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The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 µs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA.
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ADN , Simulación de Dinámica Molecular , FN-kappa B , Unión Proteica , ADN/metabolismo , Humanos , FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genética , Sitios de Unión , Cristalografía por Rayos XRESUMEN
BACKGROUND: Mean perfusion pressure (MPP) has recently emerged as a potential biomarker for personalized management of tissue perfusion in critically ill patients. However, its association with the occurrence of acute kidney injury (AKI) in septic patients and the optimal MPP range remain uncertain. Therefore, this study aims to investigate the relationship between MPP and AKI in critically ill patients with sepsis. METHODS: We identified 5867 patients with sepsis from the MIMIC-IV database who met the inclusion and exclusion criteria. The exposure variable was the first set of MPP measured within 24 h after ICU admission with invasive hemodynamic monitoring. The primary outcome was the incidence of AKI at 7 days following ICU admission according to the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines. Secondary outcomes included in-hospital mortality, lengths of ICU, and hospital stay. Optimal cut-off point for MPP were determined using the Youden index, and multivariable logistic regression was employed to examine the association between MPP and AKI. Subgroup analyses were conducted to enhance result robustness. Kaplan-Meier survival analysis was utilized to evaluate in-hospital mortality rates categorized by MPP. RESULTS: A total of 5,867 patients with sepsis were included in this study, and the overall incidence of AKI was 82.3%(4828/5867). Patients were categorized into low MPP (< 63 mmHg) and high MPP (≥ 63 mmHg) groups using the optimal ROC curve-derived cut-off point. The incidence of AKI in the low MPP group was higher than that in the high MPP group (87.6% vs. 78.3%, P < 0.001). Multivariable logistic regression analysis adjusted for confounding factors revealed that each 1 mmHg increase in MPP as a continuous variable was associated with a 2% decrease in AKI incidence within 7 days of ICU admission (OR:0.98, 95%CI:0.97-0.99, P < 0.001). When MPP was used as a categorical variable, patients in the high MPP group had a lower risk of AKI than those in the low MPP group (OR:0.71, 95%CI:0.61-0.83, P = 0.001). Subgroup analyses demonstrated a consistent association between MPP and AKI risk across all variables assessed (P for interaction all > 0.05). Kaplan-Meier curve analysis demonstrated a higher survival rate during hospitalization in the high MPP group compared to the low MPP group (Log-rank test, P < 0.0001). CONCLUSIONS: Lower levels of MPP are associated with an increased incidence of AKI at 7 days in critically ill patients with sepsis.
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Lesión Renal Aguda , Enfermedad Crítica , Mortalidad Hospitalaria , Unidades de Cuidados Intensivos , Sepsis , Humanos , Lesión Renal Aguda/epidemiología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/mortalidad , Masculino , Femenino , Persona de Mediana Edad , Sepsis/complicaciones , Estudios Retrospectivos , Anciano , Incidencia , Factores de Riesgo , Tiempo de Internación/estadística & datos numéricosRESUMEN
Freezing of gait (FoG) is one of the most distressing symptoms of Parkinson's Disease (PD), commonly occurring in patients at middle and late stages of the disease. Automatic and accurate FoG detection and prediction have emerged as a promising tool for long-term monitoring of PD and implementation of gait assistance systems. This paper reviews the recent development of FoG detection and prediction using wearable sensors, with attention on identifying knowledge gaps that need to be filled in future research. This review searched the PubMed and Web of Science databases to collect studies that detect or predict FoG with wearable sensors. After screening, 89 of 270 articles were included. The data description, extracted features, detection/prediction methods, and classification performance were extracted from the articles. As the number of papers of this area is increasing, the performance has been steadily improved. However, small datasets and inconsistent evaluation processes still hinder the application of FoG detection and prediction with wearable sensors in clinical practice.
