RESUMEN
Several studies have shown association of single nucleotide polymorphisms (SNPs) of hepcidin regulatory pathways genes with impaired iron status. The most common is in the TMPRSS6 gene. In Africa, very few studies have been reported. We aimed to investigate the correlation between the common SNPs in the transmembrane protease, serine 6 (TMPRSS6) gene and iron indicators in a sample of Egyptian children for identifying the suitable candidate for iron supplementation.Patients and methods One hundred and sixty children aged 5-13 years were included & classified into iron deficient, iron deficient anemia and normal healthy controls. All were subjected to assessment of serum iron, serum ferritin, total iron binding capacity, complete blood count, reticulocyte count, serum soluble transferrin receptor and serum hepcidin. Molecular study of TMPRSS6 genotyping polymorphisms (rs4820268, rs855791 and rs11704654) were also evaluated.Results There was an association of iron deficiency with AG of rs855791 SNP, (P = 0.01). The minor allele frequency for included children were 0.43, 0.45 & 0.17 for rs4820268, rs855791 & rs11704654 respectively. Genotype GG of rs4820268 expressed the highest hepcidin gene expression fold, the lowest serum ferroportin & iron store compared to AA and AG genotypes (p = 0.05, p = 0.05, p = 0.03 respectively). GG of rs855791 had lower serum ferritin than AA (p = 0.04), lowest iron store & highest serum hepcidin compared to AA and AG genotypes (p = 0.04, p = 0.01 respectively). Children having CC of rs11704654 had lower level of hemoglobin, serum ferritin and serum hepcidin compared with CT genotype (p = 0.01, p = 0.01, p = 0.02) respectively.Conclusion Possible contribution of SNPs (rs855791, rs4820268 and rs11704654) to low iron status.
Asunto(s)
Anemia Ferropénica , Hierro , Niño , Humanos , Hepcidinas/genética , Hepcidinas/metabolismo , Proyectos Piloto , Serina/genética , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Egipto , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Polimorfismo de Nucleótido Simple , Ferritinas , Anemia Ferropénica/genética , Proteínas de la Membrana/genéticaRESUMEN
In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.
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Encefalopatías , Ácidos Glicéricos , Serina , Humanos , Serina/genética , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/química , Encefalopatías/metabolismo , AminoácidosRESUMEN
Indole is traditionally known as a metabolite of l-tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole-deficient mutant was screened out. Via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate-binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind to l-serine (KD = 6.149 × 10-5 M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds to l-serine ABC transports. As a precursor of indole synthesis, the transmembrane transported l-serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.
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Pantoea , Mutación , Pantoea/genética , Aminoácidos/metabolismo , Indoles/metabolismo , Serina/genética , Serina/metabolismoRESUMEN
Biocontainment is a key methodology to reduce environmental risk through the deliberate release of genetically modified microorganisms. Previously, we developed a phosphite (HPO32-)-dependent biocontainment strategy, by expressing a phosphite-specific transporter HtxBCDE and phosphite dehydrogenase in bacteria devoid of their indigenous phosphate (HPO42-) transporters. This strategy did not allow Escherichia coli to generate escape mutants (EMs) in growth media containing phosphate as a phosphorus source using an assay with a detection limit of 1.9 × 10-13. In this study, we found that the coexistence of a high dose of phosphate (>0.5 mM) with phosphite in the growth medium allows the phosphite-dependent E. coli strain to generate EMs at a frequency of approximately 5.4 × 10-10. In all EMs, the mutation was a single amino acid substitution of phenylalanine to cysteine or serine at position 210 of HtxC, the transmembrane domain protein of the phosphorus compound transporter HtxBCDE. Replacement of the HtxC F210 residue with the other 17 amino acids revealed that HtxC F210 is crucial in determining substrate specificity of HtxBCDE. Based on the finding of the role of HtxC F210 as a "gatekeeper" residue for this transporter, we demonstrate that the replacement of HtxC F210 with amino acids resulting from codons that require two simultaneous point mutations to generate phosphate permissive HtxC mutants can reduce the rate of EM generation to an undetectable level. These findings also provide novel insights into the functional classification of HtxBCDE as a noncanonical ATP-binding cassette transporter in which the transmembrane domain protein participates in substrate recognition.
