RESUMEN
Deviations from the normal nucleoplasmic protein O-GlcNAcylation, as well as from normal protein sialylation and N-glycosylation in the secretory pathway, have been reported in Alzheimer's disease (AD). However, the interplay between the cytoplasmic protein O-GlcNAcylation and the secretory N-/O-glycosylation in AD has not been described. We present a comprehensive analysis of the N-, O-, and O-GlcNAc-glycomes in AD-affected brain regions as well as in AD patient serum. We detected marked differences in levels of glycan involved in both protein O-GlcNAcylation and N-/O-glycosylation between patients and healthy individuals and revealed brain region-specific glycosylation-related pathology in patients. These alterations are not general for other neurodegenerative conditions, such as frontotemporal dementia and corticobasal degeneration. The alterations in the AD glycome in the serum could potentially lead to novel glyco-based biomarkers for AD progression. Strikingly, negative interrelationship was found between the pathways of protein O-GlcNAcylation and N-/O-glycosylation, suggesting a novel intracellular cross-talk.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/patología , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Femenino , Glicosilación , Humanos , Masculino , Persona de Mediana Edad , Proteoma , Proteómica/métodosRESUMEN
It is increasingly clear that transcription factors play versatile roles in turning genes "on" or "off" depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. Here we use the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, we identified 189 down-stream targets of CHIP/LDB and show that these genes are enriched for the binding sites of APTEROUS (AP) and PANNIER (PNR), two well studied transcription factors associated with CHIP/LDB complexes. We performed extensive genetic screens and identified target genes that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing we uncovered novel roles for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, our results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Transcripción Genética , Animales , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Proteínas Nucleares/genética , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ribonucleótido Reductasas/metabolismo , Staphylococcus aureus/metabolismo , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Biología Computacional , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Ribonucleótido Reductasas/genética , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genética , Tiorredoxinas/química , Tiorredoxinas/clasificación , Tiorredoxinas/genéticaRESUMEN
Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of gamma-glutamylcysteine (gamma GC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of gamma GC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/fisiología , Glutamato-Cisteína Ligasa/química , Glutamato-Cisteína Ligasa/fisiología , Haloferax volcanii/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/clasificación , Proteínas Arqueales/genética , Cromatografía Líquida de Alta Presión , Dipéptidos/metabolismo , Regulación de la Expresión Génica Arqueal/genética , Regulación de la Expresión Génica Arqueal/fisiología , Prueba de Complementación Genética , Glutamato-Cisteína Ligasa/clasificación , Glutamato-Cisteína Ligasa/genética , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.
Asunto(s)
Glutatión Sintasa/genética , Glutatión/biosíntesis , Ligasas/genética , Listeria monocytogenes/enzimología , Listeria monocytogenes/genética , Listeriosis/microbiología , Adenosina Trifosfato/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células CACO-2 , Catálisis , Dipéptidos/metabolismo , Glutatión/metabolismo , Glutatión Sintasa/química , Glutatión Sintasa/metabolismo , Humanos , Ligasas/química , Ligasas/metabolismo , Listeria monocytogenes/crecimiento & desarrollo , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de ProteínaRESUMEN
Ribonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygen-dependent class I RNR are co-transcribed with nrdS, which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR, which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five- to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS, whereas disruption of cobN, a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS. In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.