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Trastornos Neurológicos de la Marcha , Enfermedad de Parkinson , Dispositivos Electrónicos Vestibles , Humanos , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/diagnóstico , Trastornos Neurológicos de la Marcha/diagnóstico , Trastornos Neurológicos de la Marcha/etiología , Marcha/fisiologíaRESUMEN
The mechanisms underlying subclinical hypothyroidism (SCH) remain unclear, making timely and accurate differentiation between hypothyroidism and SCH, as well as severity assessment, challenging. This study aimed to investigate the role of NFE2 like bZIP transcription factor 2 (Nrf2), gp91phox, and interleukin-17 (IL-17) in the pathogenesis of SCH. In this prospective comparative study, 105 SCH patients, 105 hypothyroidism patients, and 105 healthy individuals were enrolled from January 2022 to August 2023. SCH patients were categorized into mild-moderate and severe groups based on thyroid-stimulating hormone (TSH) levels. Levels of TSH, free T4 (FT4), free T3 (FT3), thyroglobulin antibodies (TG-Ab), thyroid peroxidase antibodies (TPO-Ab), cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-ch), and low-density lipoprotein-cholesterol (LDL-ch) were measured. Nrf2, IL-1ß, IL-6, IL-17, and gp91phox levels were tested using ELISA. Nrf2, IL-17 and gp91phox were significantly higher in SCH and hypothyroidism patients compared to the healthy controls, with hypothyroidism patients showing the highest levels. Nrf2 levels were negatively correlated with TSH, TG-Ab and IL-17, but not gp91phox. Nrf2, IL-17 and gp91phox could be used for diagnosis of SCH and severe SCH. Only TG-Ab, IL-17 and gp91phox were independent risk factors for severe SCH. This study demonstrates a negative correlation between serum Nrf2 levels and SCH severity. TG-Ab, IL-17, and gp91phox are independent risk factors, and their associations with SCH pathology suggest their potential roles in the disease mechanism. These findings provide insights into SCH pathogenesis and highlight the need for further research to elucidate their diagnostic or prognostic significance.
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The pathophysiological mechanism of hepatic fibrosis (HF) is related to the excessive activation of the DNA repair enzyme poly ADP-ribose polymerase-1 (PARP-1). The drugs, targeting PARP-1, are scarce. Therefore, the lead compound, moderately inhibiting PARP-1, with anti-HF properties should be identified. This study screened dihydrokaempferol (DHK) from herbs based on preliminary studies to intervene in a CCl4-induced liver injury and HF model in mice. In vitro, the expression levels of PARP-1-regulated related proteins and phosphorylation were examined. The binding pattern of DHK and PARP-1 was analyzed using molecular docking and molecular dynamics platforms. The results showed that DHK could significantly attenuate CCl4-induced liver injury and HF in mice. Moreover, it could also attenuate the toxic effects of CCl4 on HepG2 and inhibit α-SMA and Collagen 1/3 synthesis of LX-2 cells in-vitro. Molecular docking revealed that DHK could competitively bind to the Glu-988 and His-862 residues of the upstream DNA repair enzyme PARP-1, moderately inhibiting its overactivation. This led to maintaining NAD+ levels and energy metabolism in hepatocytes and inhibiting the activation of PARP-1-regulated downstream signaling pathways (TGF-ß1, etc.), related proteins (p-Smd2/3, etc.), and inflammatory mediators while acting indirectly. Thus, DHK could attenuate CCl4-induced liver injury and HF in mice in a different mechanism from those of the existing reported flavonoids. It was associated with inhibiting the expression of downstream pathways and related cytokines by competitively binding to PARP-1. This study might provide a basis and direction for the design and exploration of anti-HF lead compounds.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Citocinas , Animales , Ratones , Tetracloruro de Carbono/toxicidad , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/patología , Citocinas/metabolismo , Flavonoides/farmacología , Células Estrelladas Hepáticas , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/prevención & control , Simulación del Acoplamiento Molecular , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
AIM: To systematically evaluate the effect of intensive care unit diary psychotherapy on the incidence of posttraumatic stress disorder, anxiety, and depression after discharge from intensive care unit. BACKGROUND: Many studies have reported the potential advantages and risks of intensive care unit diary psychotherapy in adult patients discharged from intensive care unit, but the results are divergent. DESIGN: Systematic review and meta-analysis of prospective randomized controlled or case-controlled studies. DATA SOURCE: Databases such as Cochran Library, Pubmed, Embase, CINAHL, and ProQuest databases, China national knowledge infrastructure (CNKI) were searched for literatures published from January 2000-March 2020. REVIEW METHODS: We use the Cochrane Risk of Bias Tool for quality assessment and audit manager 5.3 software for meta-analysis. The main result is the incidence of posttraumatic stress disorder, anxiety, and depression. RESULTS: Ten studies meeting the inclusion criteria were identified, including eight randomized controlled studies and two case-controlled studies, with a total of 1,210 patients. The pooled results of this meta-analysis indicated that the intensive care unit diary could reduce the incidence of posttraumatic stress disorder, anxiety, and depression. CONCLUSION: This study showed that an intensive care unit diary could improve the psychological symptoms of adult intensive care unit patients after discharge. However, due to limitations such as publication bias and case sample size, the results should be carefully considered. Researchers need to further clarify the multidisciplinary collaborative process of intensive care unit diary therapy, the real beneficiaries, and its impact on family members' psychological status by conducting large, robust studies in the future. IMPACT: This study's findings suggest that medical staff need to re-examine the role of intensive care unit diary therapy, its standardized implementation and provide effective intervention for reducing psychological stress-related symptoms of intensive care unit patients after discharge.
