RESUMO
Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.
Assuntos
Antígenos CD , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I , Proteína do Gene 3 de Ativação de Linfócitos , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Receptor de Morte Celular Programada 1 , Animais , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Camundongos Endogâmicos C57BL , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Citotoxicidade Imunológica , Proliferação de Células , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologiaRESUMO
Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.
Assuntos
Linfócitos T CD8-Positivos , Proteína do Gene 3 de Ativação de Linfócitos , Melanoma , Receptor de Morte Celular Programada 1 , Humanos , Antígenos CD/metabolismo , Antígenos CD/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Citotoxicidade Imunológica , Proteínas de Grupo de Alta Mobilidade , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Proteína do Gene 3 de Ativação de Linfócitos/antagonistas & inibidores , Melanoma/imunologia , Melanoma/tratamento farmacológico , Melanoma/genética , Nivolumabe/uso terapêutico , Nivolumabe/farmacologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Transdução de SinaisRESUMO
Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.
Assuntos
COVID-19 , Receptor para Produtos Finais de Glicação Avançada , SARS-CoV-2 , Humanos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/patologia , COVID-19/complicações , COVID-19/virologia , Animais , Camundongos , Inflamação/metabolismo , Inflamação/patologia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/virologia , Lesão Pulmonar/imunologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Masculino , Pulmão/patologia , Pulmão/metabolismo , Pulmão/imunologia , FemininoRESUMO
Human CD8 tumor-infiltrating lymphocytes (TILs) with impaired effector functions and PD-1 expression are categorized as exhausted. However, the exhaustion-like features reported in TILs might stem from their activation rather than the consequence of T cell exhaustion itself. Using CRISPR-Cas9 and lentiviral overexpression in CD8 T cells from non-cancerous donors, we show that the T cell receptor (TCR)-induced transcription factor interferon regulatory factor 4 (IRF4) promotes cell proliferation and PD-1 expression and hampers effector functions and expression of nuclear factor κB (NF-κB)-regulated genes. While CD8 TILs with impaired interferon γ (IFNγ) production exhibit activation markers IRF4 and CD137 and exhaustion markers thymocyte selection associated high mobility group box (TOX) and PD-1, activated T cells in patients with COVID-19 do not demonstrate elevated levels of TOX and PD-1. These results confirm that IRF4+ TILs are exhausted rather than solely activated. Our study indicates, however, that PD-1 expression, low IFNγ production, and active cycling in TILs are all influenced by IRF4 upregulation after T cell activation.
Assuntos
Linfócitos T CD8-Positivos , Proliferação de Células , Fatores Reguladores de Interferon , Interferon gama , Ativação Linfocitária , Receptor de Morte Celular Programada 1 , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , COVID-19/imunologia , COVID-19/virologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , SARS-CoV-2/imunologia , NF-kappa B/metabolismo , Proteínas de Grupo de Alta MobilidadeRESUMO
Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1+ disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex.
Assuntos
Heterocromatina , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Heterocromatina/metabolismo , Heterocromatina/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Regulação Fúngica da Expressão Gênica , Epigênese Genética , Inativação Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genéticaRESUMO
Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.
Assuntos
Apirase , Linfócitos T CD8-Positivos , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Apirase/metabolismo , Apirase/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Pessoa de Meia-Idade , Ascite/imunologia , Ascite/patologia , Ascite/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Idoso , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/antagonistas & inibidores , Fator 1 de Transcrição de Linfócitos T/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Antígenos HLA-DR/metabolismo , Adulto , Exaustão das Células T , Proteínas de Grupo de Alta MobilidadeRESUMO
Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.
