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1.
J Biol Chem ; 290(12): 7586-601, 2015 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-25659432

RESUMEN

Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45·Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45·Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética
2.
J Exp Clin Cancer Res ; 42(1): 116, 2023 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-37150829

RESUMEN

BACKGROUND: Bronchial premalignant lesions (PMLs) are composed primarily of cells resembling basal epithelial cells of the airways, which through poorly understood mechanisms have the potential to progress to lung squamous cell carcinoma (LUSC). Despite ongoing efforts that have mapped gene expression and cell diversity across bronchial PML pathologies, signaling and transcriptional events driving malignancy are poorly understood. Evidence has suggested key roles for the Hippo pathway effectors YAP and TAZ and associated TEAD and TP63 transcription factor families in bronchial basal cell biology and LUSC. In this study we examine the functional association of YAP/TAZ, TEADs and TP63 in bronchial epithelial cells and PMLs. METHODS: We performed RNA-seq in primary human bronchial epithelial cells following small interfering RNA (siRNA)-mediated depletion of YAP/TAZ, TEADs or TP63, and combined these data with ChIP-seq analysis of these factors. Directly activated or repressed genes were identified and overlapping genes were profiled across gene expression data obtained from progressive or regressive human PMLs and across lung single cell RNA-seq data sets. RESULTS: Analysis of genes regulated by YAP/TAZ, TEADs, and TP63 in human bronchial epithelial cells revealed a converged transcriptional network that is strongly associated with the pathological progression of bronchial PMLs. Our observations suggest that YAP/TAZ-TEAD-TP63 associate to cooperatively promote basal epithelial cell proliferation and repress signals associated with interferon responses and immune cell communication. Directly repressed targets we identified include the MHC Class II transactivator CIITA, which is repressed in progressive PMLs and associates with adaptive immune responses in the lung. Our findings provide molecular insight into the control of gene expression events driving PML progression, including those contributing to immune evasion, offering potential new avenues for lung cancer interception. CONCLUSIONS: Our study identifies important gene regulatory functions for YAP/TAZ-TEAD-TP63 in the early stages of lung cancer development, which notably includes immune-suppressive roles, and suggest that an assessment of the activity of this transcriptional complex may offer a means to identify immune evasive bronchial PMLs and serve as a potential therapeutic target.


Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Pulmonares , Lesiones Precancerosas , Humanos , Regulación de la Expresión Génica , Neoplasias Pulmonares/genética , Lesiones Precancerosas/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAP , Factores de Transcripción de Dominio TEA
3.
Chest ; 164(4): 1028-1041, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37244587

RESUMEN

Lung cancer is the leading cause of cancer-related deaths. Early detection and diagnosis are critical, as survival decreases with advanced stages. Approximately 1.6 million nodules are incidentally detected every year on chest CT scan images in the United States. This number of nodules identified is likely much larger after accounting for screening-detected nodules. Most of these nodules, whether incidentally or screening detected, are benign. Despite this, many patients undergo unnecessary invasive procedures to rule out cancer because our current stratification approaches are suboptimal, particularly for intermediate probability nodules. Thus, noninvasive strategies are urgently needed. Biomarkers have been developed to assist through the continuum of lung cancer care and include blood protein-based biomarkers, liquid biopsies, quantitative imaging analysis (radiomics), exhaled volatile organic compounds, and bronchial or nasal epithelium genomic classifiers, among others. Although many biomarkers have been developed, few have been integrated into clinical practice as they lack clinical utility studies showing improved patient-centered outcomes. Rapid technologic advances and large network collaborative efforts will continue to drive the discovery and validation of many novel biomarkers. Ultimately, however, randomized clinical utility studies showing improved patient outcomes will be required to bring biomarkers into clinical practice.


Asunto(s)
Neoplasias Pulmonares , Nódulos Pulmonares Múltiples , Humanos , Nódulos Pulmonares Múltiples/diagnóstico , Neoplasias Pulmonares/patología , Biomarcadores , Tomografía Computarizada por Rayos X/métodos , Proteínas Sanguíneas
4.
Nat Commun ; 13(1): 7198, 2022 11 28.
Artículo en Inglés | MEDLINE | ID: mdl-36443313

