RESUMEN
The Polycomb repressor complex 2 (PRC2) is composed of the core subunits Ezh1/2, Suz12, and Eed, and it mediates all di- and tri-methylation of histone H3 at lysine 27 in higher eukaryotes. However, little is known about how the catalytic activity of PRC2 is regulated to demarcate H3K27me2 and H3K27me3 domains across the genome. To address this, we mapped the endogenous interactomes of Ezh2 and Suz12 in embryonic stem cells (ESCs), and we combined this with a functional screen for H3K27 methylation marks. We found that Nsd1-mediated H3K36me2 co-locates with H3K27me2, and its loss leads to genome-wide expansion of H3K27me3. These increases in H3K27me3 occurred at PRC2/PRC1 target genes and as de novo accumulation within what were previously broad H3K27me2 domains. Our data support a model in which Nsd1 is a key modulator of PRC2 function required for regulating the demarcation of genome-wide H3K27me2 and H3K27me3 domains in ESCs.
Asunto(s)
Proteínas Portadoras/metabolismo , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Células Madre Embrionarias de Ratones/enzimología , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Proteínas Portadoras/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , N-Metiltransferasa de Histona-Lisina , Humanos , Metilación , Ratones , Proteínas Nucleares/genética , Complejo Represivo Polycomb 2/genética , Procesamiento Proteico-PostraduccionalRESUMEN
Low-grade serous ovarian carcinoma (LGSC) is a rare and lethal subtype of ovarian cancer. LGSC is pathologically, biologically, and clinically distinct from the more common high-grade serous ovarian carcinoma (HGSC). LGSC arises from serous borderline ovarian tumours (SBTs). The mechanism of transformation for SBTs to LGSC remains poorly understood. To better understand the biology of LGSC, we performed whole proteome profiling of formalin-fixed, paraffin-embedded tissue blocks of LGSC (n = 11), HGSC (n = 19), and SBTs (n = 26). We identified that the whole proteome is able to distinguish between histotypes of the ovarian epithelial tumours. Proteins associated with the tumour microenvironment were differentially expressed between LGSC and SBTs. Fibroblast activation protein (FAP), a protein expressed in cancer-associated fibroblasts, is the most differentially abundant protein in LGSC compared with SBT. Multiplex immunohistochemistry (IHC) for immune markers (CD20, CD79a, CD3, CD8, and CD68) was performed to determine the presence of B cells, T cells, and macrophages. The LGSC FAP+ stroma was associated with greater abundance of Tregs and M2 macrophages, features not present in SBTs. Our proteomics cohort reveals that there are changes in the tumour microenvironment in LGSC compared with its putative precursor lesion, SBT. These changes suggest that the tumour microenvironment provides a supportive environment for LGSC tumourigenesis and progression. Thus, targeting the tumour microenvironment of LGSC may be a viable therapeutic strategy. © 2024 The Pathological Society of Great Britain and Ireland.
RESUMEN
Proteome coverage and accurate protein quantification are both important for evaluating biological systems; however, compromises between quantification, coverage, and mass spectrometry (MS) resources are often necessary. Consequently, experimental parameters that impact coverage and quantification must be adjusted, depending on experimental goals. Among these parameters is offline prefractionation, which is utilized in MS-based proteomics to decrease sample complexity resulting in higher overall proteome coverage upon MS analysis. Prefractionation leads to increases in required MS analysis time, although this is often mitigated by isobaric labeling using tandem-mass tags (TMT), which allow samples to be multiplexed. Here we evaluate common prefractionation schemes, TMT variants, and MS acquisition methods and their impact on protein quantification and coverage. Furthermore, we provide recommendations for experimental design depending on the experimental goals.
Asunto(s)
Proteoma , Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Proteómica/normas , Espectrometría de Masas en Tándem/métodos , Proteoma/análisis , Humanos , Fraccionamiento Químico/métodos , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.
