RESUMEN
Rationale: Pain and depression, which tend to occur simultaneously and share some common neural circuits and neurotransmitters, are highly prevalent complication in patients with advanced cancer. Exploring the underlying mechanisms is the cornerstone to prevent the comorbidity of chronic pain and depression in cancer patients. Plasticity-related gene 1 (PRG-1) protein regulates synaptic plasticity and brain functional reorganization during neuronal development or after cerebral lesion. Purinergic P2X7 receptor has been proposed as a therapeutic target for various pain and neurological disorders like depression in rodents. In this study, we investigated the roles of PRG-1 in the hippocampus in the comorbidity of pain and depressive-like behaviors in rats with bone cancer pain (BCP). Methods: The bone cancer pain rat model was established by intra-tibial cell inoculation of SHZ-88 mammary gland carcinoma cells. The animal pain behaviors were assessed by measuring the thermal withdrawal latency values by using radiant heat stimulation and mechanical withdrawal threshold by using electronic von Frey anesthesiometer, and depressive-like behavior was assessed by sucrose preference test and forced swim test. Alterations in the expression levels of PRG-1 and P2X7 receptor in hippocampus were separately detected by using western blot, immunofluorescence and immunohistochemistry analysis. The effects of intra-hippocampal injection of FTY720 (a PRG-1/PP2A interaction activator), PRG-1 overexpression or intra-hippocampal injection of A438079 (a selective competitive P2X7 receptor antagonist) were also observed. Results: Carcinoma intra-tibia injection caused thermal hyperalgesia, mechanical allodynia and depressive-like behaviors in rats, and also induced the deactivation of neurons and dendritic spine structural anomalies in the hippocampus. Western blot, immunofluorescence and immunohistochemistry analysis showed an increased expression of PRG-1 and P2X7 receptor in the hippocampus of BCP rats. Intra-hippocampal injection of FTY720 or A438079 attenuated both pain and depressive-like behaviors. Furthermore, overexpression of PRG-1 in hippocampus has similar analgesic efficacy to FTY720. In addition, they rescued neuron deactivation and dendritic spine anomalies. Conclusion: The results suggest that both PRG-1 and P2X7 receptor in the hippocampus play important roles in the development of pain and depressive-like behaviors in bone cancer condition in rats by dendritic spine regulation via P2X7R/PRG-1/PP2A pathway.
Asunto(s)
Neoplasias Óseas/complicaciones , Proteínas de Unión a Calmodulina/fisiología , Espinas Dendríticas/patología , Depresión/prevención & control , Hipocampo/fisiopatología , Manejo del Dolor/métodos , Dolor/etiología , Monoéster Fosfórico Hidrolasas/fisiología , Animales , Femenino , Ratas , Ratas Sprague-DawleyRESUMEN
Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Desmosomas/fisiología , Proteínas de Microfilamentos/fisiología , Animales , Cadherinas/química , Calcio/metabolismo , Adhesión Celular , Plasticidad de la Célula , Células Cultivadas , Desmogleína 3/metabolismo , Desmosomas/química , Humanos , RatonesRESUMEN
Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Proteínas de Plasma Seminal/fisiología , Motilidad Espermática/fisiología , Animales , Calcio/fisiología , Proteínas de Unión a Calmodulina/genética , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/fisiopatología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Mutantes , Mutagénesis , Oviductos/citología , Oviductos/fisiología , Embarazo , Proteínas de Plasma Seminal/genética , Motilidad Espermática/genética , Cola del Espermatozoide/fisiología , Transporte Espermático/genética , Transporte Espermático/fisiologíaRESUMEN
The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by calcium/calmodulin signaling in the nucleus. Recent studies have drawn attention to a new family of transcriptional regulators, the so-called calmodulin-binding transcription activator (CAMTA) proteins. CAMTAs are expressed at particularly high levels in the mouse and human brain, and we reasoned that, as calmodulin-binding transcription factors, CAMTAs may regulate the formation of long-term memory by coupling synaptic activity and calcium/calmodulin signaling to memory-related transcriptional responses. This hypothesis is supported by genetic studies that reported a correlation between Camta gene polymorphisms or mutations and cognitive capability in humans. Here, we show that acute knockdown of CAMTA1, but not CAMTA2, in the hippocampus of adult mice results in impaired performance in two memory tests, contextual fear conditioning and object-place recognition test. Short-term memory and neuronal morphology were not affected by CAMTA knockdown. Gene expression profiling in the hippocampus of control and CAMTA knockdown mice revealed a number of putative CAMTA1 target genes related to synaptic transmission and neuronal excitability. Patch clamp recordings in organotypic hippocampal slice cultures provided further evidence for CAMTA1-dependent changes in electrophysiological properties. In summary, our study provides experimental evidence that confirms previous human genetic studies and establishes CAMTA1 as a regulator of long-term memory formation.