RESUMEN

Basal-like breast cancers, an aggressive breast cancer subtype that has poor treatment options, are thought to arise from luminal mammary epithelial cells that undergo basal plasticity through poorly understood mechanisms. Using genetic mouse models and ex vivo primary organoid cultures, we show that conditional co-deletion of the LATS1 and LATS2 kinases, key effectors of Hippo pathway signaling, in mature mammary luminal epithelial cells promotes the development of Krt14 and Sox9-expressing basal-like carcinomas that metastasize over time. Genetic co-deletion experiments revealed that phenotypes resulting from the loss of LATS1/2 activity are dependent on the transcriptional regulators YAP/TAZ. Gene expression analyses of LATS1/2-deleted mammary epithelial cells notably revealed a transcriptional program that associates with human basal-like breast cancers. Our study demonstrates in vivo roles for the LATS1/2 kinases in mammary epithelial homeostasis and luminal-basal fate control and implicates signaling networks induced upon the loss of LATS1/2 activity in the development of basal-like breast cancer.


Asunto(s)
Carcinoma , Proteínas Serina-Treonina Quinasas , Humanos , Animales , Ratones , Proteínas Serina-Treonina Quinasas/genética , Genes Reguladores , Transducción de Señal , Células Epiteliales , Proteínas Supresoras de Tumor/genética
5.
Sci Rep ; 12(1): 18168, 2022 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-36307504

RESUMEN

SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in the airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments have not been fully characterized using matched samples from large cohorts. Gene expression data from 793 nasal and 1673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking status (current versus former) was the only clinical factor significantly and reproducibly associated with VE gene expression. The expression of ACE2 and TMPRSS2 was higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the gene expression data confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose, these genes are not predominantly expressed in cell populations modulated by smoking. In individuals with elevated lung cancer risk, smoking-induced VE gene expression changes in the nose likely have minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.


Asunto(s)
COVID-19 , Neoplasias Pulmonares , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Detección Precoz del Cáncer , Peptidil-Dipeptidasa A/metabolismo , Neoplasias Pulmonares/metabolismo , Bronquios/metabolismo , Fumar/efectos adversos , Fumar/genética
6.
Cell Rep ; 36(2): 109347, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34260916

RESUMEN

Proper lung function relies on the precise balance of specialized epithelial cells that coordinate to maintain homeostasis. Herein, we describe essential roles for the transcriptional regulators YAP/TAZ in maintaining lung epithelial homeostasis, reporting that conditional deletion of Yap and Wwtr1/Taz in the lung epithelium of adult mice results in severe defects, including alveolar disorganization and the development of airway mucin hypersecretion. Through in vivo lineage tracing and in vitro molecular experiments, we reveal that reduced YAP/TAZ activity promotes intrinsic goblet transdifferentiation of secretory airway epithelial cells. Global gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggest that YAP/TAZ act cooperatively with TEA domain (TEAD) transcription factors and the NuRD complex to suppress the goblet cell fate program, directly repressing the SPDEF gene. Collectively, our study identifies YAP/TAZ as critical factors in lung epithelial homeostasis and offers molecular insight into the mechanisms promoting goblet cell differentiation, which is a hallmark of many lung diseases.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linaje de la Célula , Células Caliciformes/citología , Células Caliciformes/metabolismo , Homeostasis , Pulmón/citología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Proteínas Señalizadoras YAP , Adulto , Animales , Células Cultivadas , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Vía de Señalización Hippo , Humanos , Metaplasia , Ratones , Ratones Noqueados , Mucina 5AC/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción de Dominio TEA/metabolismo
7.
Transl Psychiatry ; 11(1): 504, 2021 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-34601489

RESUMEN

Transcriptomic changes in specific brain regions can influence the risk of alcohol use disorder (AUD), but the underlying mechanism is not fully understood. We investigated AUD-associated miRNA-mRNA regulatory networks in multiple brain regions by analyzing transcriptomic changes in two sets of postmortem brain tissue samples and ethanol-exposed human embryonic stem cell (hESC)-derived cortical interneurons. miRNA and mRNA transcriptomes were profiled in 192 tissue samples (Set 1) from eight brain regions (amygdala, caudate nucleus, cerebellum, hippocampus, nucleus accumbens, prefrontal cortex, putamen, and ventral tegmental area) of 12 AUD and 12 control European Australians. Nineteen differentially expressed miRNAs (fold-change>2.0 & P < 0.05) and 97 differentially expressed mRNAs (fold-change>2.0 & P < 0.001) were identified in one or multiple brain regions of AUD subjects. AUD-associated miRNA-mRNA regulatory networks in each brain region were constructed using differentially expressed and negatively correlated miRNA-mRNA pairs. AUD-relevant pathways (including CREB Signaling, IL-8 Signaling, and Axonal Guidance Signaling) were potentially regulated by AUD-associated brain miRNA-mRNA pairs. Moreover, miRNA and mRNA transcriptomes were mapped in additional 96 tissue samples (Set 2) from six of the above eight brain regions of eight AUD and eight control European Australians. Some of the AUD-associated miRNA-mRNA regulatory networks were confirmed. In addition, miRNA and mRNA transcriptomes were analyzed in hESC-derived cortical interneurons with or without ethanol exposure, and ethanol-influenced miRNA-mRNA regulatory networks were constructed. This study provided evidence that alcohol could induce concerted miRNA and mRNA expression changes in reward-related or alcohol-responsive brain regions. We concluded that altered brain miRNA-mRNA regulatory networks might contribute to AUD development.