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Trastornos por Estrés Postraumático , Adulto , Ansiedad , China , Humanos , Incidencia , Unidades de Cuidados Intensivos , Estudios Prospectivos , Trastornos por Estrés Postraumático/epidemiología , Trastornos por Estrés Postraumático/terapia , SobrevivientesRESUMEN
Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.
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Coenzimas/química , Proteínas de Unión al ADN/química , FN-kappa B/química , Factor de Transcripción ReIA/química , Línea Celular , Núcleo Celular/química , Núcleo Celular/genética , Coenzimas/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Proteínas Ribosómicas/química , Factor de Transcripción ReIA/genética , Proteína p53 Supresora de Tumor/químicaRESUMEN
The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.
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ADN/metabolismo , Selectina E/genética , Regiones Promotoras Genéticas , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , ADN/genética , Regulación de la Expresión Génica , Ratones , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Factor de Transcripción ReIA/químicaRESUMEN
Length of hospital stay (LOS) of asthma can be a reflection of the disease burden faced by patients, and it is also sensitive to air pollution. This study aims at estimating and validating the effects of air pollution and readmission on the LOS for those who have asthma, considering their readmission history, minimum temperature, and threshold effects of air pollutants. In addition, sex, age, and season were also constructed for stratification to achieve more precise and specific results. The results show that no significant effects of PM2.5 and NO2 on LOS were observed in any of the patients, but there were significant effects of PM2.5 and NO2 on LOS when a stratifying subgroup analysis was performed. The effect of PM10 on LOS was found to be lower than that of PM2.5 and higher than that of NO2 . SO2 did not have a significant effect on LOS for patients with asthma in our study. Our study confirmed that the adverse effects of air pollutants (such as PM10 ) on LOS for patients with asthma existed; in addition, these effects vary for different stratifications. We measured the effects of air pollutants on the LOS for patients with asthma, and this study offers policy makers quantitative evidence that can support relevant policies for health care resource management and ambient air pollutants control.
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OBJECTIVE: This study aimed to determine whether a search index could provide insight into trends in asthma admission in China. An Internet search index is a powerful tool to monitor and predict epidemic outbreaks. However, whether using an internet search index can significantly improve asthma admissions forecasts remains unknown. The long-term goal is to develop a surveillance system to help early detection and interventions for asthma and to avoid asthma health care resource shortages in advance. METHODS: In this study, we used a search index combined with air pollution data, weather data, and historical admissions data to forecast asthma admissions using machine learning. RESULTS: Results demonstrated that the best area under the curve in the test set that can be achieved is 0.832, using all predictors mentioned earlier. CONCLUSION: A search index is a powerful predictor in asthma admissions forecast, and a recent search index can reflect current asthma admissions with a lag-effect to a certain extent. The addition of a real-time, easily accessible search index improves forecasting capabilities and demonstrates the predictive potential of search index.