Asunto(s)
Antígenos CD , Mastocitos , Ratones , Animales , Antígenos CD/metabolismo , Proteómica , Ratones Transgénicos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Inmunoglobulina E/metabolismoRESUMEN
Targeted and semitargeted mass spectrometry-based approaches are reliable methods to consistently detect and quantify low abundance proteins including proteins of clinical significance. Despite their potential, the development of targeted and semitargeted assays is time-consuming and often requires the purchase of costly libraries of synthetic peptides. To improve the efficiency of this rate-limiting step, we developed PeptideRanger, a tool to identify peptides from protein of interest with physiochemical properties that make them more likely to be suitable for mass spectrometry analysis. PeptideRanger is a flexible, extensively annotated, and intuitive R package that uses a random forest model trained on a diverse data set of thousands of MS experiments spanning a variety of sample types profiled with different chromatography setups and instruments. To support a variety of applications and to leverage rapidly growing public MS databases, PeptideRanger can readily be retrained with experiment-specific data sets and customized to prioritize and filter peptides based on selected properties.
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Péptidos , Proteómica , Proteómica/métodos , Péptidos/análisis , Espectrometría de Masas/métodos , ProteínasRESUMEN
Clear cell ovarian carcinoma (CCOC) is the second most common subtype of epithelial ovarian carcinoma. Late-stage CCOC is not responsive to gold-standard chemotherapy and results in suboptimal outcomes for patients. In-depth molecular insight is urgently needed to stratify the disease and drive therapeutic development. We conducted global proteomics for 192 cases of CCOC and compared these with other epithelial ovarian carcinoma subtypes. Our results showed distinct proteomic differences in CCOC compared with other epithelial ovarian cancer subtypes including alterations in lipid and purine metabolism pathways. Furthermore, we report potential clinically significant proteomic subgroups within CCOC, suggesting the biologic plausibility of stratified treatment for this cancer. Taken together, our results provide a comprehensive understanding of the CCOC proteomic landscape to facilitate future understanding and research of this disease. © 2022 The Pathological Society of Great Britain and Ireland.
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Adenocarcinoma de Células Claras , Neoplasias Ováricas , Femenino , Humanos , Carcinoma Epitelial de Ovario/patología , Proteoma , Proteómica , Adenocarcinoma de Células Claras/patología , Neoplasias Ováricas/metabolismoRESUMEN
Sex is a modulator of health that has been historically overlooked in biomedical research. Recognizing this knowledge gap, funding agencies now mandate the inclusion of sex as a biological variable with the goal of stimulating efforts to illuminate the molecular underpinnings of sex biases in health and disease. DNA methylation (DNAm) is a strong molecular candidate for mediating such sex biases; however, a robust and well characterized annotation of sex differences in DNAm is yet to emerge. Beginning with a large (n = 3795) dataset of DNAm profiles from normative adult whole blood samples, we identified, validated and characterized autosomal sex-associated co-methylated genomic regions (sCMRs). Strikingly, sCMRs showed consistent sex differences in DNAm over the life course and a subset were also consistent across cell, tissue and cancer types. sCMRs included sites with known sex differences in DNAm and links to health conditions with sex biased effects. The robustness of sCMRs enabled the generation of an autosomal DNAm-based predictor of sex with 96% accuracy. Testing this tool on blood DNAm profiles from individuals with sex chromosome aneuploidies (Klinefelter [47,XXY], Turner [45,X] and 47,XXX syndrome) revealed an intimate relationship between sex chromosomes and sex-biased autosomal DNAm.
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Metilación de ADN , Trastornos de los Cromosomas Sexuales del Desarrollo Sexual/genética , Procesos de Determinación del Sexo/genética , Cromosomas/genética , Islas de CpG , Femenino , Humanos , MasculinoRESUMEN
The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach, we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo by mitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/biosíntesis , Transformación Celular Neoplásica/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias Experimentales/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Estrés del Retículo Endoplásmico , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Ratones , Ratones Noqueados , Ratones Desnudos , Células 3T3 NIH , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Oxidación-Reducción , Estrés Oxidativo , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a-Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3-positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1-phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.