Assuntos
Cromatina , Proteínas de Grupo de Alta Mobilidade , Nucleossomos , Regiões Promotoras Genéticas , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Cromatina/metabolismo , Cromatina/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células HeLa , Montagem e Desmontagem da Cromatina , Células HEK293 , Elongação da Transcrição Genética , Terminação da Transcrição GenéticaRESUMO
When cells are stressed, DNA from energy-producing mitochondria can leak out and drive inflammatory immune responses if not cleared. Cells employ a quality control system called autophagy to specifically degrade damaged components. We discovered that mitochondrial transcription factor A (TFAM)-a protein that binds mitochondrial DNA (mtDNA)-helps to eliminate leaked mtDNA by interacting with the autophagy protein LC3 through an autolysosomal pathway (we term this nucleoid-phagy). TFAM contains a molecular zip code called the LC3 interacting region (LIR) motif that enables this binding. Although mutating TFAM's LIR motif did not affect its normal mitochondrial functions, more mtDNA accumulated in the cell cytoplasm, activating inflammatory signalling pathways. Thus, TFAM mediates autophagic removal of leaked mtDNA to restrict inflammation. Identifying this mechanism advances understanding of how cells exploit autophagy machinery to selectively target and degrade inflammatory mtDNA. These findings could inform research on diseases involving mitochondrial damage and inflammation.
Assuntos
Autofagia , DNA Mitocondrial , Proteínas de Ligação a DNA , Inflamação , Mitocôndrias , Proteínas Mitocondriais , Fatores de Transcrição , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Animais , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Ligação Proteica , Citoplasma/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Células HEK293 , Camundongos Endogâmicos C57BL , Proteínas de Grupo de Alta MobilidadeRESUMO
Mitochondrial transcription factor A, TFAM, is essential for mitochondrial function. We examined the effects of overexpressing the TFAM gene in mice. Two types of transgenic mice were created: TFAM heterozygous (TFAM Tg) and homozygous (TFAM Tg/Tg) mice. TFAM Tg/Tg mice were smaller and leaner notably with longer lifespans. In skeletal muscle, TFAM overexpression changed gene and protein expression in mitochondrial respiratory chain complexes, with down-regulation in complexes 1, 3, and 4 and up-regulation in complexes 2 and 5. The iMPAQT analysis combined with metabolomics was able to clearly separate the metabolomic features of the three types of mice, with increased degradation of fatty acids and branched-chain amino acids and decreased glycolysis in homozygotes. Consistent with these observations, comprehensive gene expression analysis revealed signs of mitochondrial stress, with elevation of genes associated with the integrated and mitochondrial stress responses, including Atf4, Fgf21, and Gdf15. These found that mitohormesis develops and metabolic shifts in skeletal muscle occur as an adaptive strategy.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Grupo de Alta Mobilidade , Longevidade , Camundongos Transgênicos , Proteínas Mitocondriais , Músculo Esquelético , Fatores de Transcrição , Animais , Camundongos , Músculo Esquelético/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Longevidade/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Masculino , Metabolômica/métodos , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Regulação da Expressão GênicaRESUMO
Alveolar macrophages (AMs) seed in lung during embryogenesis and become mature in perinatal period. Establishment of acclimatization to environmental challenges is important, whereas the detailed mechanisms that drive metabolic adaptation of AMs remains to be elucidated. Here, we showed that energy metabolism of AMs was transformed from glycolysis prenatally to oxidative phosphorylation (OXPHOS) postnatally accompanied by up-regulated expression of mitochondrial transcription factor A (TFAM). TFAM deficiency disturbed mitochondrial stability and decreased OXPHOS, which finally impaired AM maintenance and function, but not AM embryonic development. Mechanistically, Tfam-deletion resulted in impaired mitochondrial respiration and decreased ATP production, which triggered endoplasmic reticulum (ER) stress to cause B cell lymphoma 2 ovarian killer (BOK) accumulation and abnormal distribution of intracellular Ca2+, eventually led to induce AM apoptotic death. Thus, our data illustrated mitochondrial-dependent OXPHOS played a key role in orchestrating AM postnatal metabolic adaptation.
