RESUMEN
Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.
Asunto(s)
Proteínas 14-3-3/fisiología , Túbulos Renales/fisiología , Proteínas 14-3-3/metabolismo , Adenosina/farmacología , Alopurinol/farmacología , Antioxidantes/farmacología , Soluciones Cardiopléjicas/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Criopreservación/métodos , Deferoxamina/farmacología , Glutatión/farmacología , Humanos , Insulina/farmacología , Túbulos Renales/citología , Túbulos Renales/metabolismo , Mitocondrias/metabolismo , Mitocondrias/fisiología , Preservación de Órganos/métodos , Soluciones Preservantes de Órganos/farmacología , Fosforilación Oxidativa , Estrés Oxidativo/fisiología , Rafinosa/farmacología , Sideróforos/farmacologíaRESUMEN
Recent studies suggest that royal jelly (RJ) and its related substances may have antiaging properties. However, the molecular mechanisms underlying the beneficial effects remain elusive. We report that the effects of RJ and enzyme-treated RJ (eRJ) on life span and health span in Caenorhabditis elegans (C elegans) are modulated by the sophisticated interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. Dietary supplementation with RJ or eRJ increased C. elegans life span in a dose-dependent manner. The RJ and eRJ consumption increased the tolerance of C elegans to oxidative stress, ultraviolet irradiation, and heat shock stress. Our genetic analyses showed that RJ/eRJ-mediated life-span extension requires insulin/IGF-1 signaling and the activities of DAF-16, SIR-2.1, HCF-1, and FTT-2, a 14-3-3 protein. Earlier studies reported that DAF-16/FOXO, SIR-2.1/SIRT1, FTT-2, and HCF-1 have extensive interplays in worms and mammals. Our present findings suggest that RJ/eRJ-mediated promotion of longevity and stress resistance in C elegans is dependent on these conserved interplays. From an evolutionary point of view, this study not only provides new insights into the molecular mechanisms of RJ's action on health span promotion in C elegans, but also has imperative implications in using RJ/eRJ as nutraceuticals to delay aging and age-related disorders.
Asunto(s)
Proteínas 14-3-3/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Ácidos Grasos/farmacología , Factores de Transcripción Forkhead/fisiología , Factor C1 de la Célula Huésped/fisiología , Longevidad/efectos de los fármacos , Sirtuinas/fisiología , Animales , Caenorhabditis elegans , Estrés Fisiológico/efectos de los fármacosRESUMEN
14-3-3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14-3-3 gene family members (TFT1-TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild-type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⺠flux along their root tip and activity of plasma membrane Hâº-ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane Hâº-ATPase to release more protons under LP.
Asunto(s)
Proteínas 14-3-3/fisiología , Adaptación Fisiológica , Fósforo/metabolismo , Proteínas de Plantas/fisiología , Solanum lycopersicum/fisiología , Estrés Fisiológico , Plantas Modificadas GenéticamenteRESUMEN
This study investigates the scar-reducing efficacy of topical application of stratifin and acetylsalicylic acid (ASA) in a rabbit ear model. A total of five New Zealand white rabbits with four wounds per ear were examined. Either recombinant stratifin (0.002%) or ASA (0.5%) incorporated in carboxymethyl cellulose gel was topically applied on each wound at postwounding Day 5. Scars were harvested at postwounding Day 28 for histological analysis. The wounds treated with stratifin and ASA showed 82 and 73% reduction in scar volume, respectively, compared with that of untreated controls. A reduction of 57 and 41% in total tissue cellularity along with 79 and 91% reduction in infiltrated CD3+ T cells were observed in response to treatment with stratifin and ASA, respectively, compared with those of untreated controls. Wounds treated with stratifin showed a 2.8-fold increase in matrix metalloproteinase-1 expression, which resulted in a 48% decrease in collagen density compared with those of untreated controls. Qualitative wound assessment showed a reduced hypertrophic scarring in stratifin and ASA-treated wounds when compared with the controls. This study showed that topical application of either stratifin or ASA-impregnated carboxymethyl cellulose gel reduced hypertrophic scar formation following dermal injuries in a rabbit ear fibrotic model.
