RESUMEN
The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.
Asunto(s)
Discapacidades del Desarrollo/genética , Regulación del Desarrollo de la Expresión Génica , Microcefalia/genética , Micrognatismo/genética , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Adolescente , Secuencia de Aminoácidos , Animales , Niño , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Embrión no Mamífero , Femenino , Humanos , Lisina/análogos & derivados , Lisina/genética , Lisina/metabolismo , Masculino , Microcefalia/metabolismo , Microcefalia/patología , Micrognatismo/metabolismo , Micrognatismo/patología , Factores de Iniciación de Péptidos/deficiencia , Péptidos/genética , Péptidos/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espermidina/farmacología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
BACKGROUND AND PURPOSE: Small conductance calcium-activated potassium (KCa 2.x) channels have a widely accepted canonical function in regulating cellular excitability. In this study, we address a potential non-canonical function of KCa 2.x channels in breast cancer cell survival, using in vitro models. EXPERIMENTAL APPROACH: The expression of all KCa 2.x channel isoforms was initially probed using RT-PCR, Western blotting and microarray analysis in five widely studied breast cancer cell lines. In order to assess the effect of pharmacological blockade and siRNA-mediated knockdown of KCa 2.x channels on these cell lines, we utilized MTS proliferation assays and also followed the corresponding expression of apoptotic markers. KEY RESULTS: All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were highly sensitive to KCa 2.x channel blockade. UCL1684 caused cytotoxicity, with LD50 values in the low nanomolar range, in all cell lines. The role of KCa 2.x channels was confirmed using pharmacological inhibition and siRNA-mediated knockdown. This reduced cell viability and also reduced expression of Bcl-2 but increased expression of active caspase-7 and caspase-9. Complementary to these results, a variety of cell lines can be protected from apoptosis induced by staurosporine using the KCa 2.x channel activator CyPPA. CONCLUSIONS AND IMPLICATIONS: In addition to a well-established role for KCa 2.x channels in migration, blockade of these channels was potently cytotoxic in breast cancer cell lines, pointing to modulation of KCa 2.x channels as a potential therapeutic approach to breast cancer.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Técnicas de Silenciamiento del Gen , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/deficiencia , Alcanos/toxicidad , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/biosíntesis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Dosificación Letal Mediana , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/deficiencia , Pirazoles/farmacología , Pirimidinas/farmacología , Compuestos de Quinolinio/toxicidad , ARN Interferente Pequeño/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/biosíntesis , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Estaurosporina/antagonistas & inhibidores , Estaurosporina/farmacologíaRESUMEN
The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1(-/-) MT(-/-) ZnT4(-/-) cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1(-/-) MT(-/-) ZnT4(-/-) cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1(-/-) MT(-/-) ZnT4(-/-) cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.
Asunto(s)
Fosfatasa Alcalina/genética , Proteínas Aviares/genética , Proteínas de Transporte de Catión/genética , Metalotioneína/genética , Vías Secretoras/genética , Transducción de Señal/genética , Zinc/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Proteínas Aviares/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/deficiencia , Línea Celular Transformada , Pollos/genética , Pollos/metabolismo , Citoplasma/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Metalotioneína/deficiencia , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Transporte de ProteínasRESUMEN
Ca(2+)/calmodulin-dependent protein kinase Iδ (CaMKIδ) is expressed ubiquitously, but little is known about its physiological functions. Recently, we cloned and characterized two splice variants of zebrafish (Danio rerio) CaMKIδ (CaMKIδ-S/L). In the present study we cloned a new CaMKIδ isoform, CaMKIδ-LL, encoded by a different gene from CaMKIδ-S/L. While the catalytic domain of CaMKIδ-LL showed 86% identity that of CaMKIδ-S/L, it had a unique C-terminal sequence. To clarify the functional role of CaMKIδ-LL, we investigated the biological significance of this new isoform during zebrafish embryogenesis. Although CaMKIδ-LL exhibited essentially the same catalytic properties and substrate specificities as the other CaMKIδ isoforms, it showed different temporal and spatial expression. During zebrafish embryogenesis, RT-PCR analysis detected CaMKIδ-LL expression after 48 h post-fertilization. Western blotting in adult zebrafish demonstrated that CaMKIδ-LL is expressed in the brain, the eye, and, abundantly, in fins. Knockdown of CaMKIδ-LL expression using morpholino-based antisense oligonucleotides resulted in an increase in abnormal embryos with small fins and underdeveloped cartilage. These phenotypes were rescued by co-injection with recombinant CaMKIδ-LL. These results clearly indicated that CaMKIδ-LL plays an important role in the generation of cartilage and fins during zebrafish embryogenesis.
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Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/deficiencia , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Técnicas de Silenciamiento del Gen , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Clonación Molecular , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
INTRODUCTION: Neuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary goal of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects. METHODS: Serum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU. RESULTS: NMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux. CONCLUSIONS: NMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors.
Asunto(s)
Artritis/inmunología , Autoanticuerpos/inmunología , Neuropéptidos/inmunología , Receptores de Neurotransmisores/inmunología , Animales , Artritis/genética , Artritis/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Calcio/inmunología , Calcio/metabolismo , Femenino , Masculino , Mastocitos/inmunología , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Neuropéptidos/deficiencia , Neuropéptidos/genética , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Receptores de Neurotensina/deficiencia , Receptores de Neurotensina/genética , Receptores de Neurotensina/inmunología , Receptores de Neurotransmisores/deficiencia , Receptores de Neurotransmisores/genética , Bazo/inmunología , Bazo/metabolismo , Bazo/patologíaRESUMEN
Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.