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Degradation of I kappaB (κB) inhibitors is critical to activation of dimeric transcription factors of the NF-κB family. There are two types of IκB inhibitors: the prototypical IκBs (IκBα, IκBß, and IκBε), which form low-molecular-weight (MW) IκB:NF-κB complexes that are highly stable, and the precursor IκBs (p105/IκBγ and p100/IκBδ), which form high-MW assemblies, thereby suppressing the activity of nearly half the cellular NF-κB [Savinova OV, Hoffmann A, Ghosh G (2009) Mol Cell 34(5):591-602]. The identity of these larger assemblies and their distinct roles in NF-κB inhibition are unknown. Using the X-ray crystal structure of the C-terminal domain of p100/IκBδ and functional analysis of structure-guided mutants, we show that p100/IκBδ forms high-MW (IκBδ)4:(NF-κB)4 complexes, referred to as kappaBsomes. These IκBδ-centric "kappaBsomes" are distinct from the 2:2 complexes formed by IκBγ. The stability of the IκBδ tetramer is enhanced upon association with NF-κB, and hence the high-MW assembly is essential for NF-κB inhibition. Furthermore, weakening of the IκBδ tetramer impairs both its association with NF-κB subunits and stimulus-dependent processing into p52. The unique ability of p100/IκBδ to stably interact with all NF-κB subunits by forming kappaBsomes demonstrates its importance in sequestering NF-κB subunits and releasing them as dictated by specific stimuli for developmental programs.
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Proteínas I-kappa B , Complejos Multiproteicos , Subunidad p52 de NF-kappa B , Proteínas , Proteolisis , Células 3T3 , Animales , Cristalografía por Rayos X , Humanos , Proteínas I-kappa B/química , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Subunidad p52 de NF-kappa B/química , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.
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Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , Cromatografía en Gel , Cristalografía por Rayos X , Activación Enzimática , Células HEK293 , Humanos , Modelos Moleculares , Fosforilación , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Relación Estructura-Actividad , TransfecciónRESUMEN
Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the REL homology domain of p65 on the response to IL-1ß in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.
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Proteínas F-Box/metabolismo , Regulación de la Expresión Génica/inmunología , Lisina/metabolismo , FN-kappa B/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Western Blotting , Inmunoprecipitación de Cromatina , Citocinas/farmacología , Cartilla de ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Interleucina-1beta/farmacología , Histona Demetilasas con Dominio de Jumonji , Metilación , Análisis por Micromatrices , Mutagénesis Sitio-Dirigida , Mutación/genética , FN-kappa B/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1ß, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that â¼85% of the NF-κB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.
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Regulación de la Expresión Génica/genética , Procesamiento Proteico-Postraduccional/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Factor de Transcripción ReIA/metabolismo , Western Blotting , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunoprecipitación , Luciferasas , Metilación , Análisis por Micromatrices , Oligonucleótidos/genética , Espectrometría de Masas en TándemRESUMEN
The signaling module that specifies nuclear factor-κΒ (NF-κB) activation is a three-component system: NF-κB, inhibitor of NF-κΒ (IκΒ), and IκΒ kinase complex (IKK). IKK receives upstream signals from the surface or inside the cell and converts itself into a catalytically active form, leading to the destruction of IκB in the inhibited IκB:NF-κB complex, leaving active NF-κB free to regulate target genes. Hidden within this simple module are family members that all can undergo various modifications resulting in expansion of functional spectrum. Three-dimensional structures representing all three components are now available. These structures have allowed us to interpret cellular observations in molecular terms and at the same time helped us to bring forward new concepts focused towards understanding the specificity in the NF-κB activation pathway.
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FN-kappa B/química , Animales , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKß function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.