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Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/fisiología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Animales , Diferenciación Celular , Inmunoprecipitación , Espectrometría de Masas , Ratones , Unión ProteicaRESUMEN
Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Piridinas/uso terapéutico , Sarcoma de Ewing/tratamiento farmacológico , Factores de Transcripción/metabolismo , Acetilación , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Metástasis de la Neoplasia , Estrés Oxidativo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologíaRESUMEN
Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of fcp1 mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in fcp1 and cdk8Δ mutants and was required for the suppression of fcp1 mutant growth defects by loss of CDK8 under oxidative stress conditions. Targeted analysis revealed that mutating FCP1 decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes (TRX2 and TSA1) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of TRX2 and TSA1 observed in the fcp1 mutant. Interestingly, loss of CDK8 robustly normalized the mRNA levels of Skn7-dependent genes in the fcp1 mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of CDK8 could overcome transcriptional and/or posttranscriptional alterations in the fcp1 mutant through its regulatory effect on Skn7. Furthermore, our work also implicated FCP1 and CDK8 in the broader response to environmental stressors in yeast.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Estrés Oxidativo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/genética , Peroxidasas/genética , Peroxidasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Estabilidad Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Despite being the most common childhood bone tumor, the genomic characterization of osteosarcoma remains incomplete. In particular, very few osteosarcoma metastases have been sequenced to date, critical to better understand mechanisms of progression and evolution in this tumor. We performed an integrated whole genome and exome sequencing analysis of paired primary and metastatic pediatric osteosarcoma specimens to identify recurrent genomic alterations. Sequencing of 13 osteosarcoma patients including 13 primary, 10 metastatic, and 3 locally recurring tumors revealed a highly heterogeneous mutational landscape, including cases of hypermutation and microsatellite instability positivity, but with virtually no recurrent alterations except for mutations involving the tumor suppressor genes RB1 and TP53. At the germline level, we detected alterations in multiple cancer related genes in the majority of the cohort, including those potentially disrupting DNA damage response pathways. Metastases retained only a minimal number of short variants from their corresponding primary tumors, while copy number alterations showed higher conservation. One recurrently amplified gene, KDR, was highly expressed in advanced cases and associated with poor prognosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Osteosarcoma/genética , Osteosarcoma/secundario , Secuenciación Completa del Genoma , Factores de Edad , Colombia Británica , Variaciones en el Número de Copia de ADN , Femenino , Amplificación de Genes , Dosificación de Gen , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Inestabilidad de Microsatélites , Mutación , Fenotipo , Polimorfismo de Nucleótido Simple , Transcriptoma , Estados Unidos , Secuenciación del ExomaRESUMEN
RNA polymerase II (RNAPII) contains a unique C-terminal domain that is composed of heptapeptide repeats and which plays important regulatory roles during gene expression. RNAPII is responsible for the transcription of most protein-coding genes, a subset of non-coding genes, and retrotransposons. Retrotransposon transcription is the first step in their multiplication cycle, given that the RNA intermediate is required for the synthesis of cDNA, the material that is ultimately incorporated into a new genomic location. Retrotransposition can have grave consequences to genome integrity, as integration events can change the gene expression landscape or lead to alteration or loss of genetic information. Given that RNAPII transcribes retrotransposons, we sought to investigate if the RNAPII-CTD played a role in the regulation of retrotransposon gene expression. Importantly, we found that the RNAPII-CTD functioned to maintaining genome integrity through inhibition of retrotransposon gene expression, as reducing CTD length significantly increased expression and transposition rates of Ty1 elements. Mechanistically, the increased Ty1 mRNA levels in the rpb1-CTD11 mutant were partly due to Cdk8-dependent alterations to the RNAPII-CTD phosphorylation status. In addition, Cdk8 alone contributed to Ty1 gene expression regulation by altering the occupancy of the gene-specific transcription factor Ste12. Loss of STE12 and TEC1 suppressed growth phenotypes of the RNAPII-CTD truncation mutant. Collectively, our results implicate Ste12 and Tec1 as general and important contributors to the Cdk8, RNAPII-CTD regulatory circuitry as it relates to the maintenance of genome integrity.