Asunto(s)
Fosfitos , Fosfitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cisteína , Proteínas Bacterianas/metabolismo , Mutación , Transportadoras de Casetes de Unión a ATP/genética , Fosfatos/metabolismo , Fósforo/metabolismo , Serina/genética , Fenilalanina/genéticaRESUMEN
AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.
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Cardiomiopatía Dilatada , Terapia Molecular Dirigida , Miocitos Cardíacos , Inhibidores de Proteínas Quinasas , Serina , Troponina T , Factor de Transcripción Activador 4/metabolismo , Adenosina Trifosfato/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Carbazoles/farmacología , Carbazoles/uso terapéutico , Cardiomiopatía Dilatada/tratamiento farmacológico , Cardiomiopatía Dilatada/genética , Evaluación Preclínica de Medicamentos/métodos , Glucosa/metabolismo , Glicina/biosíntesis , Glicina/genética , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Células Madre Pluripotentes Inducidas/fisiología , Mutación , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Fosfoglicerato-Deshidrogenasa/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Serina/antagonistas & inhibidores , Serina/biosíntesis , Serina/genética , Troponina T/genética , Troponina T/metabolismoRESUMEN
Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.
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Fibras de la Dieta/metabolismo , Hidrolasas/metabolismo , Oryza , Proteínas de Vegetales Comestibles/metabolismo , Serina/metabolismo , Suplementos Dietéticos , Almacenamiento de Alimentos , Hidrolasas/genética , Anotación de Secuencia Molecular , Análisis por Matrices de Proteínas , Serina/genética , LevadurasRESUMEN
OBJECTIVE: To study the correlation between the phosphorylation of keratin 18 (K18) and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin (OXA) and investigate its possible mechanism. METHODS: HCT116 cells were transfected with empty plasmid, wild-type K18 expression plasmid and 33, 52 phosphorylation site mutated K18 (Ser33/52A) expression plasmid separately, and all cells were then treated with 60 µmol/L OXA, followed by supplementation of autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide (PI) double staining combined with flow cytometry, calcein-AM/PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation, autophagy related proteins microtubule associated protein 1 light chain 3 (LC3) and beclin-1. RESULTS: Transfection of Ser33/52A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA, the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group, while the apoptosis rate of Ser33/52A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group, the autophagy of K18 plasmid transfected group was significantly promoted, while the autophagy in Ser33/52A plasmid transfected group was significantly inhibited. CONCLUSION: K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Queratina-18/metabolismo , Compuestos Organoplatinos/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Antineoplásicos/farmacología , Apoptosis/genética , Autofagia/genética , Western Blotting , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Citometría de Flujo , Células HCT116 , Humanos , Queratina-18/genética , Microscopía Fluorescente , Mutación , Oxaliplatino , Fosforilación , Serina/genética , Serina/metabolismo , Sirolimus/farmacología , TransfecciónRESUMEN
An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50-90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.
Asunto(s)
Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Microbiana/genética , Isoniazida/uso terapéutico , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Antituberculosos/farmacología , Codón , Análisis Mutacional de ADN , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mutación Puntual , Serina/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Water movement across cellular membranes is mostly regulated by aquaporins. A tonoplast intrinsic protein PgTIP1 from Panax ginseng has been found to play an important role in plant growth and development, and also in the response of plants to abiotic stress. However, the regulation of its function and activity remains unknown. To answer this question, mutated forms of PgTIP1 were made by replacing Ser(128) with Ala (named S128A) or Asp (named S128D), and also by replacing Thr(54) with Ala (named T54A) or Asp (named T54D). Then, wild type or mutated PgTIP1 was expressed in yeast and water transport was monitored in protoplasts. The substitution of Ser(128) abolished the water channel activity of PgTIP1, while the substitution of Thr(54) did not inhibit its activity. Moreover, the overexpression of PgTIP1 but not S128A or S128D in Arabidopsis significantly increased plant growth as determined by biomass production, it also had a beneficial effect on salt stress tolerance. Importantly, the overexpression of PgTIP1 led to the altered expression of stress-related genes, which made the plants more tolerant to salt stress. Our results demonstrated that PgTIP1 conferred faster growth and enhanced tolerance to salt in Arabidopsis, and that its biological activity related to Ser(128) residue.
