal attention network, while between-community eigenvector centrality also increased significantly for the DMN, ECN, and SN. However, within-community eigenvector centrality remained stable across all functional networks during early childhood. These results suggest that while centrality of cross-community interactions in early childhood functional networks increases, centrality within communities remains stable. Finally, mediation analysis was conducted to explore the relationships between age, brain dynamic graph metrics, and both global and local efficiency based on community structure. The results indicated that the dynamic graph metrics of the SN primarily mediated the relationship between age and the decrease in global efficiency, while those of the DMN, language network, ECN, dorsal attention network, and SN primarily mediated the relationship between age and the increase in local efficiency. This pattern suggests a developmental trajectory in early childhood from global information integration to local information segregation, with the SN playing a pivotal role in this transformation. This study provides novel insights into the mechanisms by which early childhood brain functional development impacts information transmission efficiency through cross-community adjustments in functional networks.

17.
Anal Chem ; 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39042763

RESUMEN

Sensitive and reliable microRNA imaging in living cells has significant implications for clinical diagnosis and monitoring. Catalytic DNA circuits have emerged as potent tools for tracking these intracellular biomarkers and probing the corresponding biochemical processes. However, their utility is hindered by the low resistance to external interference, leading to undesired off-site activation and consequent signal leakage. Therefore, achieving the endogenous control of the DNA circuit's activation is preferable to the reliable target analysis in living cells. In this study, we attempted to address this challenge by engineering a simple yet effective endogenous glutathione (GSH)-regulated hybridization chain reaction (HCR) circuit for acquiring high-contrast miRNA imaging. Initially, the HCR hairpin reactants were blocked by the engineered disulfide-integrated DNA duplex, thus effectively passivating their sensing function. And the precaged HCR hairpin was liberated by the cell-specific GSH molecule, thus initiating the HCR system for selectively amplified detection of microRNA-21 (miR-21). This approach prevented unwanted signal leakage before exposure into target cells, thus ensuring robust miR-21 imaging with high accuracy and reliability in specific tumor cells. Moreover, the endogenously responsive HCR circuit established a link between the small regulatory factors and miRNA, thereby enhancing the signal gain. In summary, the endogenously activatable DNA circuit represents a versatile toolbox for robust bioanalysis and exploration of potential signaling pathways in living cells.

18.
Anal Chem ; 96(23): 9666-9675, 2024 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-38815126

RESUMEN

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.


Asunto(s)
ADN Catalítico , MicroARNs , MicroARNs/análisis , MicroARNs/metabolismo , ADN Catalítico/metabolismo , ADN Catalítico/química , Animales , Ratones , Humanos , Metilación de ADN , Imagen Óptica
19.
Mol Carcinog ; 63(6): 1038-1050, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38411361

RESUMEN

Heat shock protein 90 (Hsp90) is a tumor marker that accelerates cancer growth by disrupting protein homeostasis. However, concerns such as low clinical efficacy and drug resistance continue to be obstacles to the successful marketing of Hsp90 inhibitors. The cytoprotective function of autophagy has been identified as one of the mechanisms by which tumor cells gain resistance to chemotherapy. JD-02 was identified as a new Hsp90 inhibitor that suppressed colorectal cancer (CRC) growth by lowering client protein levels in vivo and in vitro. We found that JD-02 increased cellular autophagy, which inhibited apoptosis. JD-02 enhanced cytoprotective autophagy and regulated apoptotic suppression by increasing intracellular reactive oxygen species and inhibiting SRC protein levels, as demonstrated by quantitative proteomics, bioinformatic analysis, western blotting, and flow cytometry. This effect was reversed by autophagy inhibition. Therefore, due to the synergistic effects of Hsp90 and autophagy inhibitors in efficiently activating apoptotic pathways, they could potentially serve as promising therapeutic options for CRC.


Asunto(s)
Apoptosis , Autofagia , Neoplasias Colorrectales , Proteínas HSP90 de Choque Térmico , Especies Reactivas de Oxígeno , Humanos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Autofagia/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Animales , Ratones , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación Celular/efectos de los fármacos , Familia-src Quinasas/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Ratones Desnudos , Antineoplásicos/farmacología , Transducción de Señal/efectos de los fármacos , Ratones Endogámicos BALB C
20.
New Phytol ; 241(1): 227-242, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37853545

RESUMEN

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Celulosa/metabolismo , Regulación de la Expresión Génica de las Plantas , Fotosíntesis , Estomas de Plantas/metabolismo
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