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1.
J Lipid Res ; 65(9): 100607, 2024 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-39067520

RESUMEN

Blood plasma is one of the most commonly analyzed and easily accessible biological samples. Here, we describe an automated liquid-liquid extraction platform that generates accurate, precise, and reproducible samples for metabolomic, lipidomic, and proteomic analyses from a single aliquot of plasma while minimizing hands-on time and avoiding contamination from plasticware. We applied mass spectrometry to examine the metabolome, lipidome, and proteome of 90 plasma samples to determine the effects of age, time of day, and a high-fat diet in mice. From 25 µl of mouse plasma, we identified 907 lipid species from 16 different lipid classes and subclasses, 233 polar metabolites, and 344 proteins. We found that the high-fat diet induced only mild changes in the polar metabolome, upregulated apolipoproteins, and induced substantial shifts in the lipidome, including a significant increase in arachidonic acid and a decrease in eicosapentaenoic acid content across all lipid classes.

2.
Elife ; 112022 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-35119359

RESUMEN

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-ß2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.


Asunto(s)
Senescencia Celular , Transición Epitelial-Mesenquimal , Anciano , Envejecimiento/fisiología , Línea Celular , Senescencia Celular/genética , Fibroblastos/metabolismo , Humanos
3.
PLoS One ; 11(11): e0166438, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27875550

RESUMEN

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.


Asunto(s)
Proteínas de Drosophila/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histonas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Inhibidores Enzimáticos/farmacología , Estudio de Asociación del Genoma Completo , Histonas/genética , Humanos , Metilación/efectos de los fármacos , Análisis de Secuencia de ADN
4.
Nat Chem Biol ; 12(7): 531-8, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27214401

RESUMEN

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.


Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteína 2 de Unión a Retinoblastoma/metabolismo , Relación Estructura-Actividad
5.
Mol Cell Proteomics ; 14(4): 1148-58, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25680960

RESUMEN

Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a "one-pot" hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.


Asunto(s)
Histonas/metabolismo , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Coloración y Etiquetado/métodos , Cromatografía Liquida , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Histona Demetilasas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Péptidos/metabolismo , Propionatos/metabolismo , ARN Interferente Pequeño/metabolismo , Estándares de Referencia , Tripsina/metabolismo
6.
Nat Cell Biol ; 15(1): 61-71, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23263283

RESUMEN

The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell-cycle exit and apoptosis, is a major determinant of size control. The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). We performed a split-TEV-based genome-wide RNAi screen for modulators of Hpo signalling. We characterize the Drosophila salt-inducible kinases (Sik2 and Sik3) as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser 413. As Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hpo pathway and systemic growth control.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas 14-3-3/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Nucleares/metabolismo , Tamaño de los Órganos , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Transactivadores/metabolismo , Alas de Animales/enzimología , Alas de Animales/crecimiento & desarrollo , Proteínas Señalizadoras YAP
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