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1.
Nat Rev Mol Cell Biol ; 25(1): 46-64, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37710009

RESUMEN

The forkhead box protein O (FOXO, consisting of FOXO1, FOXO3, FOXO4 and FOXO6) transcription factors are the mammalian orthologues of Caenorhabditis elegans DAF-16, which gained notoriety for its capability to double lifespan in the absence of daf-2 (the gene encoding the worm insulin receptor homologue). Since then, research has provided many mechanistic details on FOXO regulation and FOXO activity. Furthermore, conditional knockout experiments have provided a wealth of data as to how FOXOs control development and homeostasis at the organ and organism levels. The lifespan-extending capabilities of DAF-16/FOXO are highly correlated with their ability to induce stress response pathways. Exogenous and endogenous stress, such as cellular redox stress, are considered the main drivers of the functional decline that characterizes ageing. Functional decline often manifests as disease, and decrease in FOXO activity indeed negatively impacts on major age-related diseases such as cancer and diabetes. In this context, the main function of FOXOs is considered to preserve cellular and organismal homeostasis, through regulation of stress response pathways. Paradoxically, the same FOXO-mediated responses can also aid the survival of dysfunctional cells once these eventually emerge. This general property to control stress responses may underlie the complex and less-evident roles of FOXOs in human lifespan as opposed to model organisms such as C. elegans.


Asunto(s)
Caenorhabditis elegans , Transducción de Señal , Animales , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Transducción de Señal/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Envejecimiento/genética , Longevidad/genética , Mamíferos/metabolismo
2.
Mol Cell ; 83(22): 4141-4157.e11, 2023 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-37977121

RESUMEN

Biomolecular condensates have emerged as a major organizational principle in the cell. However, the formation, maintenance, and dissolution of condensates are still poorly understood. Transcriptional machinery partitions into biomolecular condensates at key cell identity genes to activate these. Here, we report a specific perturbation of WNT-activated ß-catenin condensates that disrupts oncogenic signaling. We use a live-cell condensate imaging method in human cancer cells to discover FOXO and TCF-derived peptides that specifically inhibit ß-catenin condensate formation on DNA, perturb nuclear ß-catenin condensates in cells, and inhibit ß-catenin-driven transcriptional activation and colorectal cancer cell growth. We show that these peptides compete with homotypic intermolecular interactions that normally drive condensate formation. Using this framework, we derive short peptides that specifically perturb condensates and transcriptional activation of YAP and TAZ in the Hippo pathway. We propose a "monomer saturation" model in which short interacting peptides can be used to specifically inhibit condensate-associated transcription in disease.


Asunto(s)
Neoplasias , beta Catenina , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Transducción de Señal , Vía de Señalización Hippo , Péptidos/genética
3.
Nat Rev Mol Cell Biol ; 14(2): 83-97, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23325358

RESUMEN

Forkhead box O (FOXO) transcription factors are involved in the regulation of the cell cycle, apoptosis and metabolism. In model organisms, FOXO activity also affects stem cell maintenance and lifespan as well as age-related diseases, such as cancer and diabetes. Multiple upstream pathways regulate FOXO activity through post-translational modifications and nuclear-cytoplasmic shuttling of both FOXO and its regulators. The diversity of this upstream regulation and the downstream effects of FOXOs suggest that they function as homeostasis regulators to maintain tissue homeostasis over time and coordinate a response to environmental changes, including growth factor deprivation, metabolic stress (starvation) and oxidative stress.


