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1.
Basic Res Cardiol ; 115(5): 51, 2020 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-32699940

RESUMEN

The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca2+ leakage, basal phosphorylation of Ca2+-cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.


Asunto(s)
Arritmias Cardíacas/etiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Cardiomegalia/etiología , AMP Cíclico/metabolismo , Acoplamiento Excitación-Contracción , Factor-23 de Crecimiento de Fibroblastos , Proteínas Klotho , Masculino , Ratones , Nefrectomía , Cultivo Primario de Células , Ratas Wistar
2.
Haematologica ; 102(5): 883-894, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28232371

RESUMEN

While c-Myc dysregulation is constantly associated with highly proliferating B-cell tumors, nuclear factor (NF)-κB addiction is found in indolent lymphomas as well as diffuse large B-cell lymphomas, either with an activated B-cell like phenotype or associated with the Epstein-Barr virus. We raised the question of the effect of c-Myc in B cells with NF-κB activated by three different inducers: Epstein-Barr virus-latency III program, TLR9 and CD40. Induction of c-Myc overexpression increased proliferation of Epstein-Barr virus-latency III immortalized B cells, an effect that was dependent on NF-κB. Results from transcriptomic signatures and functional studies showed that c-Myc overexpression increased Epstein-Barr virus-latency III-driven proliferation depending on NF-κB. In vitro, induction of c-Myc increased proliferation of B cells with TLR9-dependant activation of MyD88, with decreased apoptosis. In the transgenic λc-Myc mouse model with c-Myc overexpression in B cells, in vivo activation of MyD88 by TLR9 induced splenomegaly related to an increased synthesis phase (S-phase) entry of B cells. Transgenic mice with both continuous CD40 signaling in B cells and the λc-Myc transgene developed very aggressive lymphomas with characteristics of activated diffuse large B-cell lymphomas. The main characteristic gene expression profile signatures of these tumors were those of proliferation and energetic metabolism. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype, activated B-cell like diffuse large B-cell lymphomas. This would explain why NF-κB is associated with both indolent and aggressive lymphomas, and opens new perspectives on the possibility of combinatory therapies targeting both the c-Myc proliferating program and NF-κB activation pathways in diffuse large B-cell lymphomas.


Asunto(s)
Linfocitos B/metabolismo , Transformación Celular Viral , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Apoptosis/genética , Linfocitos B/virología , Antígenos CD40/genética , Antígenos CD40/metabolismo , Línea Celular Transformada , Proliferación Celular/genética , Perfilación de la Expresión Génica/métodos , Herpesvirus Humano 4/fisiología , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal/genética
3.
J Immunol ; 189(1): 181-90, 2012 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-22661084

RESUMEN

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1-containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Antígeno B7-H1/fisiología , Herpesvirus Humano 4/inmunología , Células Asesinas Naturales/inmunología , Lisosomas/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Viral/fisiología , Subgrupos de Linfocitos T/inmunología , Latencia del Virus/inmunología , Subgrupos de Linfocitos B/patología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/biosíntesis , Transporte Biológico Activo/inmunología , Muerte Celular/inmunología , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Supervivencia Celular/inmunología , Regulación hacia Abajo/inmunología , Humanos , Lisosomas/inmunología , ARN Mensajero/genética
4.
Nat Commun ; 15(1): 4885, 2024 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-38849353

RESUMEN

Inherited cardiomyopathies are common cardiac diseases worldwide, leading in the late stage to heart failure and death. The most promising treatments against these diseases are small molecules directly modulating the force produced by ß-cardiac myosin, the molecular motor driving heart contraction. Omecamtiv mecarbil and Mavacamten are two such molecules that completed phase 3 clinical trials, and the inhibitor Mavacamten is now approved by the FDA. In contrast to Mavacamten, Omecamtiv mecarbil acts as an activator of cardiac contractility. Here, we reveal by X-ray crystallography that both drugs target the same pocket and stabilize a pre-stroke structural state, with only few local differences. All-atom molecular dynamics simulations reveal how these molecules produce distinct effects in motor allostery thus impacting force production in opposite way. Altogether, our results provide the framework for rational drug development for the purpose of personalized medicine.


Asunto(s)
Simulación de Dinámica Molecular , Contracción Miocárdica , Urea , Contracción Miocárdica/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Urea/análogos & derivados , Urea/farmacología , Urea/química , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Miosinas Ventriculares/metabolismo , Miosinas Ventriculares/química , Miosinas Ventriculares/genética , Animales , Bencilaminas , Uracilo/análogos & derivados
5.
bioRxiv ; 2023 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-38014327

RESUMEN

Inherited cardiomyopathies are amongst the most common cardiac diseases worldwide, leading in the late-stage to heart failure and death. The most promising treatments against these diseases are small-molecules directly modulating the force produced by ß-cardiac myosin, the molecular motor driving heart contraction. Two of these molecules that produce antagonistic effects on cardiac contractility have completed clinical phase 3 trials: the activator Omecamtiv mecarbil and the inhibitor Mavacamten. In this work, we reveal by X-ray crystallography that both drugs target the same pocket and stabilize a pre-stroke structural state, with only few local differences. All atoms molecular dynamics simulations reveal how these molecules can have antagonistic impact on the allostery of the motor by comparing ß-cardiac myosin in the apo form or bound to Omecamtiv mecarbil or Mavacamten. Altogether, our results provide the framework for rational drug development for the purpose of personalized medicine.