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Hipocampo/fisiología , Memoria a Largo Plazo/fisiología , Transactivadores/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Células Cultivadas , Condicionamiento Clásico , Dendritas/fisiología , Miedo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Ratones Endogámicos C57BL , Células Piramidales/citología , Reconocimiento en Psicología , Transmisión Sináptica , Transactivadores/genéticaRESUMEN
SG2NA in association with striatin and zinedin forms a striatin family of WD-40 repeat proteins. This family of proteins functions as scaffold in different signal transduction pathways. They also act as a regulatory subunit of protein phosphatase 2A. We have shown that SG2NA which evolved first in the metazoan evolution among the striatin family members expresses different isoforms generated out of alternative splicing. We have also shown that SG2NA protects cells from oxidative stress by recruiting DJ-1 and Akt to mitochondria and membrane in the post-mitotic neuronal cells. DJ-1 is both cancer and Parkinson's disease related protein. In the present study we have shown that SG2NA protects DJ-1 from proteasomal degradation in cancer cells. Hence, downregulation of SG2NA reduces DJ-1/Akt colocalization in cancer cells resulting in the reduction of anchorage dependent and independent growth. Thus SG2NA enhances cancer cell survival. Reactive oxygen species enhances SG2NA, DJ-1 and Akt trimerization. Removal of the reactive oxygen species by N-acetyl-cysteine thus reduces cancer cell growth.
Asunto(s)
Autoantígenos/fisiología , Proteínas de Unión a Calmodulina/fisiología , Supervivencia Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/metabolismo , Peroxirredoxinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Activación Enzimática , Humanos , Ratones , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Proteína Desglicasa DJ-1 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Vascular calcification is a risk factor for cerebral and cardiovascular events and has a high prevalence among elderly. To finding ways of prevent or cure vascular calcification may leads to not only a healthy longevity but also medical expenditure reduction. However, the molecular mechanism underlying this pathogenic process is still obscure. To clarify the mechanism of vascular calcification, the development of animal models that exhibit extensive and robust vascular calcification is an important issue for research in vascular biology.
Asunto(s)
Envejecimiento/patología , Calcificación Vascular/genética , Envejecimiento/genética , Animales , Proteína Morfogenética Ósea 2/fisiología , Proteínas de Unión a Calmodulina/fisiología , Enfermedades Cardiovasculares/etiología , Diferenciación Celular/genética , Trastornos Cerebrovasculares/etiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Modelos Animales de Enfermedad , Humanos , Ratones , Músculo Liso Vascular/citología , Osteoblastos/citología , Osteoprotegerina/fisiología , Ligando RANK/fisiología , Factores de Riesgo , Calcificación Vascular/patologíaRESUMEN
Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas de Unión a Calmodulina/fisiología , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/citología , Cuerpos Polares del Huso/fisiología , Proteína Quinasa CDC2/fisiología , Citocinesis , MitosisRESUMEN
Recently several potential susceptibility genes for major psychiatric disorders (schizophrenia and major depression) such as disrupted-in-schizophrenia 1(DISC1), dysbindin and pituitary adenylate cyclase-activating polypeptide (PACAP) have been reported. DISC1 is involved in neural development directly via adhesion molecules or via its binding partners of DISC1 such as elongation protein ζ-1 (FEZ1), DISC1-binding zinc-finger protein (DBZ) and kendrin. PACAP also regulates neural development via stathmin 1 or via regulation of the DISC1-DBZ binding. Dysbindin is also involved in neural development by regulating centrosomal microtubule network formation. All such molecules examined to date are involved in neural development. Thus, these findings provide new molecular insights into the mechanisms of neural development and neuropsychiatric disorders. On the other hand, in addition to neurons, both DISC and DBZ have been detected in oligodendrocytes and implicated in regulating oligodendrocyte differentiation. DISC1 inhibits the differentiation of oligodendrocyte precursor cells into oligodendrocytes, while DBZ has a positive regulatory role in oligodendrocyte differentiation. Evidence suggesting that disturbance of oligodendrocyte development causes major depression is also described.