Asunto(s)
Factores de Transcripción Forkhead/fisiología , Homeostasis/genética , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Homeostasis/fisiología , Humanos , Modelos Biológicos , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/genética , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Relación Estructura-Actividad
4.
NMR Biomed ; 36(4): e4882, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36451530

RESUMEN

Patient-derived cancer cells cultured in vitro are a cornerstone of cancer metabolism research. More recently, the introduction of organoids has provided the research community with a more versatile model system. Physiological structure and organization of the cell source tissue are maintained in organoids, representing a closer link to in vivo tumor models. High-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) is a commonly applied analytical approach for metabolic profiling of intact tissue, but its use has not been reported for organoids. The aim of the current work was to compare the performance of HR MAS MRS and extraction-based nuclear magnetic resonance (NMR) in metabolic profiling of wild-type and tumor progression organoids (TPOs) from human colon cancer, and further to investigate how the sequentially increased genetic alterations of the TPOs affect the metabolic profile. Sixteen metabolites were reliably identified and quantified both in spectra based on NMR of extracts and HR MAS MRS of intact organoids. The metabolite concentrations from the two approaches were highly correlated (r = 0.94), and both approaches were able to capture the systematic changes in metabolic features introduced by the genetic alterations characteristic of colorectal cancer progression (e.g., increased levels of lactate and decreased levels of myo-inositol and phosphocholine with an increasing number of mutations). The current work highlights that HR MAS MRS is a well-suited method for metabolic profiling of intact organoids, with the additional benefit that the nondestructive nature of HR MAS enables subsequent recovery of the organoids for further analyses based on nucleic acids or proteins.


Asunto(s)
Neoplasias Colorrectales , Metabolómica , Humanos , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Metaboloma
5.
Nature ; 543(7645): 424-427, 2017 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-28273069

RESUMEN

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.


Asunto(s)
Autorrenovación de las Células , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Intestino Delgado/citología , Intestino Delgado/metabolismo , Células Madre/citología , Animales , Diferenciación Celular , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Glucólisis , Homeostasis , Ácido Láctico/metabolismo , Ratones , Mitocondrias/metabolismo , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismo , Fosforilación Oxidativa , Células de Paneth/citología , Células de Paneth/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Células Madre/fisiología , Proteína Wnt3A/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
6.
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
7.
Proc Natl Acad Sci U S A ; 116(52): 26580-26590, 2019 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-31818951

RESUMEN

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

8.
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
9.
J Biol Chem ; 294(4): 1128-1141, 2019 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-30530489

RESUMEN

Lysine methylation is an important post-translational modification that is also present on mitochondrial proteins, but the mitochondrial lysine-specific methyltransferases (KMTs) responsible for modification are in most cases unknown. Here, we set out to determine the function of human family with sequence similarity 173 member B (FAM173B), a mitochondrial methyltransferase (MTase) reported to promote chronic pain. Using bioinformatics analyses and biochemical assays, we found that FAM173B contains an atypical, noncleavable mitochondrial targeting sequence responsible for its localization to mitochondria. Interestingly, CRISPR/Cas9-mediated KO of FAM173B in mammalian cells abrogated trimethylation of Lys-43 in ATP synthase c-subunit (ATPSc), a modification previously reported as ubiquitous among metazoans. ATPSc methylation was restored by complementing the KO cells with enzymatically active human FAM173B or with a putative FAM173B orthologue from the nematode Caenorhabditis elegans Interestingly, lack of Lys-43 methylation caused aberrant incorporation of ATPSc into the ATP synthase complex and resulted in decreased ATP-generating ability of the complex, as well as decreased mitochondrial respiration. In summary, we have identified FAM173B as the long-sought KMT responsible for methylation of ATPSc, a key protein in cellular ATP production, and have demonstrated functional significance of ATPSc methylation. We suggest renaming FAM173B to ATPSc-KMT (gene name ATPSCKMT).


Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina/metabolismo , Mitocondrias/enzimología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Animales , Línea Celular , Biología Computacional , Células HeLa , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metilación , Ratones , Mitocondrias/metabolismo
10.
Metabolomics ; 16(9): 99, 2020 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-32915321

RESUMEN

Direct infusion untargeted mass spectrometry-based metabolomics allows for rapid insight into a sample's metabolic activity. However, analysis is often complicated by the large array of detected m/z values and the difficulty to prioritize important m/z and simultaneously annotate their putative identities. To address this challenge, we developed MetaboShiny, a novel R/RShiny-based metabolomics package featuring data analysis, database- and formula-prediction-based annotation and visualization. To demonstrate this, we reproduce and further explore a MetaboLights metabolomics bioinformatics study on lung cancer patient urine samples. MetaboShiny enables rapid and rigorous analysis and interpretation of direct infusion untargeted mass spectrometry-based metabolomics data.