Asunto(s)
Alcoholismo , MicroARNs , Australia , Encéfalo , Redes Reguladoras de Genes , Humanos , MicroARNs/genética , ARN Mensajero
8.
Res Sq ; 2021 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-34729557

RESUMEN

Background : SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments has not been fully characterized using matched samples from large cohorts. Results : Gene expression data from 793 nasal and 1,673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking was the only clinical factor significantly and reproducibly associated with VE gene expression. ACE2 and TMPRSS2 expression were higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48 + secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the RNA-seq confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. Conclusions : The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose these genes are not predominantly expressed in cell populations modulated by smoking. Smoking-induced VE gene expression changes in the nose likely has minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.

9.
J Alzheimers Dis ; 60(4): 1611-1620, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29036819

RESUMEN

BACKGROUND: Digital Clock Drawing Test (dCDT) technology enables the examination of detailed neurocognitive behavior as behavior unfolds in real time; a capability that cannot be obtained using a traditional pen and paper testing format. OBJECTIVE: Parameters obtained from the dCDT were used to investigate neurocognitive constructs related to higher-order neurocognitive decision making and information processing speed. The current research sought to determine the effect of age as related to combined motor and non-motor components of drawing, and higher-order decision making latencies. METHODS: A large group of stroke- and dementia- free Framingham Heart Study participants were administered the dCDT to command and copy with hands set for "10 after 11". Six age groups (age range 28-98) were constructed. RESULTS: Differences between age groups were found for total time to completion, total pen stroke count, and higher-order decision making latencies in both command and copy test conditions. CONCLUSION: Longer age-related decision making latencies may reflect a greater need for working memory and increased self-monitoring in older subjects. These latency measures have potential to serve as neurocognitive biomarkers of Alzheimer's disease and other insidious neurodegenerative disorders.


Asunto(s)
Toma de Decisiones , Evaluación Geriátrica/métodos , Destreza Motora , Pruebas Neuropsicológicas , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades Cardiovasculares/epidemiología , Femenino , Humanos , Estudios Longitudinales , Masculino , Massachusetts , Persona de Mediana Edad , Análisis Multivariante , Tiempo de Reacción
10.
PLoS One ; 10(2): e0116093, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25706313

RESUMEN

Simian virus 40 (SV40) serves as an important model organism for studying eukaryotic DNA replication. Its helicase, Large T-antigen (Tag), is a multi-functional protein that interacts with multiple host proteins, including the ubiquitous ssDNA binding protein Replication Protein A (RPA). Tag recruits RPA, actively loads it onto the unwound DNA, and together they promote priming of the template. Although interactions of Tag with RPA have been mapped, no interaction between Tag and the N-terminal protein interaction domain of the RPA 70kDa subunit (RPA70N) has been reported. Here we provide evidence of direct physical interaction of Tag with RPA70N and map the binding sites using a series of pull-down and mutational experiments. In addition, a monoclonal anti-Tag antibody, the epitope of which overlaps with the binding site, blocks the binding of Tag to RPA70N. We use NMR chemical shift perturbation analysis to show that Tag uses the same basic cleft in RPA70N as multiple of DNA damage response proteins. Mutations in the binding sites of both RPA70N and Tag demonstrate that specific charge reversal substitutions in either binding partner strongly diminish the interaction. These results expand the known repertoire of contacts between Tag and RPA, which mediate the many critical roles of Tag in viral replication.


Asunto(s)
Antígenos Virales de Tumores/metabolismo , ADN Helicasas/metabolismo , ADN Viral , Proteína de Replicación A/metabolismo , Virus 40 de los Simios/inmunología , Replicación del ADN/fisiología , Replicación Viral/fisiología
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