Assuntos
Pulmão , Macrófagos Alveolares , Mitocôndrias , Fosforilação Oxidativa , Animais , Macrófagos Alveolares/metabolismo , Mitocôndrias/metabolismo , Camundongos , Pulmão/metabolismo , Adaptação Fisiológica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estresse do Retículo Endoplasmático , Camundongos Knockout , Apoptose , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Feminino , Glicólise , Trifosfato de Adenosina/metabolismo , Proteínas de Grupo de Alta MobilidadeRESUMO
HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.
Assuntos
Proteínas de Grupo de Alta Mobilidade , Camundongos Endogâmicos C57BL , Animais , Masculino , Camundongos , beta-Galactosidase/metabolismo , beta-Galactosidase/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Óperon Lac/genética , Camundongos Transgênicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Under stress conditions, ribosome biogenesis is downregulated. This process requires that expression of ribosomal RNA, ribosomal protein, and ribosome biogenesis genes be controlled in a coordinated fashion. The mechanistic Target of Rapamycin Complex 1 (mTORC1) participates in sensing unfavorable conditions to effect the requisite change in gene expression. In Saccharomyces cerevisiae, downregulation of ribosomal protein genes involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which was implicated in communicating mTORC1 inhibition and hence was perceived as a repressor. We focus here on two ribosomal biogenesis genes, encoding Utp22p and the high mobility group protein Hmo1p, both of which are required for communication of mTORC1 inhibition to target genes. Crf1p functions as an activator on these genes as evidenced by reduced mRNA abundance and RNA polymerase II occupancy in a crf1Δ strain. Inhibition of mTORC1 has distinct effects on expression of HMO1 and UTP22; for example, on UTP22, but not on HMO1, the presence of Crf1p promotes the stable depletion of Ifh1p. Our data suggest that Crf1p functions as a weak activator, and that it may be required to prevent re-binding of Ifh1p to some gene promoters after mTORC1 inhibition in situations when Ifh1p is available. We propose that the inclusion of genes encoding proteins required for mTORC1-mediated downregulation of ribosomal protein genes in the same regulatory circuit as the ribosomal protein genes serves to optimize transcriptional responses during mTORC1 inhibition.
Assuntos
Regulação Fúngica da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , TransativadoresRESUMO
Recent studies have highlighted the pivotal roles of T cell transcription factors TCF-1 and TOX in modulating the immune response in cancer, with TCF-1 maintaining CD8+ T cell stemness and TOX promoting T cell exhaustion. The prognostic significance of these factors in lung adenocarcinoma (LUAD) remains a critical area of investigation. The retrospective study included 191 patients with LUAD who underwent surgery, of whom 83% were in stages II and III. These patients were divided into exploratory (n = 135) and validation (n = 56) groups based on the time of diagnosis. Multiplex fluorescence immunohistochemistry was used to examine the infiltration levels of CD8+ T cells, TCF1+ CD8+ T cells, and TOX+ CD8+ T cells. The percentage of CD8+ T cells in tumor was markedly lower than that in stroma (p < 0.05). In tumor-draining lymph nodes (TDLNs) invaded by tumor, the proportion of stem-like TCF1+ CD8+ T cells was significantly decreased (p < 0.01). Importantly, higher infiltration levels of CD8+ T cells and TCF1+ CD8+ T cells were associated with improved disease-free survival (DFS) (p = 0.009 and p = 0.006, respectively) and overall survival (OS) (p = 0.018 and p = 0.010, respectively). This study underscores the potential of TCF1+ CD8+ T cells as prognostic biomarkers in LUAD, providing insights into the tumor immune microenvironment and guiding future therapeutic strategies.