Asunto(s)
Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/metabolismo , Panax/genética , Proteínas de Plantas/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Sequías , Expresión Génica , Proteínas de la Membrana/genética , Mutación , Fenotipo , Fosforilación , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Tolerancia a la Sal , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/fisiología , Serina/genética , Cloruro de Sodio/farmacología , Estrés FisiológicoRESUMEN
The Disrupted-in-Schizophrenia 1 (DISC1) gene has been thought as a putative susceptibility gene for various psychiatric disorders, and DISC1 Ser704Cys is associated with variations of brain morphology and function. Moreover, our recent diffusion magnetic resonance imaging (dMRI) study reported that DISC1 Ser704Cys was associated with information transfer efficiency in the brain anatomical network. However, the effects of the DISC1 gene on functional brain connectivity and networks, especially for thalamic-prefrontal circuit, which are disrupted in various psychiatric disorders, are largely unknown. Using a functional connectivity density (FCD) mapping method based on functional magnetic resonance imaging data in a large sample of healthy Han Chinese subjects, we first investigated the association between DISC1 Ser704Cys and short- and long-range FCD hubs. Compared with Ser homozygotes, Cys-allele individuals had increased long-range FCD hubs in the bilateral thalami. The functional and anatomical connectivity of the thalamus to the prefrontal cortex was further analyzed. Significantly increased thalamic-prefrontal functional connectivity and decreased thalamic-prefrontal anatomical connectivity were found in DISC1 Cys-allele carriers. Our findings provide consistent evidence that the DISC1 Ser704Cys polymorphism influences the thalamic-prefrontal circuits in humans and may provide new insights into the neural mechanisms that link DISC1 and the risk for psychiatric disorders.
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Vías Aferentes/anatomía & histología , Cisteína/genética , Proteínas del Tejido Nervioso/genética , Corteza Prefrontal/anatomía & histología , Serina/genética , Tálamo/anatomía & histología , Adolescente , Adulto , Vías Aferentes/irrigación sanguínea , Mapeo Encefálico , Distribución de Chi-Cuadrado , Femenino , Humanos , Imagenología Tridimensional , Imagen por Resonancia Magnética , Masculino , Oxígeno/sangre , Corteza Prefrontal/irrigación sanguínea , Tálamo/irrigación sanguínea , Adulto JovenAsunto(s)
Candida albicans/genética , Farmacorresistencia Fúngica/genética , Fluconazol/uso terapéutico , Mutación Missense , Esterol 14-Desmetilasa/genética , Sustitución de Aminoácidos , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Sistema Enzimático del Citocromo P-450/genética , Humanos , Pruebas de Sensibilidad Microbiana , Fenilalanina/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina/genéticaRESUMEN
The voltage-gated Ca(2+) channel ß-subunit is a member of the membrane-associated guanylate kinase family and modulates kinetic properties of the Ca(2+) channels, such as their voltage-dependent activation and inactivation rates. Two cDNA clones were identified to encode each ß-subunit isotype of the voltage-gated Ca(2+) channel of Clonorchis sinensis, CsCavß1 and CsCavß2, which consist of 606 and 887 amino acids, respectively. CsCavß1 was found to be similar to the ß-subunit containing two conserved serine residues that constitute the consensus protein kinase C phosphorylation site in the ß-interaction domain (BID). CsCavß2 had cysteine and alanine residues instead of the two serine residues conserved in BID and was homologous to variant ß-subunit of Schistosoma mansoni and Schistosoma japonicum. CsCavß1 and CsCavß2 were almost equally expressed in the adults and metacercariae, but were more expressed in adult C. sinensis than in metacercariae. Collectively, our findings suggest that substitution of the two serine residues in BID of CsCavß2 may render C. sinensis sensitive to praziquantel.