Asunto(s)
Depresión/genética , Proteínas Asociadas a la Distrofina/genética , Predisposición Genética a la Enfermedad/genética , Proteínas del Tejido Nervioso/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Esquizofrenia/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de Unión a Calmodulina/fisiología , Diferenciación Celular/genética , Proteínas de Unión al ADN/fisiología , Disbindina , Proteínas Asociadas a la Distrofina/fisiología , Humanos , Proteínas del Tejido Nervioso/fisiología , Neurogénesis/genética , Oligodendroglía/citología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Unión Proteica , Estatmina/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The precise molecular mechanisms leading to disturbance of Ca(2+)/calmodulin-dependent intracellular signalling in cardiac hypertrophy remains unclear. As an endogenous calmodulin regulator protein, the pathophysiology role of PEP-19 during cardiac hypertrophy was investigated in the present study. We here demonstrated that PEP-19 protein levels are significantly elevated in the aortic banding model in vivo and angiotensin II-induced cardiomyocyte hypertrophy in vitro. Consistent with inhibitory actions of PEP-19 on cardiomyocyte hypertrophy, induction of CaMKII and calcineurin activation as well as hypertrophy-related genes including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was significantly inhibited by PEP-19 transfection. Moreover, PEP-19 partially ameliorates angiotensin II-induced elevation of phospho-phospholamban (Thr-17) and sarcoplasmic reticulum Ca(2+) release in cardiomyocytes. Together, our results suggest that PEP-19 attenuates angiotensin II-induced cardiomyocyte hypertrophy via suppressing the disturbance of CaMKII and calcineurin signaling.
Asunto(s)
Angiotensina II/efectos adversos , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Cardiomegalia/genética , Expresión Génica , Miocitos Cardíacos/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Animales , Factor Natriurético Atrial/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Señalización del Calcio , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patología , Células Cultivadas , Hipertrofia/genética , Masculino , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/metabolismo , Ratas Sprague-Dawley , Retículo Sarcoplasmático/metabolismoRESUMEN
Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Cuerpos Polares del Huso/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Citocinesis , Mitosis , Transporte de Proteínas , Schizosaccharomyces/citologíaRESUMEN
Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin-based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell-cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Mitosis/fisiología , Miosinas/fisiología , Huso Acromático/fisiología , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Línea Celular , Línea Celular Tumoral , Segregación Cromosómica , Perros , Células HEK293 , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Mitosis/genética , Miosinas/genética , Miosinas/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.