Assuntos
Adenocarcinoma de Pulmão , Linfócitos T CD8-Positivos , Fator 1-alfa Nuclear de Hepatócito , Neoplasias Pulmonares , Linfócitos do Interstício Tumoral , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/mortalidade , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/mortalidade , Prognóstico , Masculino , Pessoa de Meia-Idade , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Idoso , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Estudos Retrospectivos , Microambiente Tumoral/imunologia , Intervalo Livre de Doença , Proteínas de Grupo de Alta Mobilidade/metabolismo , TransativadoresRESUMO
Objective: This study aims to explore the effect of YiQi GuBen capsule on improving mitochondrial dysfunction in an animal model of asthma.Methods: The mice (n = 8) were divided into four groups including control (NC), ovalbumin (OVA), dexamethasone (OVA + DEX), and YiQi GuBen (OVA + YQGB) groups. Firstly, we established an OVA-induced mouse asthma model except for the NC group, which then were treated with dexamethasone and YiQi GuBen capsule. Subsequently, HE staining and Masson staining were used for pathological analysis of mice lung tissues. Next, we used transmission electron microscopy (TEM) to observe the effect of the Yiqi Guben capsule on the ultrastructure of mitochondria. Flow cytometry was used to analyze the ROS level, membrane potential, and the number of mitochondria in lung tissue. Moreover, we analyzed the copy number of mitochondrial DNA (mtDNA) and the expression levels of activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM).Results: The results of the pathological analysis showed that after treatment with the YiQi GuBen capsule, the lung tissue damage was significantly reduced. In addition, we observed that the ultrastructural damage of mitochondria was improved. Flow cytometry proved that after treatment with the YiQi GuBen capsule, the level of ROS in the mitochondria was effectively reduced, while the mitochondrial membrane potential decreased and the number increased significantly. Moreover, we found that the copy number of mtDNA was significantly increased and the expression levels of PGC-1α and TFAM were significantly upgraded.Conclusion: This study suggests YiQi GuBen capsule can effectively improve mitochondrial dysfunction in the OVA-induced mouse model.
Assuntos
Asma , DNA Mitocondrial , Medicamentos de Ervas Chinesas , Pulmão , Mitocôndrias , Ovalbumina , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Espécies Reativas de Oxigênio , Animais , Asma/tratamento farmacológico , Asma/patologia , Medicamentos de Ervas Chinesas/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Pulmão/efeitos dos fármacos , Pulmão/patologia , Espécies Reativas de Oxigênio/metabolismo , DNA Mitocondrial/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Feminino , Dexametasona/farmacologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Cápsulas , Proteínas de Grupo de Alta MobilidadeRESUMO
In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
Assuntos
Cromatina , DNA Topoisomerases Tipo II , Substâncias Intercalantes , RNA Polimerase II , Humanos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Carbazóis , Cromatina/metabolismo , Dicetopiperazinas , DNA/metabolismo , DNA/química , Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/metabolismo , Substâncias Intercalantes/farmacologia , Substâncias Intercalantes/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase I/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Inibidores da Topoisomerase II/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Aclarubicina/farmacologiaRESUMO
RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismoRESUMO
Retinoic acid-induced 1 (RAI1) encodes a transcriptional regulator critical for brain development and function. RAI1 haploinsufficiency in humans causes a syndromic autism spectrum disorder known as Smith-Magenis syndrome (SMS). The neuroanatomical distribution of RAI1 has not been quantitatively analyzed during the development of the prefrontal cortex, a brain region critical for cognitive function and social behaviors and commonly implicated in autism spectrum disorders, including SMS. Here, we performed comparative analyses to uncover the evolutionarily convergent and divergent expression profiles of RAI1 in major cell types during prefrontal cortex maturation in common marmoset monkeys (Callithrix jacchus) and mice (Mus musculus). We found that while RAI1 in both species is enriched in neurons, the percentage of excitatory neurons that express RAI1 is higher in newborn mice than in newborn marmosets. By contrast, RAI1 shows similar neural distribution in adult marmosets and adult mice. In marmosets, RAI1 is expressed in several primate-specific cell types, including intralaminar astrocytes and MEIS2-expressing prefrontal GABAergic neurons. At the molecular level, we discovered that RAI1 forms a protein complex with transcription factor 20 (TCF20), PHD finger protein 14 (PHF14), and high mobility group 20A (HMG20A) in the marmoset brain. In vitro assays in human cells revealed that TCF20 regulates RAI1 protein abundance. This work demonstrates that RAI1 expression and protein interactions are largely conserved but with some unique expression in primate-specific cells. The results also suggest that altered RAI1 abundance could contribute to disease features in disorders caused by TCF20 dosage imbalance.
