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1.
EMBO J ; 37(5)2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29363506

RESUMEN

The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes providing them with the ability to induce a permanent S-phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post-translational mechanisms. Here, we show that atypical E2Fs can be directly phosphorylated by checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that 14-3-3 protein isoforms interact with both E2Fs in a Chk1-dependent manner. Strikingly, Chk1 phosphorylation and 14-3-3-binding did not relocate or degrade atypical E2Fs, but instead, 14-3-3 is recruited to E2F7/8 target gene promoters to possibly interfere with transcription. We observed that high levels of 14-3-3 strongly correlate with upregulated transcription of atypical E2F target genes in human cancer. Thus, we reveal that Chk1 and 14-3-3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to DNA-damaging therapeutic reagents.


Asunto(s)
Proteínas 14-3-3/metabolismo , Puntos de Control del Ciclo Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Factor de Transcripción E2F7/antagonistas & inhibidores , Neoplasias/patología , Proteínas Represoras/antagonistas & inhibidores , Apoptosis/fisiología , Línea Celular Tumoral , Supervivencia Celular/genética , Replicación del ADN/genética , Factor de Transcripción E2F7/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Biosíntesis de Proteínas/genética , Proteínas Represoras/metabolismo
2.
Am J Hum Genet ; 105(2): 283-301, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31353023

RESUMEN

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Hipotonía Muscular/patología , Mutación , Trastornos del Neurodesarrollo/patología , Saccharomyces cerevisiae/crecimiento & desarrollo , Adolescente , Edad de Inicio , Niño , Preescolar , Femenino , Células HeLa , Heterocigoto , Humanos , Masculino , Hipotonía Muscular/enzimología , Hipotonía Muscular/genética , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
3.
Mol Cell ; 49(4): 730-42, 2013 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-23333309

RESUMEN

Forkhead box O (FOXO; DAF-16 in worms) transcription factors, which are of vital importance in cell-cycle control, stress resistance, tumor suppression, and organismal lifespan, are largely regulated through nucleo-cytoplasmic shuttling. Insulin signaling keeps FOXO/DAF-16 cytoplasmic, and hence transcriptionally inactive. Conversely, as in loss of insulin signaling, reactive oxygen species (ROS) can activate FOXO/DAF-16 through nuclear accumulation. How ROS regulate the nuclear translocation of FOXO/DAF-16 is largely unknown. Cysteine oxidation can stabilize protein-protein interactions through the formation of disulfide-bridges when cells encounter ROS. Using a proteome-wide screen that identifies ROS-induced mixed disulfide-dependent complexes, we discovered several interaction partners of FOXO4, one of which is the nuclear import receptor transportin-1. We show that disulfide formation with transportin-1 is required for nuclear localization and the activation of FOXO4/DAF-16 induced by ROS, but not by the loss of insulin signaling. This molecular mechanism for nuclear shuttling is conserved in C. elegans and directly connects redox signaling to the longevity protein FOXO/DAF-16.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Factores de Transcripción/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Cistina/metabolismo , Factores de Transcripción Forkhead , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , beta Carioferinas/fisiología
4.
Int J Mol Sci ; 21(21)2020 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-33114768

RESUMEN

Body fluids contain many populations of extracellular vesicles (EV) that differ in size, cellular origin, molecular composition, and biological activities. EV in seminal plasma are in majority originating from prostate epithelial cells, and hence are also referred to as prostasomes. Nevertheless, EV are also contributed by other accessory sex glands, as well as by the testis and epididymis. In a previous study, we isolated EV from seminal plasma of vasectomized men, thereby excluding contributions from the testis and epididymis, and identified two distinct EV populations with diameters of 50 and 100 nm, respectively. In the current study, we comprehensively analyzed the protein composition of these two EV populations using quantitative Liquid Chromatography-Mass Spectrometry (LC-MS/MS). In total 1558 proteins were identified. Of these, ≈45% was found only in the isolated 100 nm EV, 1% only in the isolated 50 nm EV, and 54% in both 100 nm and 50 nm EV. Gene ontology (GO) enrichment analysis suggest that both originate from the prostate, but with distinct biogenesis pathways. Finally, nine proteins, including KLK3, KLK2, MSMB, NEFH, PSCA, PABPC1, TGM4, ALOX15B, and ANO7, with known prostate specific expression and alternate expression levels in prostate cancer tissue were identified. These data have potential for the discovery of EV associated prostate cancer biomarkers in blood.


Asunto(s)
Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Próstata/metabolismo , Proteómica/métodos , Semen/metabolismo , Tamaño de la Célula , Cromatografía Liquida , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Masculino , Especificidad de Órganos , Espectrometría de Masas en Tándem
5.
Gastroenterology ; 152(6): 1462-1476.e10, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28130067

RESUMEN

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. METHODS: To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15NH4Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. RESULTS: In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. CONCLUSIONS: In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients.