6.
Sci Rep ; 13(1): 85, 2023 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-36596813

RESUMEN

Biallelic pathogenic variants in the SLC34A3 gene, encoding for the NPT2c cotransporter, cause Hereditary Hypophosphatemic Rickets with Hypercalciuria (HHRH). However, the associated phenotype is highly variable. In addition, mice deleted for Slc34a3 exhibit a different phenotype compared to humans, without urinary phosphate leakage. The mechanisms by which SLC34A3 variants disrupt phosphate/calcium metabolism are un-completely understood. In this study we explored these mechanisms in vitro using SLC34A3 variants identified in patients with urinary phosphate leakage. We analyzed the consequences of these variants on NPT2c function and the link with the phenotype of the patients. We studied 20 patients with recurrent nephrolithiasis and low serum phosphate concentration harboring variants in the SLC34A3 gene. Half of the patients carried homozygous or composite heterozygous variants. Three patients had in addition variants in SLC34A1 and SLC9A3R1 genes. All these patients benefited from a precise analysis of their phenotype. We generated 13 of these mutants by site-directed mutagenesis. Then we carried out transient transfections of these mutants in HEK cells and measured their phosphate uptake capacity under different conditions. Among the 20 patients included, 3 had not only mutations in NPT2c but also in NPT2a or NHERF1 genes. Phosphate uptake was decreased in 8 NPT2c mutants studied and normal for 5. Four variants were initially categorized as variants of uncertain significance. Expression of the corresponding mutants showed that one did not modify phosphate transport, two reduced it moderately and one abolished it. Co-transfection of the NPT2c mutants with the wild-type plasmid of NPT2c or NPT2a did not reveal dominant negative effect of the mutants on NPT2c-mediated phosphate transport. A detailed analysis of patient phenotypes did not find a link between the severity of the disorder and the level of phosphate transport impairment. NPT2c mutations classified as ACMG3 identified in patients with renal phosphate leak should be characterized by in vitro study to check if they alter NPT2c-mediated phosphate transport since phosphate uptake capacity may not be affected. In addition, research for mutations in NHERF1 and NPT2a genes should always be associated to NPT2c sequencing.


Asunto(s)
Raquitismo Hipofosfatémico Familiar , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc , Animales , Humanos , Ratones , Raquitismo Hipofosfatémico Familiar/genética , Raquitismo Hipofosfatémico Familiar/patología , Riñón/metabolismo , Mutación , Fenotipo , Fosfatos/metabolismo
7.
Nat Commun ; 14(1): 6732, 2023 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-37872146

RESUMEN

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.


Asunto(s)
Actinas , Cadenas Pesadas de Miosina , Humanos , Células HeLa , Dimerización , Actinas/metabolismo , Cadenas Pesadas de Miosina/metabolismo
8.
Stem Cell Reports ; 16(12): 2958-2972, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34739847

RESUMEN

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) ß-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteína Nodal/metabolismo , Proproteína Convertasa 9/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular , Regulación de la Expresión Génica , Humanos , Mutación con Pérdida de Función , Proteína Nodal/genética , Fosforilación , Proproteína Convertasa 9/química , Proproteína Convertasa 9/deficiencia , Proproteína Convertasa 9/genética , Unión Proteica , Dominios Proteicos , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Regulación hacia Arriba
9.
Nat Chem ; 9(10): 1025-1033, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28937680

RESUMEN

Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Hierro/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Piranos/farmacología , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Homeostasis/efectos de los fármacos , Humanos , Lisosomas/química , Conformación Molecular , Células Madre Neoplásicas/metabolismo , Piranos/química , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo
10.
Artículo en Inglés | MEDLINE | ID: mdl-27352424

RESUMEN

Phosphate is a key component of dental mineral composition. The physiological role of membrane proteins of dental cells is suspected to be crucial for mineralization mechanisms. Contrary to published data related to calcium, data on regulation of phosphate flux through membrane of mineralizing cells are scarce. To address this lack of data, we studied the expression of six membranous phosphate transporters in two dental cell lines: a rat odontoblastic cell line (M2H4) and a mouse ameloblastic cell line (ALC) for which we optimized the mineralizing culture conditions.


Asunto(s)
Proteínas de Transporte de Fosfato/biosíntesis , Calcificación de Dientes/fisiología , Diente/metabolismo , Ameloblastos/citología , Animales , Línea Celular , Ratones , Odontoblastos/citología , Ratas
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