Asunto(s)
Biología Computacional , Metabolómica/métodos , Programas Informáticos , Curaduría de Datos , Interpretación Estadística de Datos , Bases de Datos Factuales , Humanos , Neoplasias Pulmonares/metabolismo , Aprendizaje Automático , Espectrometría de Masas en Tándem
11.
Am J Pathol ; 188(9): 1956-1972, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30030980

RESUMEN

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in cancer. Tumors are potentially sensitive to PI3K pathway inhibitors, but reliable diagnostic tests that assess functional PI3K activity are lacking. Because PI3K pathway activity negatively regulates forkhead box-O (FOXO) transcription factor activity, FOXO target gene expression is inversely correlated with PI3K activity. A knowledge-based Bayesian computational model was developed to infer PI3K activity in cancer tissue samples from FOXO target gene mRNA levels and validated in cancer cell lines treated with PI3K inhibitors. However, applied to patient tissue samples, FOXO was often active in cancer types with expected active PI3K. SOD2 was differentially expressed between FOXO-active healthy and cancer tissue samples, indicating that cancer-associated cellular oxidative stress alternatively activated FOXO. To enable correct interpretation of active FOXO in cancer tissue, threshold levels for normal SOD2 expression in healthy tissue were defined above which FOXO activity is oxidative stress induced and below which PI3K regulated. In slow-growing luminal A breast cancer and low Gleason score prostate cancer, FOXO was active in a PI3K-regulated manner, indicating inactive PI3K. In aggressive luminal B, HER2, and basal breast cancer, FOXO was increasingly inactive or actively induced by oxidative stress, indicating PI3K activity. We provide a decision tree that facilitates functional PI3K pathway activity assessment in tissue samples from patients with cancer for therapy response prediction and prognosis.


Asunto(s)
Neoplasias de la Mama/metabolismo , Biología Computacional/métodos , Factores de Transcripción Forkhead/metabolismo , Bases del Conocimiento , Fosfatidilinositol 3-Quinasas/metabolismo , Superóxido Dismutasa/metabolismo , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Pruebas Diagnósticas de Rutina , Femenino , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Superóxido Dismutasa/genética
12.
Mol Syst Biol ; 14(6): e8227, 2018 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-29945941

RESUMEN

Intestinal organoids accurately recapitulate epithelial homeostasis in vivo, thereby representing a powerful in vitro system to investigate lineage specification and cellular differentiation. Here, we applied a multi-omics framework on stem cell-enriched and stem cell-depleted mouse intestinal organoids to obtain a holistic view of the molecular mechanisms that drive differential gene expression during adult intestinal stem cell differentiation. Our data revealed a global rewiring of the transcriptome and proteome between intestinal stem cells and enterocytes, with the majority of dynamic protein expression being transcription-driven. Integrating absolute mRNA and protein copy numbers revealed post-transcriptional regulation of gene expression. Probing the epigenetic landscape identified a large number of cell-type-specific regulatory elements, which revealed Hnf4g as a major driver of enterocyte differentiation. In summary, by applying an integrative systems biology approach, we uncovered multiple layers of gene expression regulation, which contribute to lineage specification and plasticity of the mouse small intestinal epithelium.