Asunto(s)
Proteínas Asociadas a Microtúbulos/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Saco Vitelino/irrigación sanguínea , Saco Vitelino/enzimología , Animales , Autofagia/genética , Autofagia/fisiología , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Endodermo/irrigación sanguínea , Endodermo/citología , Endodermo/enzimología , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mesodermo/irrigación sanguínea , Mesodermo/citología , Mesodermo/enzimología , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Neovascularización Fisiológica/genética , Embarazo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Saco Vitelino/citología , Saco Vitelino/embriologíaRESUMEN
Control of organ size by cell expansion and cell proliferation is a fundamental process during development, but the importance of BIG in this process is still poorly understood. Here, we report the isolation and characterization of a new allele mutant of BIG in Arabidopsis: big-j588. The mutant displayed small aerial organs that were characterized by reduced cell size in the epidermis and short roots with decreased cell numbers. The big-j588 axr1 double and big-j588 arf7 arf19 triple mutants displayed more severe defects in leaf expansion and root elongation than their parents, implying BIG is involved in auxin-dependent organ growth. Genetic analysis suggests that BIG may act synergistically with PIN1 to affect leaf growth. The PIN1 protein level decreased in both the root cells and the tips of leaf pavement cell lobes of big-j588. Further analysis showed that the auxin maxima in the roots and the leaves of big-j588 decreased. Therefore, we concluded that the small leaves and the short roots of big-j588 were associated with reduction of auxin maxima. Overall, our study suggested that BIG is required for Arabidopsis organ growth via auxin action.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Proteínas de Unión a Calmodulina/fisiología , Ácidos Indolacéticos/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proliferación Celular , Proteínas de Transporte de Membrana/metabolismo , Mutación , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 ((132)LLINRKGELKLADFGLARAFGIP(154)) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP.
Asunto(s)
Aminopeptidasas/metabolismo , Proteínas de Unión a Calmodulina , Núcleo Celular/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/fisiología , Dictyostelium , Aminopeptidasas/antagonistas & inhibidores , Antígenos de Protozoos/metabolismo , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/fisiología , Núcleo Celular/enzimología , Quinasa 5 Dependiente de la Ciclina/química , Quinasa 5 Dependiente de la Ciclina/genética , Dictyostelium/enzimología , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Dictyostelium/ultraestructura , Farmacorresistencia Microbiana , Inmunoprecipitación , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteínas Protozoarias/metabolismo , Puromicina/farmacología , Eliminación de Secuencia , Tripanocidas/farmacologíaRESUMEN
BACKGROUND: Caldesmon (CaD), a major actin-associated protein, is found in smooth muscle and non-muscle cells. Smooth muscle caldesmon, h-CaD, is a multifunctional protein, and non-muscle cell caldesmon, l-CaD, plays a role in cytoskeletal architecture and dynamics. h-CaD is thought to be an useful marker for smooth muscle tumors, but the role(s) of l-CaD has not been examined in tumors. METHODS: Primary colon cancer and liver metastasis tissues were obtained from colon cancer patients. Prior to chemoradiotherapy (CRT), normal and cancerous tissues were obtained from rectal cancer patients. Whole-tissue protein extracts were analyzed by 2-DE-based proteomics. Expression and phosphorylation level of main cellular signaling proteins were determined by western blot analysis. Cell proliferation after CaD siRNA transfection was monitored by MTT assay. RESULTS: The expression level of l-CaD was significantly increased in primary colon cancer and liver metastasis tissues compared to the level in the corresponding normal tissues. In cancerous tissues obtained from the patients showing poor response to CRT (Dworak grade 4), the expression of l-CaD was increased compared to that of good response group (Dworak grade 1). In line with, l-CaD positive human colon cancer cell lines were more resistant to 5-fluorouracil (5-FU) and radiation treatment compared to l-CaD negative cell lines. Artificial suppression of l-CaD increased susceptibility of colon cancer cells to 5-FU, and caused an increase of p21 and c-PARP, and a decrease of NF-kB and p-mTOR expression. CONCLUSION: Up-regulated expression of l-CaD may have a role for increasing metastatic property and decreasing CRT susceptibility in colorectal cancer cells.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Anciano , Antimetabolitos Antineoplásicos/farmacología , Biomarcadores de Tumor/fisiología , Western Blotting , Proteínas de Unión a Calmodulina/fisiología , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Femenino , Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Fosforilación , ARN Interferente Pequeño , Regulación hacia ArribaRESUMEN