Assuntos
Transtorno do Espectro Autista , Síndrome de Smith-Magenis , Transativadores , Animais , Camundongos , Transtorno do Espectro Autista/genética , Callithrix , Neurônios GABAérgicos , Proteínas de Grupo de Alta Mobilidade , Fatores de Transcrição/genética , Transativadores/genéticaRESUMO
Background: Atherosclerosis, a chronic inflammatory disease, poses a significant risk for cardiovascular disorders. Meanwhile, emerging evidence suggests that long noncoding RNAs (lncRNAs) play pivotal roles in diverse cardiovascular conditions. Nonetheless, the functional implications of lncRNAs in atherosclerosis remain largely unexplored. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess lncRNA HOTAIR and miR-19a-3p expression levels in patients with atherosclerosis and macrophage-derived foam cells. The release of inflammatory factors was evaluated using enzyme-linked immunosorbent assay (ELISA), while lipid uptake by foam cells was assessed through Oil Red O staining. Additionally, the targeting relationship between lncRNA HOTAIR and miR-19a-3p was validated via a Luciferase reporter assay. Results: LncRNA HOTAIR exhibited downregulation in the plasma of atherosclerosis patients and was found to be inhibited by ox-LDL in human macrophage-derived foam cells. Overexpression of HOTAIR effectively reduced lipid uptake and suppressed the inflammatory response by downregulating the expression of TNF-α and IL-6 during foam cell formation. Mechanistically, HOTAIR mitigated foam cell formation by repressing the expression of miR-19a-3p. Conclusions: In conclusion, our findings, in conjunction with previous studies, elucidate the role of HOTAIR in atherosclerosis. Specifically, we demonstrate that HOTAIR plays a role in alleviating foam cell formation and suppressing the inflammatory response by inhibiting miR-19a-3p in the context of atherosclerosis. Our results suggest the involvement of the TNF-α/miR-19a/HBP1/MIF pathway in mediating these effects. These findings contribute to a better understanding of atherosclerosis's molecular mechanisms and highlight the potential therapeutic implications of targeting HOTAIR and its associated pathways.
Assuntos
Aterosclerose , Doenças Cardiovasculares , MicroRNAs , RNA Longo não Codificante , Humanos , Aterosclerose/genética , Células Espumosas , Proteínas de Grupo de Alta Mobilidade , MicroRNAs/genética , Proteínas Repressoras , RNA Longo não Codificante/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Histone chaperone FACT (facilitates chromatin transcription) is well known to promote chromatin recovery during transcription. However, the mechanism how FACT regulates genome-wide chromatin accessibility and transcription factor binding has not been fully elucidated. Through loss-of-function studies, we show here that FACT component Ssrp1 is required for DNA replication and DNA damage repair and is also essential for progression of cell phase transition and cell proliferation in mouse embryonic fibroblast cells. On the molecular level, absence of the Ssrp1 leads to increased chromatin accessibility, enhanced CTCF binding, and a remarkable change in dynamic range of gene expression. Our study thus unequivocally uncovers a unique mechanism by which FACT complex regulates transcription by coordinating genome-wide chromatin accessibility and CTCF binding.
Assuntos
Fator de Ligação a CCCTC , Cromatina , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade , Chaperonas de Histonas , Animais , Camundongos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Replicação do DNA , Chaperonas de Histonas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Células NIH 3T3 , Reparo do DNARESUMO
RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. RSC utilizes its ATPase subunit, Sth1, to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such, when RSC is depleted, nucleosomes encroach NDRs, leading to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to those observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 (sth1-K501R) exhibited a severe reduction in TBP binding, but, surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. The Pol II occupancies further increased upon depleting endogenous Sth1 in the catalytic-dead mutant, suggesting that the inactive Sth1 contributes to Pol II accumulation in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones, FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could indicate severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.