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/genética , ARN Polimerasa II/genética , Retroelementos/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Alelos , Quinasa 8 Dependiente de Ciclina/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Proteínas de Unión al ADN/biosíntesis , Regulación Fúngica de la Expresión Génica , Genoma , Mutación , Fenotipo , Estructura Terciaria de Proteína , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Citocinas/antagonistas & inhibidores , Linfocitos T/inmunología , Animales , Antígenos CD19/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Soluble oligomers of amyloid-ß (Aß) impair synaptic plasticity, perturb neuronal energy homeostasis, and are implicated in Alzheimer's disease (AD) pathogenesis. Therefore, significant efforts in AD drug discovery research aim to prevent the formation of Aß oligomers or block their neurotoxicity. The eukaryotic elongation factor-2 kinase (eEF2K) plays a critical role in synaptic plasticity, and couples neurotransmission to local dendritic mRNA translation. Recent evidence indicates that Aß oligomers activate neuronal eEF2K, suggesting a potential link to Aß induced synaptic dysfunction. However, a detailed understanding of the role of eEF2K in AD pathogenesis, and therapeutic potential of eEF2K inhibition in AD, remain to be determined. Here, we show that eEF2K activity is increased in postmortem AD patient cortex and hippocampus, and in the hippocampus of aged transgenic AD mice. Furthermore, eEF2K inhibition using pharmacological or genetic approaches prevented the toxic effects of Aß42 oligomers on neuronal viability and dendrite formation in vitro. We also report that eEF2K inhibition promotes the nuclear factor erythroid 2-related factor (NRF2) antioxidant response in neuronal cells, which was crucial for the beneficial effects of eEF2K inhibition in neurons exposed to Aß42 oligomers. Accordingly, NRF2 knockdown or overexpression of the NRF2 inhibitor, Kelch-Like ECH-Associated Protein-1 (Keap1), significantly attenuated the neuroprotection associated with eEF2K inhibition. Finally, genetic deletion of the eEF2K ortholog efk-1 reduced oxidative stress, and improved chemotaxis and serotonin sensitivity in C. elegans expressing human Aß42 in neurons. Taken together, these findings highlight the potential utility of eEF2K inhibition to reduce Aß-mediated oxidative stress in AD.
Asunto(s)
Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Quinasa del Factor 2 de Elongación/deficiencia , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidad , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/enzimología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/toxicidad , Especies Reactivas de OxígenoRESUMEN
Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed â¼11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape.
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Interferencia de ARN , Animales , Cromatina/metabolismo , Epistasis Genética , Técnicas de Silenciamiento del Gen , RatonesRESUMEN
The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/genética , ARN Polimerasa II/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Mutación , Mio-Inositol-1-Fosfato Sintasa/genética , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled. The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates.
RESUMEN
PURPOSE: Ewing sarcoma is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. Although the survival rate of patients diagnosed with localized disease is approximately 70%, this decreases to approximately 30% for patients with metastatic disease and only approximately 10% for treatment-refractory disease, which have not changed for decades. Therefore, new therapeutic strategies are urgently needed for metastatic and refractory Ewing sarcoma. EXPERIMENTAL DESIGN: This study analyzed 19 unique Ewing sarcoma patient- or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared with a reference standard of 12 immortalized non-Ewing sarcoma cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to complement the surfaceome data. All models were analyzed by Tandem Mass Tags-based mass spectrometry to quantify identified proteins. RESULTS: The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known Ewing sarcoma-associated proteins, including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in Ewing sarcoma tumors compared with other childhood sarcomas and normal tissues. CONCLUSIONS: Our comprehensive proteomic characterization of the Ewing sarcoma surfaceome provides a rich resource of surface-expressed proteins in Ewing sarcoma. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in Ewing sarcoma. See related commentary by Bailey, p. 934.
Asunto(s)
Neoplasias Óseas , Sarcoma de Ewing , Sarcoma , Niño , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/terapia , Proteínas de la Membrana , Proteoma , Proteómica , Neoplasias Óseas/genética , Neoplasias Óseas/terapia , Inmunoterapia , Antígenos de Neoplasias , OxidorreductasasRESUMEN
The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.