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Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Ingeniería de Proteínas/métodos , Cristalografía por Rayos X , Activación Enzimática/genética , Humanos , Quinasa I-kappa B/metabolismo , Fosforilación/genética , Unión Proteica/genética , Multimerización de Proteína/genética , Especificidad por Sustrato/genéticaRESUMEN
Monogenic diabetes caused by GATA6 mutations were almost described as neonatal diabetes, and the phenotypic spectrum has expanded since then. Our study underscores the broad phenotypic spectrum by reporting a de novo GATA6 mutation in a family. Furthermore, we reviewed related literature to summarize the clinical and genetic characteristics of monogenic diabetes with GATA6 mutations (n = 39) in order to improve clinicians' understanding of the disease. We conclude that the GATA6 missense mutation (c. 749G > T, p. Gly250Val) is not reported presently, characterized by adult-onset diabetes with pancreatic dysplasia and located in transcriptional activation region. Carries with GATA6 mutations (n = 55) have a variable spectrum of diabetes, ranging from neonatal (72.7%), childhood-onset (20%) to adults-onset (7.5%). 83.5% of patients with abnormal pancreatic development. Heart and hepatobillary defects are the most common abnormalities of extrapancreatic features. Most mutations with GATA6 are loss of function (LOF, 71.8%) and located in functional region. Functional studies mostly support loss-of-function as the pathophysiological mechanism. In conclusion, there are various types of diabetes with GATA6 mutations, which can also occur in adult diabetes. Phenotypic defects with GATA6 mutations are most frequently malformations of pancreas and heart. This highlights the importance of comprehensive clinical evaluation of identified carriers to evaluate their full phenotypic spectrum.
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Diabetes Mellitus Tipo 2 , Cardiopatías Congénitas , Recién Nacido , Adulto , Humanos , Niño , Cardiopatías Congénitas/genética , Páncreas/anomalías , Mutación , Fenotipo , Factor de Transcripción GATA6/genéticaRESUMEN
An uncommon and dangerous disease with a fatality rate of more than 95% is caused by the amoeba known as Balamuthia mandrillaris. Here, we discuss the treatment of a patient who underwent a renal transplant and contracted the amoeba B. mandrillaris. The patient had a sudden onset of high fever on the 13th day after renal transplantation; on the morning of the 16th postoperative day, the patient's condition worsened and he was transferred to the ICU for treatment; on the 17th postoperative day, the patient was given mechanical ventilation; and on the 20th postoperative day, he underwent a lumbar large-pool puncture, combined with intrathecal injection of the administered medication. In order to prevent further deterioration of the patient's condition, the main aspects of care for this patient included close monitoring of changes in the patient's condition and early detection of risk factors; prompt emergency care for the patient's seizures; close monitoring of the efficacy and side effects of the patient's medication; and precise medication administration; improved hemodynamic monitoring while administering CRRT to the patient, as well as performing exercises on the patient's limb and respiratory functions. On the 32nd postoperative day, a tracheotomy is performed following thorough monitoring and care. The ventilator was turned off on postoperative day 34, and a venturi mask was installed for tracheotomy-cannula-based oxygen administration. On surgical day 40, the intrathecal injections halted and the lumbar pool drainage tube was removed. On postoperative day 46, the patient was stabilized and transferred from the intensive care unit to the organ transplant unit for extra care. This study strictly complied with the Helsinki Congress and the Istanbul Declaration regarding donor source.
Asunto(s)
Balamuthia mandrillaris , Unidades de Cuidados Intensivos , Trasplante de Riñón , Humanos , Trasplante de Riñón/efectos adversos , Masculino , Amebiasis , Respiración Artificial , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
SR proteins promote spliceosome formation by recognizing exonic splicing enhancers (ESEs) during pre-mRNA splicing. Each SR protein binds diverse ESEs using strategies that are yet to be elucidated. Here, we show that the RNA-binding domain (RBD) of SRSF1 optimally binds to decameric purine rich ESE sequences although locations of purines are not stringently specified. The presence of uracils either within or outside of the recognition site is detrimental for binding with SRSF1. The entire RBD, comprised of two RRMs and a glycine-rich linker, is essential for ESE binding. Mutation within each segment reduced or nearly abolished binding, suggesting that these segments mediate cooperative binding. The linker plays a decisive role in organizing ESE binding. The flanking basic regions of the linker appear to communicate with each other in bringing the two RRMs close together to form the complex with RNA. Our study thus suggests semi-conservative adaptable interaction between ESE and SRSF1, and such binding mode is not only essential for the recognition of plethora of physiological ESE sequences but may also be essential for the interaction with various factors during the spliceosome assembly.