Calmodulin (CaM), a key Ca2+ sensor, regulates diverse cellular processes by modulating the activity of a variety of enzymes and proteins. However, little is known about the biological function of CaM in plant development. In this study, an ASYMMETRIC LEAVES1 (AS1) transcription factor was isolated as a CaM-binding protein. AS1 contains two putative CaM-binding domains (CaMBDs) at the N-terminus. Using domain mapping analysis, both predicted domains were identified as authentic Ca2+ -dependent CaMBDs. We identified three hydrophobic amino acid residues for CaM binding, Trp49 in CaMBDI, and Trp81 and Phe103 in CaMBDII. The interactions of AS1 with CaM were verified in yeast and plant cells. Based on electrophoretic mobility shift assays, CaM inhibited the DNA-binding activity of the AS1/AS2 complex to two cis-regulatory motifs in the KNAT1 promoter. Furthermore, CaM relieved the suppression of KNAT1 transcription by AS1 not only in transient expression assays of protoplasts but also by the overexpression of a CaM-binding negative form of AS1 in as1 mutant plant. Our study suggests that CaM, a calcium sensor, can be involved in the transcriptional control of meristem cell-specific genes by the inhibition of AS1 under the condition of higher levels of Ca2+ in plants.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Proteínas de Unión a Calmodulina/fisiología , Calmodulina/fisiología , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/fisiología , Agrobacterium tumefaciens/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Mapeo Cromosómico , Escherichia coli/genética , Biblioteca de Genes , Proteínas de Homeodominio/genética , Meristema/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Nicotiana/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
We recently identified a novel IQ motif-containing protein family, IQM, which shares sequence homology with a pea heavy metal-induced protein 6 and a ribosome inactivating protein, trichosanthin. Distinct expression patterns for each gene suggest that each IQM family member may play a different role in plant development and response to environmental cues. However functions of the IQM family members remain to be analyzed. IQM1 bound with calmodulin 5 (CaM5) in yeast two-hybrid assay via its IQ-motif. The CaM binding was Ca(2+)-independent in vitro, and was also observed in bimolecular fluorescence complementation analyses in onion epidermal cells. IQM1 was found to express strongly in guard cells and the cortex of roots. The T-DNA insertion mutants of IQM1 displayed a smaller stomatal aperture, a decreased water loss rate and a shorter primary root. Moreover, iqm1 did not change its stomatal aperture when treated with light, dark, ABA and chitin obviously. Microarray analyses showed that 243 and 28 genes were up- and down-regulated by more than twofold in iqm1-1, respectively. Interesting, 34 of 117 and 7 of 30 chitin-responsive transcriptional factor and ubiquitin ligase genes were up-regulated, respectively. Stomatal guard cells of iqm1-1 also showed enhanced expression of genes involved in production and signaling of reactive oxygen species (ROS). Consistently, increased ROS level was observed in the iqm1 guard cells.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Proteínas de Unión a Calmodulina/fisiología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Señalización del Calcio , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Quitina/metabolismo , ADN Bacteriano/genética , Hongos/patogenicidad , Perfilación de la Expresión Génica , Genes de Plantas , Movimiento/fisiología , Mutagénesis Insercional , Estomas de Plantas/genética , Estomas de Plantas/fisiología , Dominios y Motivos de Interacción de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
Diminishing global fresh water availability has focused research to elucidate mechanisms of water use in poplar, an economically important species. A GT-2 family trihelix transcription factor that is a determinant of water use efficiency (WUE), PtaGTL1 (GT-2 like 1), was identified in Populus tremula × P. alba (clone 717-IB4). Like other GT-2 family members, PtaGTL1 contains both N- and C-terminal trihelix DNA binding domains. PtaGTL1 expression, driven by the Arabidopsis thaliana AtGTL1 promoter, suppressed the higher WUE and drought tolerance phenotypes of an Arabidopsis GTL1 loss-of-function mutation (gtl1-4). Genetic suppression of gtl1-4 was associated with increased stomatal density due to repression of Arabidopsis STOMATAL DENSITY AND DISTRIBUTION1 (AtSDD1), a negative regulator of stomatal development. Electrophoretic mobility shift assays (EMSA) indicated that a PtaGTL1 C-terminal DNA trihelix binding fragment (PtaGTL1-C) interacted with an AtSDD1 promoter fragment containing the GT3 box (GGTAAA), and this GT3 box was necessary for binding. PtaGTL1-C also interacted with a PtaSDD1 promoter fragment via the GT2 box (GGTAAT). PtaSDD1 encodes a protein with 60% primary sequence identity with AtSDD1. In vitro molecular interaction assays were used to determine that Ca(2+)-loaded calmodulin (CaM) binds to PtaGTL1-C, which was predicted to have a CaM-interaction domain in the first helix of the C-terminal trihelix DNA binding domain. These results indicate that, in Arabidopsis and poplar, GTL1 and SDD1 are fundamental components of stomatal lineage. In addition, PtaGTL1 is a Ca(2+)-CaM binding protein, which infers a mechanism by which environmental stimuli can induce Ca(2+) signatures that would modulate stomatal development and regulate plant water use.