Asunto(s)
Canales de Calcio/metabolismo , Clonorchis sinensis/genética , Proteínas del Helminto/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/genética , Clonación Molecular , Clonorchis sinensis/metabolismo , Secuencia Conservada , ADN Complementario/genética , Proteínas del Helminto/genética , Datos de Secuencia Molecular , Fosforilación , Filogenia , Praziquantel , Alineación de Secuencia , Serina/genéticaRESUMEN
This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Familia de Multigenes , Fosfoglicerato-Deshidrogenasa/metabolismo , Plastidios/enzimología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolómica/métodos , Microscopía Confocal , Datos de Secuencia Molecular , Mutación , Fosfoglicerato-Deshidrogenasa/clasificación , Fosfoglicerato-Deshidrogenasa/genética , Fosforilación , Filogenia , Componentes Aéreos de las Plantas/enzimología , Componentes Aéreos de las Plantas/genética , Componentes Aéreos de las Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Polen/enzimología , Polen/genética , Polen/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/enzimología , Semillas/genética , Semillas/metabolismo , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismoRESUMEN
We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.
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Evolución Molecular , Fertilidad/genética , Fosfoproteínas/genética , Procesamiento Proteico-Postraduccional , Espermatozoides/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Secuencia Conservada , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica , Humanos , Masculino , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Fosforilación , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genéticaRESUMEN
Vitamin E is a fat-soluble vitamin with antioxidant properties. Tocopherols are the predominant form of vitamin E found in the diet and in supplements and have garnered interest for their potential cancer therapeutic and preventive effects, such as the dephosphorylation of Akt, a serine/threonine kinase with a pivotal role in cell growth, survival, and metabolism. Dephosphorylation of Akt at Ser473 substantially reduces its catalytic activity and inhibits downstream signaling. We found that the mechanism by which α-tocopherol and γ-tocopherol facilitate this site-specific dephosphorylation of Akt was mediated through the pleckstrin homology (PH) domain-dependent recruitment of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1) to the plasma membrane. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain-containing proteins, which may underlie the preferential recruitment of these proteins to membranes containing tocopherols. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. By describing a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, we provide insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vitamina E/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Desnudos , Microscopía Confocal , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Serina/genética , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Vitamina E/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , alfa-Tocoferol/metabolismo , alfa-Tocoferol/farmacología , gamma-Tocoferol/metabolismo , gamma-Tocoferol/farmacologíaRESUMEN
Selenium (Se) is an essential redox-active trace element with close connections to cancer. Most of Se's biological functions have been attributed to the antioxidant properties of Se-containing proteins. However, the relative contribution of selenoproteins and small Se compounds in cancer protection is still a matter of debate. The tumor suppressor p53 is the most frequently mutated gene in human cancer and is often referred to as the "guardian of the genome". In response to genomic stresses, p53 causes cell cycle arrest to allow time for genomic damage to be repaired before cell division or induces apoptosis to eliminate irreparably damaged cells. Selenoprotein W (SEPW1) is a highly conserved small thioredoxin-like protein required for cell cycle progression. The present work shows that SEPW1 facilitates the G1 to S-phase transition by down-regulating expression of the cyclin-dependent kinase inhibitor p21. SEPW1 controls p21 by modulating levels of the p53 transcription factor, and this is associated with changes in phosphorylation of Ser-33 in p53. More work is needed to identify the mechanism by which SEPW1 regulates phosphorylation of Ser-33 and the kinase or phosphatase enzymes involved.