Asunto(s)
Amoníaco/metabolismo , Glutamina/biosíntesis , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Urea/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Proteínas en la Dieta/administración & dosificación , Expresión Génica , Hepatocitos , Hígado/enzimología , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteoma , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores
6.
J Biol Chem ; 291(46): 24121-24132, 2016 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-27703004

RESUMEN

Gene-specific transcription factors (GSTFs) control gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. Mutations in the GSTFs Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4 are frequently associated with colon and rectal carcinomas. These proteins play an important role in bone morphogenic protein (BMP) and transforming growth factor ß (TGF-ß) signaling pathways controlling cell fate and proliferation. To study the protein interactome of the SMAD protein family we generated a quantitative proteomics pipeline that allows for inducible expression of GFP-tagged SMAD proteins followed by affinity purification and quantitative mass spectrometry analysis. Data are available via ProteomeXchange with identifier PXD004529. The nuclear importin IPO5 was identified as a novel interacting protein of SMAD1. Overexpression of IPO5 in various cell lines specifically increases nuclear localization of BMP receptor-activated SMADs (R-SMADs) confirming a functional relationship between IPO5 and BMP but not TGF-ß R-SMADs. Finally, we provide evidence that variation in length of the lysine stretch of the nuclear localization sequence is a determinant for importin specificity.


Asunto(s)
Núcleo Celular/metabolismo , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , beta Carioferinas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas/genética , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Proteómica , Proteína Smad1/genética , Proteína Smad2/genética , Proteína Smad4/genética , beta Carioferinas/genética
7.
Biochem Soc Trans ; 42(4): 971-8, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25109988

RESUMEN

Until recently, ROS (reactive oxygen species) were often seen as merely damaging agents. However, small, but significant, amounts of hydrogen peroxide (H2O2) are also being produced upon, for instance, NADPH-oxidase activation in response to growth factor signalling and as a by-product of mitochondrial respiration. H2O2 perturbs the local cellular redox state and this results in specific and reversible cysteine oxidation in target proteins, thereby translating the redox state into a signal that ultimately leads to an appropriate cellular response. This phenomenon of signalling through cysteine oxidation is known as redox signalling and has recently been shown to be involved in a wide range of physiological processes. Cysteine residue oxidation can lead to a range of post-translational modifications, one of which is the formation of intermolecular disulfides. In the present mini-review we will give a number of examples of proteins regulated by intermolecular disulfides and discuss a recently developed method to screen for these interactions. The consequences of the regulation of the FOXO4 (forkhead box O4) transcription factor by formation of intermolecular disulfides with both TNPO1 (transportin 1) and p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein] are discussed in more detail.


Asunto(s)
Cisteína/metabolismo , Disulfuros/metabolismo , Factores de Transcripción Forkhead/metabolismo , Animales , Cisteína/química , Factores de Transcripción Forkhead/genética , Humanos , Oxidación-Reducción , Transducción de Señal
8.
Cell Stem Cell ; 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38754429

RESUMEN

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

9.
Front Immunol ; 14: 1251134, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38332916

RESUMEN

Background and aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2. Methods: Anti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays. Results: Anti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx. Conclusions: A subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated.


Asunto(s)
Colangitis , Enfermedad Relacionada con Inmunoglobulina G4 , Humanos , Anexinas , Autoanticuerpos , Autoantígenos , Ácidos y Sales Biliares , Colangitis/inmunología , Cromatografía Liquida , Galectina 3/inmunología , Inmunoglobulina G , Prohibitinas/inmunología , Espectrometría de Masas en Tándem
10.
Cell Rep ; 42(6): 112583, 2023 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-37267106

RESUMEN

Upon antigen-specific T cell receptor (TCR) engagement, human CD4+ T cells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extramitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient T cells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation. Furthermore, T cell activation results in the nuclear translocation of PDH and its association with both the p300 acetyltransferase and histone H3K27ac. These data support the tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4+ T cell activation. Targeting this pathway may provide a therapeutic approach to specifically regulate antigen-driven T cell activation.


Asunto(s)
Ensamble y Desensamble de Cromatina , Histonas , Humanos , Histonas/metabolismo , Acetilcoenzima A/metabolismo , Linfocitos T CD4-Positivos/metabolismo
11.
Mol Cell Proteomics ; 8(7): 1566-78, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19321433

RESUMEN

An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that approximately 500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development.


Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Isótopos/metabolismo , Coloración y Etiquetado/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida/métodos , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/citología , Oocitos/fisiología , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Activación Transcripcional
12.
Antioxidants (Basel) ; 10(10)2021 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-34679713

RESUMEN

Reversible cysteine oxidation plays an essential role in redox signaling by reversibly altering protein structure and function. Cysteine oxidation may lead to intra- and intermolecular disulfide formation, and the latter can drastically stabilize protein-protein interactions in a more oxidizing milieu. The activity of the tumor suppressor p53 is regulated at multiple levels, including various post-translational modification (PTM) and protein-protein interactions. In the past few decades, p53 has been shown to be a redox-sensitive protein, and undergoes reversible cysteine oxidation both in vitro and in vivo. It is not clear, however, whether p53 also forms intermolecular disulfides with interacting proteins and whether these redox-dependent interactions contribute to the regulation of p53. In the present study, by combining (co-)immunoprecipitation, quantitative mass spectrometry and Western blot we found that p53 forms disulfide-dependent interactions with several proteins under oxidizing conditions. Cysteine 277 is required for most of the disulfide-dependent interactions of p53, including those with 14-3-3θ and 53BP1. These interaction partners may play a role in fine-tuning p53 activity under oxidizing conditions.

13.
Antioxidants (Basel) ; 10(4)2021 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-33923941

RESUMEN

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

14.
Nat Commun ; 12(1): 84, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33398012

RESUMEN

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin ß4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.


Asunto(s)
Isquemia/patología , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Dependovirus/metabolismo , Regulación de la Expresión Génica , Humanos , Isquemia/genética , Ratones Noqueados , Modelos Biológicos , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Neovascularización Fisiológica/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Timosina/análogos & derivados , Timosina/genética , Timosina/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética
15.
J Extracell Vesicles ; 9(1): 1791450, 2020 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-32944179

RESUMEN

Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment.

16.
Cells ; 9(4)2020 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-32316189

RESUMEN

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein-protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Transporte Biológico Activo/genética , Hepatocitos/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cromatografía Liquida , Técnicas de Silenciamiento del Gen , Humanos , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Unión Proteica , Simportadores/genética , Espectrometría de Masas en Tándem
17.
Redox Biol ; 28: 101316, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31539802

RESUMEN

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-ß sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.


Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Cisteína/química , Amiloide/química , Ciclo Celular , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Células HEK293 , Humanos , Modelos Moleculares , Oxidación-Reducción , Multimerización de Proteína , Estructura Secundaria de Proteína
18.
Mol Cancer Res ; 18(4): 537-548, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31911540

RESUMEN

Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The BRAF gene is frequently involved in oncogenic gene fusions, with fusion frequencies of 0.2%-3% throughout different cancers. However, BRAF fusions rarely occur in the same gene configuration, potentially challenging personalized therapy design. In particular, the impact of the wide variety of fusion partners on the oncogenic role of BRAF during tumor growth and drug response is unknown. Here, we used patient-derived colorectal cancer organoids to functionally characterize and cross-compare BRAF fusions containing various partner genes (AGAP3, DLG1, and TRIM24) with respect to cellular behavior, downstream signaling activation, and response to targeted therapies. We demonstrate that 5' fusion partners mainly promote canonical oncogenic BRAF activity by replacing the auto-inhibitory N-terminal region. In addition, the 5' partner of BRAF fusions influences their subcellular localization and intracellular signaling capacity, revealing distinct subsets of affected signaling pathways and altered gene expression. Presence of the different BRAF fusions resulted in varying sensitivities to combinatorial inhibition of MEK and the EGF receptor family. However, all BRAF fusions conveyed resistance to targeted monotherapy against the EGF receptor family, suggesting that BRAF fusions should be screened alongside other MAPK pathway alterations to identify patients with metastatic colorectal cancer to exclude from anti-EGFR-targeted treatment. IMPLICATIONS: Although intracellular signaling and sensitivity to targeted therapies of BRAF fusion genes are influenced by their 5' fusion partner, we show that all investigated BRAF fusions confer resistance to clinically relevant EGFR inhibition.


Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Diferenciación Celular/fisiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Terapia Molecular Dirigida , Fusión de Oncogenes , Organoides , Inhibidores de Proteínas Quinasas/farmacología
19.
Hepatol Commun ; 2(12): 1550-1566, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30556041

RESUMEN

Cholestasis-induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)-mediated transcriptional down-regulation of the bile acid importer Na+-taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding or bile duct ligation in FXR-/- mice after 7 or 3 days, respectively. Novel NTCP-interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co-immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP-mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis-induced ER stress. Calnexin down-regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress-induced down-regulation of calnexin provides an additional mechanism to dampen NTCP-mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis.

20.
Nat Struct Mol Biol ; 25(12): 1119-1127, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30510221

RESUMEN

TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.


Asunto(s)
Chaperonina con TCP-1/fisiología , Modelos Moleculares , Factor de Transcripción TFIID/química , Chaperonina con TCP-1/metabolismo , Cristalografía por Rayos X , Células HeLa , Humanos , Espectrometría de Masas , Dominios Proteicos , Factores Asociados con la Proteína de Unión a TATA/química , Factor de Transcripción TFIID/metabolismo , Transcripción Genética
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