Asunto(s)
Biología Computacional , Intestinos/citología , Organogénesis , Organoides/citología , Animales , Regulación de la Expresión Génica , Ratones , Organogénesis/genética , Células Madre
13.
Rheumatology (Oxford) ; 58(12): 2305-2314, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31131409

RESUMEN

OBJECTIVE: A considerable body of evidence supports a role for type-I IFN in the pathogenesis of primary SS (pSS). As plasmacytoid dendritic cells (pDCs) are a major source of type-I IFN, we investigated their molecular regulation by measuring expression of a large set of miRNAs. METHODS: pDCs were isolated from peripheral blood of pSS patients (n = 30) and healthy controls (n = 16) divided into two independent cohorts (discovery and replication). Screening of 758 miRNAs was assessed by an OpenArray quantitative PCR-based technique; replication of a set of identified miRNAs was performed by custom array. Functional annotation of miRNA targets was performed using pathway enrichment. Novel targets of miR-29a and miR-29c were identified using a proteomic approach (stable isotope labelling with amino acids in cell culture). RESULTS: In the discovery cohort, 20 miRNAs were differentially expressed in pSS pDCs compared with healthy control pDCs. Of these, differential expression of 10 miRNAs was confirmed in the replication cohort. The dysregulated miRNAs were involved in phosphoinositide 3-kinase-Ak strain transforming and mammalian target of rapamycin signalling, as well as regulation of cell death. In addition, a set of novel protein targets of miR-29a and miR-29c were identified, including five targets that were regulated by both miRs. CONCLUSION: The dysregulated miRNome in pDCs of patients with pSS is associated with aberrant regulation of processes at the centre of pDC function, including type-I IFN production and cell death. As miR-29a and miR-29c are pro-apoptotic factors and several of the novel targets identified here are regulators of apoptosis, their downregulation in patients with pSS is associated with enhanced pDC survival.


Asunto(s)
Células Dendríticas/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Síndrome de Sjögren/genética , Adulto , Anciano , Células Cultivadas , Células Dendríticas/patología , Regulación hacia Abajo , Femenino , Humanos , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Proteómica/métodos , ARN/genética , Transducción de Señal , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología
14.
Mol Cell ; 41(4): 445-57, 2011 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-21329882

RESUMEN

Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNA screening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.


Asunto(s)
Neoplasias Colorrectales/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Regulación hacia Abajo , Retroalimentación Fisiológica , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HCT116 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
15.
Gut ; 67(4): 728-735, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-28765476

RESUMEN

OBJECTIVE: Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan immune-mediated disease that predominantly affects the biliary tract (IgG4-associated cholangitis, IAC) and pancreas (autoimmune pancreatitis, AIP). We recently identified highly expanded IgG4+ B-cell receptor clones in blood and affected tissues of patients with IAC/AIP suggestive of specific (auto)antigenic stimuli involved in initiating and/or maintaining the inflammatory response. This study aimed to identify (auto)antigen(s) that are responsible for the clonal expansion of IgG4+ B cells in IgG4-RD. DESIGN: We screened sera of patients with IAC/AIP (n=50), in comparison to control sera of patients with primary sclerosing cholangitis (PSC) and pancreatobiliary malignancies (n=47), for reactivity against human H69 cholangiocyte lysates on immunoblot. Subsequently, target antigens were immunoprecipitated and analysed by mass spectrometry. RESULTS: Prominent reactivity against a 56 kDa protein was detected in human H69 cholangiocyte lysates exposed to sera of nine patients with IAC/AIP. Affinity purification and mass spectrometry analysis identified annexin A11, a calcium-dependent phospholipid-binding protein. Annexin A11-specific IgG4 and IgG1 antibodies were only detected in serum of patients with IgG4-RD of the biliary tract/pancreas/salivary glands and not in disease mimickers with PSC and pancreatobiliary malignancies. Epitope analysis showed that two annexin A11 epitopes targeted by IgG1 and IgG4 autoantibodies were shared between patients with IAC/AIP and IgG4 antibodies blocked binding of IgG1 antibodies to the shared annexin A11 epitopes. CONCLUSION: Our data suggest that IgG1-mediated pro-inflammatory autoreactivity against annexin A11 in patients with IgG4-RD may be attenuated by formation of annexin A11-specific IgG4 antibodies supporting an anti-inflammatory role of IgG4 in IgG4-RD.