Asunto(s)
Neoplasias de la Mama/patología , Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Selenoproteína W/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , Femenino , Silenciador del Gen , Humanos , Fosforilación , ARN Interferente Pequeño/genética , Selenio/metabolismo , Selenoproteína W/genética , Serina/genética , Serina/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Phosphatase of regenerating liver 3 (PRL-3) is suggested as a biomarker and therapeutic target in several cancers. It has a well-established causative role in cancer metastasis. However, little is known about its natural substrates, pathways, and biological functions, and only a few protein substrates have been suggested so far. To improve our understanding of the substrate specificity and molecular determinants of PRL-3 activity, the wild-type (WT) protein, two supposedly catalytically inactive mutants D72A and C104S, and the reported hyperactive mutant A111S were tested in vitro for substrate specificity and activity toward phosphopeptides and phosphoinositides (PIPs), their structural stability, and their ability to promote cell migration using stable HEK293 cell lines. We discovered that WT PRL-3 does not dephosphorylate the tested phosphopeptides in vitro. However, as shown by two complementary biochemical assays, PRL-3 is active toward the phosphoinositide PI(4,5)P(2). Our experimental results substantiated by molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. The C104S variant was shown to be not only catalytically inactive but also structurally destabilized and unable to promote cell migration, whereas WT PRL-3 promotes cell migration. The D72A mutant is structurally stable and does not dephosphorylate the unnatural substrate 3-O-methylfluorescein phosphate (OMFP). However, we observed residual in vitro activity of D72A against PI(4,5)P(2), and in accordance with this, it exhibits the same cellular phenotype as WT PRL-3. Our analysis of the A111S variant shows that the hyperactivity toward the unnatural OMFP substrate is not apparent in dephosphorylation assays with phosphoinositides: the mutant is completely inactive against PIPs. We observed significant structural destabilization of this variant. The cellular phenotype of this mutant equals that of the catalytically inactive C104S mutant. These results provide a possible explanation for the absence of the conserved Ser of the PTP catalytic motif in the PRL family. The correlation of the phosphatase activity toward PI(4,5)P(2) with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P(2) dephosphorylation by PRL-3 and its role in cell migration.
Asunto(s)
Movimiento Celular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiología , Fosfatidilinositoles/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/fisiología , Secuencia de Aminoácidos , Catálisis , Movimiento Celular/genética , Cisteína/genética , Activación Enzimática/genética , Variación Genética , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutación/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , Fosfatidilinositoles/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Serina/genética , Especificidad por Sustrato/genéticaRESUMEN
Zinc is abundant in the central nervous system and regulates pain, but the underlying mechanisms are unknown. In vitro studies have shown that extracellular zinc modulates a plethora of signaling membrane proteins, including NMDA receptors containing the NR2A subunit, which display exquisite zinc sensitivity. We created NR2A-H128S knock-in mice to investigate whether Zn2+-NR2A interaction influences pain control. In these mice, high-affinity (nanomolar) zinc inhibition of NMDA currents was lost in the hippocampus and spinal cord. Knock-in mice showed hypersensitivity to radiant heat and capsaicin, and developed enhanced allodynia in inflammatory and neuropathic pain models. Furthermore, zinc-induced analgesia was completely abolished under both acute and chronic pain conditions. Our data establish that zinc is an endogenous modulator of excitatory neurotransmission in vivo and identify a new mechanism in pain processing that relies on NR2A NMDA receptors. The study also potentially provides a molecular basis for the pain-relieving effects of dietary zinc supplementation.
Asunto(s)
Neuronas/efectos de los fármacos , Dolor/tratamiento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo , Oligoelementos/farmacología , Estimulación Acústica , Análisis de Varianza , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fuerza de la Mano/fisiología , Hipocampo/citología , Histidina/genética , Técnicas In Vitro , Larva , Locomoción/efectos de los fármacos , Locomoción/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Dolor/etiología , Dolor/fisiopatología , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Estimulación Física , Unión Proteica/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/genética , Receptores de N-Metil-D-Aspartato/genética , Reflejo/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante/métodos , Serina/genética , Olfato/efectos de los fármacos , Olfato/genética , Médula Espinal/citología , Estadísticas no Paramétricas , Percepción del Tacto/efectos de los fármacos , Percepción del Tacto/genética , Oligoelementos/uso terapéutico , Xenopus , Zinc/farmacología , Zinc/uso terapéuticoRESUMEN
Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.