Asunto(s)
Proteínas 14-3-3/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Aspirina/uso terapéutico , Biomarcadores de Tumor/uso terapéutico , Cicatriz Hipertrófica/prevención & control , Exonucleasas/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Proteínas 14-3-3/farmacología , Proteínas 14-3-3/fisiología , Administración Cutánea , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Vendajes , Biomarcadores de Tumor/farmacología , Biomarcadores de Tumor/fisiología , Carboximetilcelulosa de Sodio/uso terapéutico , Cicatriz Hipertrófica/etiología , Cicatriz Hipertrófica/patología , Colágeno/efectos de los fármacos , Colágeno/fisiología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Exonucleasas/farmacología , Exonucleasas/fisiología , Exorribonucleasas , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Geles , Metaloproteinasa 1 de la Matriz/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/fisiología , Conejos , Índice de Severidad de la EnfermedadRESUMEN
Ten years ago, par-1 and par-3 were cloned as two of the six par genes essential for the asymmetric division of the Caenorhabditis elegans zygote. PAR-1 is a protein kinase, whereas PAR-3 is a PDZ-domain-containing scaffold protein. Work over the past decade has shown that they are part of an evolutionarily conserved PAR-aPKC system involved in cell polarity in various biological contexts. Recent progress has illustrated the common principle that the PAR-aPKC system is the molecular machinery that converts initial polarity cues in the establishment of complementary membrane domains along the polarity axis. In most cases, this is achieved by mutually antagonistic interactions between the aPKC-PAR-3-PAR-6 complex and PAR-1 or PAR2 located opposite. However, accumulating evidence has also revealed that mechanisms by which the asymmetrically localized components of the PAR-aPKC system are linked with other cellular machinery for developing polarity are divergent depending on the cell type.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Polaridad Celular/fisiología , Proteína Quinasa C/fisiología , Proteínas/fisiología , Transducción de Señal , Proteínas 14-3-3/fisiología , Animales , Proteínas del Dominio Armadillo/metabolismo , Caenorhabditis elegans , Drosophila , Proteínas de Drosophila/fisiología , Uniones Intercelulares/fisiología , Microtúbulos/fisiología , Unión Proteica , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
Individual members of the RGK family of Ras-related GTPases, which comprise Rad, Gem/Kir, Rem and Rem2, have been implicated in important functions such as the regulation of voltage-gated calcium channel activity and remodeling of cell shape. The GTPase Kir/Gem inhibits the activity of calcium channels by interacting with the beta-subunit and also regulates cytoskeleton dynamics by inhibiting the Rho-Rho kinase pathway. In addition, Kir/Gem interacts with 14-3-3 and calmodulin, but the significance of this interaction on Kir/Gem function is poorly understood. Here, we present a comprehensive analysis of the binding of 14-3-3 and calmodulin to Kir/Gem. We show that 14-3-3, in conjunction with calmodulin, regulates the subcellular distribution of Kir/Gem between the cytoplasm and the nucleus. In addition, 14-3-3 and calmodulin binding modulate Kir/Gem-mediated cell shape remodeling and downregulation of calcium channel activity. Competition experiments show that binding of 14-3-3, calmodulin and calcium channel beta-subunits to Kir/Gem is mutually exclusive, providing a rationale for the observed regulatory effects of 14-3-3 and calmodulin on Kir/Gem localization and function.
Asunto(s)
Proteínas 14-3-3/fisiología , Canales de Calcio/metabolismo , Calmodulina/fisiología , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas de Unión al GTP Monoméricas/biosíntesis , Animales , Células COS , Calcio/metabolismo , Canales de Calcio/química , Calmodulina/química , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Dimerización , Electrofisiología , GTP Fosfohidrolasas/metabolismo , Glutatión Transferasa/metabolismo , Inmunohistoquímica , Microscopía Fluorescente , Modelos Biológicos , Mutación , Células PC12 , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Fracciones Subcelulares , TransfecciónRESUMEN
The 14-3-3 protein is one of the best candidates for coordinating all plant metabolic pathways. To verify this suggestion transgenic potato plants with repression of one (J4 and J5 plants), two (G1 plants), and six (G3 plants) constitutive 14-3-3 protein isoforms as well as plants overexpressing the 14-3-3 protein were studied. Reduction in the 14-3-3 protein level in the J4 and J5 transformants, the G1 transformants, and the G3 transformants was close to 29, 41.5, 38, and 55%, respectively. In the case of the 14-3-3 overexpressing plants (J2), a 30% increase in protein content was detected. Changes in nitrate reductase (NR), sucrose phosphate synthase (SPS), and starch synthase (SS) activities in the transgenic plants perfectly reflect the overall 14-3-3 protein level. The highest increase in enzyme activities was observed for the G3 plants and the lowest for the J4 transformants. The same was detected for the measured metabolites. The highest increase in the protein, starch, and sucrose levels was detected in the tubers from the G3 transgenic plants. Because there was almost no change in the isoform ratio in the transgenic plants when compared to the control, it is suggested that it is the overall content of the 14-3-3 protein, rather than the content of particular isoforms, which plays a crucial role in the regulation of enzyme activities and thus in metabolite synthesis. The properties of the 14-3-3 overexpressing plants are very similar to those of the control ones, suggesting that the protein is in excess in the nontransformants and a further increase in its content is not recognized by cell metabolism. A considerable influence of the 14-3-3 protein level on potato plant metabolism was demonstrated. This effect was observed in key metabolic enzyme activities and metabolite content as well. A high variability between mean values, representing individual transgenes, with respect to nitrate reductase, sucrose phosphate synthase, and starch synthase activities in the examined genotypes was noted. These changes were closely correlated with metabolite levels, among them protein, starch, reducing sugars, and sucrose. The results obtained for the five types of transgenic potato plants in comparison with the control were statistically assessed using discriminate function and cluster analyses.
Asunto(s)
Proteínas 14-3-3/fisiología , Metabolismo de los Hidratos de Carbono , Nitratos/metabolismo , Plantas Modificadas Genéticamente/enzimología , Solanum tuberosum/enzimología , Proteínas 14-3-3/análisis , Proteínas 14-3-3/genética , Expresión Génica , Nitrato-Reductasa , Nitrato Reductasas/metabolismo , Proteínas de Plantas/análisis , Tubérculos de la Planta/enzimología , Isoformas de Proteínas/análisis , Solanum tuberosum/genética , TransfecciónRESUMEN
Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.