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Calcio/química , Proteínas de Unión a Calmodulina/fisiología , Calmodulina/química , Genes de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/fisiología , Arabidopsis/genética , Proteínas de Unión a Calmodulina/genética , Núcleo Celular/metabolismo , Sequías , Modelos Genéticos , Mutación , Fenotipo , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Agua/químicaRESUMEN
Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable 'prion domain' enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer's disease and Huntington's disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion domains of RNA-binding proteins could underlie the classical non-cell-autonomous emanation of neurodegenerative pathology from originating epicenters to neighboring portions of the nervous system. This article is part of a Special Issue entitled RNA-Binding Proteins.
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Enfermedades Neurodegenerativas/genética , Priones/genética , Proteínas de Unión al ARN/genética , Algoritmos , Esclerosis Amiotrófica Lateral/genética , Animales , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/patología , Humanos , Enfermedades Neurodegenerativas/metabolismo , Priones/metabolismo , Priones/fisiología , Proteína EWS de Unión a ARN , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/fisiologíaRESUMEN
OBJECTIVE: To investigate expression and α1-adrenergic regulation of caldesmon in the human prostate. Caldesmon is an important mediator and regulator of contraction in different smooth muscle types. However, this has not been investigated in the prostate to date. The activity of caldesmon may be tightly regulated by serine-789 phosphorylation. MATERIALS AND METHODS: Prostate tissue was obtained from patients undergoing radical prostatectomy. Caldesmon expression was studied by Western blot analysis and immunohistochemistry. The adrenergic regulation of caldesmon phosphorylation was investigated by Western blot analyses with a site- and phosphospecific antibody. RESULTS: Caldesmon expression was detectable by Western blot analysis in all investigated samples of human prostates (n = 8 patients). Immunoreactivity after staining with a caldesmon antibody was strong in smooth muscle cells, but not observed in glandular or epithelial cells (n = 5 patients). In double fluorescence staining, caldesmon co-localized with α1A-adrenoceptors and α-smooth muscle actin (n = 6 patients). Stimulation of prostate tissue with noradrenaline (30 µM, n = 6 patients) or the α1-adrenergic agonist phenylephrine (10 µM, n = 6 patients) resulted in progressive phosphorylation of caldesmon at serine-789. Noradrenaline-induced caldesmon phosphorylation was 1.5 ± 0.2-fold after 5 minutes (P<.04 vs basal phosphorylation), and 1.6 ± 0.2-fold after 10 minutes (P<.04). Phenylephrine-induced caldesmon phosphorylation was 1.7 ± 0.2-fold after 10 minutes (P<.02 vs basal phosphorylation), and 2.4 ± 0.6-fold after 20 minutes (P<.05). CONCLUSIONS: Caldesmon is an effector of α1-adrenoceptors in the human prostate. Caldesmon activation may be of importance for α1-adrenergic prostate contraction, and during therapy with α1-blockers.