Asunto(s)
Anexinas/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Inmunoglobulina G/inmunología , Factores Inmunológicos/inmunología , Centros Médicos Académicos , Anciano , Anciano de 80 o más Años , Enfermedades Autoinmunes/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Colangitis/diagnóstico , Colangitis/inmunología , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Pancreatitis/diagnóstico , Pancreatitis/inmunología
16.
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
17.
EMBO Rep ; 16(4): 456-66, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25648147

RESUMEN

FOXO transcription factors are considered bona fide tumor suppressors; however, recent studies showed FOXOs are also required for tumor survival. Here, we identify FOXOs as transcriptional activators of IDH1. FOXOs promote IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise stimulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation and inhibits TET enzymes and histone demethylases. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion.


Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Isocitrato Deshidrogenasa/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Línea Celular , Proliferación Celular , Ciclo del Ácido Cítrico/genética , Activación Enzimática , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Glutaratos/metabolismo , Células HeLa , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Intrones , Isocitrato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , NADP/metabolismo , Unión Proteica , Transducción de Señal , Factores de Transcripción/genética , Transcripción Genética
18.
J Inherit Metab Dis ; 40(6): 883-891, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28801717

RESUMEN

Pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.


Asunto(s)
Serina/metabolismo , Vitamina B 6/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Glicina/sangre , Glicina/metabolismo , Humanos , Fosfato de Piridoxal/sangre , Fosfato de Piridoxal/metabolismo , Piridoxina/sangre , Serina/sangre , Vitamina B 6/sangre , Deficiencia de Vitamina B 6/sangre , Deficiencia de Vitamina B 6/metabolismo
19.
Biochem J ; 469(2): 289-98, 2015 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-25990325

RESUMEN

Activity of FOXO (forkhead box O) transcription factors is inhibited by growth factor-PI3K (phosphoinositide 3-kinase)-PKB (protein kinase B)/Akt signalling to control a variety of cellular processes including cell cycle progression. Through comparative analysis of a number of microarray datasets we identified a set of genes commonly regulated by FOXO proteins and PI3K-PKB/Akt, which includes CTDSP2 (C-terminal domain small phosphatase 2). We validated CTDSP2 as a genuine FOXO target gene and show that ectopic CTDSP2 can induce cell cycle arrest. We analysed transcriptional regulation after CTDSP2 expression and identified extensive regulation of genes involved in cell cycle progression, which depends on the phosphatase activity of CTDSP2. The most notably regulated gene is the CDK (cyclin-dependent kinase) inhibitor p21(Cip1/Waf1) and in the present study we show that p21(Cip1/Waf1) is partially responsible for the cell cycle arrest through decreasing cyclin-CDK activity. Our data suggest that CTDSP2 induces p21(Cip1/Waf1) through increasing the activity of Ras. As has been described previously, Ras induces p21(Cip1/Waf1) through p53-dependent and p53-independent pathways and indeed both p53 and MEK inhibition can mitigate the CTDSP2-induced p21(Cip1/Waf1) mRNA up-regulation. In support of Ras activation by CTDSP2, depletion of endogenous CTDSP2 results in reduced Ras activity and thus CTDSP2 seems to be part of a larger set of genes regulated by FOXO proteins, which increase growth factor signalling upon FOXO activation.


Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas ras/metabolismo , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transcripción Genética/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas ras/genética
20.
EMBO J ; 30(16): 3298-308, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21743441

RESUMEN

Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain-sDMA interactions, piRNAs and piRNA targets.


Asunto(s)
Chaperonas Moleculares/fisiología , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/metabolismo , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Elementos Transponibles de ADN/genética , Femenino , Sustancias Macromoleculares , Masculino , Oocitos/metabolismo , Oocitos/ultraestructura , Ovario/metabolismo , Mapeo de Interacción de Proteínas , Interferencia de ARN , Proteínas de Unión al ARN/química , Fracciones Subcelulares/metabolismo , Testículo/metabolismo , Transcripción Genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo
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