RESUMEN
O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3ß/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.
Asunto(s)
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas , Modelos Moleculares , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/metabolismo , ProteómicaRESUMEN
O-Linked ß-N-acetylglucosamine (O-GlcNAc) is a critical post-translational modification (PTM) of thousands of intracellular proteins. Reversible O-GlcNAcylation governs many aspects of cell physiology and is dysregulated in numerous human diseases. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling remain poorly understood, including the biochemical mechanisms through which O-GlcNAc transduces information. Recent work from many laboratories, including our own, has revealed that O-GlcNAc, like other intracellular PTMs, can control its substrates' functions by inhibiting or inducing protein-protein interactions. This dynamic regulation of multiprotein complexes exerts diverse downstream signaling effects in a range of processes, cell types, and organisms. Here, we review the literature about O-GlcNAc-regulated protein-protein interactions and suggest important questions for future studies in the field.
Asunto(s)
Acetilglucosamina/metabolismo , Bioquímica/métodos , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Transducción de Señal , Acetilglucosamina/química , Aminoacilación , Animales , Bioquímica/tendencias , Humanos , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Dysregulation of the proteasome has been documented in a variety of human diseases such as Alzheimer, muscle atrophy, cataracts etc. Proteolytic activity of 26 S proteasome is ATP- and ubiquitin-dependent. O-GlcNAcylation of Rpt2, one of the AAA ATPases in the 19 S regulatory cap, shuts off the proteasome through the inhibition of ATPase activity. Thus, through control of the flux of glucose into O-GlcNAc, the function of the proteasome is coupled to glucose metabolism. In the present study we found another metabolic control of the proteasome via cAMP-dependent protein kinase (PKA). Contrary to O-Glc-NAcylation, PKA activated proteasomes both in vitro and in vivo in association with the phosphorylation at Ser(120) of another AAA ATPase subunit, Rpt6. Mutation of Ser(120) to Ala blocked proteasome function. The stimulatory effect of PKA and the phosphorylation of Rpt6 were reversible by protein phosphatase 1 gamma. Thus, hormones using the PKA system can also regulate proteasomes often in concert with glucose metabolism. This finding might lead to novel strategies for the treatment of proteasome-related diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/fisiología , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , AMP Cíclico/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Isoquinolinas/farmacología , Riñón/metabolismo , Proteínas con Dominio LIM , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , Ratas , Factor de Transcripción Sp1/metabolismo , Sulfonamidas/farmacología , Factores de Transcripción/metabolismoRESUMEN
Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors. Increased O-GlcNAc levels suppress transcriptional expression of coactivators and of the nuclear hormone receptors themselves. Surprisingly, increased O-GlcNAc levels are also associated with increased transcription of genes encoding corepressor proteins NCoR and SMRT. The association of the enzyme O-GlcNAc transferase with these corepressors contributes to specific regulation of nuclear hormone receptors by O-GlcNAc. Overall, transcriptional inhibition is related to the integrated effect of O-GlcNAc by direct modification of critical elements of the transcriptome and indirectly through O-GlcNAc modification of the proteasome.
Asunto(s)
Regulación de la Expresión Génica , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Humanos , Glándulas Mamarias Humanas/embriología , Glándulas Mamarias Humanas/enzimología , Ratones , Ratones Transgénicos , Modelos Biológicos , N-Acetilglucosaminiltransferasas/fisiología , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Proteínas Represoras/metabolismo , TransfecciónRESUMEN
NCOAT is a bifunctional nucleo-cytoplasmic protein with both O-GlcNAcase and histone acetyltransferase domains. The O-GlcNAcase domain catalyzes the removal of O-linked GlcNAc modifications from proteins and we have found that it resides in the N-terminal third of NCOAT. The recognition of the substrate GlcNAc suggests that the O-GlcNAcase is related in structure and catalytic mechanism to chitinases, hexosaminidases and hyaluronidases. These families of glycosidases all possess a catalytic doublet of carboxylate-containing residues, with one providing an acid-base function, and the second acting to orient and use the N-acetyl group of GlcNAc during catalysis. Indeed, we show that the O-GlcNAcase also possesses the catalytic doublet motif shared among these enzymes and that these two essential residues are aspartic acids at positions 175 and 177, respectively, in mouse NCOAT. In addition, a conserved cysteine at 166 and a conserved aspartic acid at 174 were also found to be necessary for fully efficient enzymatic activity. Given this information, we propose that the O-GlcNAcase active site resembles those of the above glycosidases which carry out the hydrolysis of GlcNAc linkages in a substrate-assisted acid-base manner.
Asunto(s)
Acetilglucosaminidasa/química , Acetilglucosaminidasa/genética , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Mutagénesis , Mutación , beta-N-AcetilhexosaminidasasRESUMEN
Streptozotocin (STZ) is a 2-deoxy-d-glucopyranose derivative of a class of drugs known as alkylnitrosoureas, and is an established diabetogenic agent whose cytotoxic affects on pancreatic beta-cells has been partially explained by the presence of its N-methyl-N-nitrosourea side chain, which has the ability to release nitric oxide as well as donate methyl groups to nucleotides in DNA. It has also been observed that STZ administration results in a rise in the level of O-GlcNAcylated proteins within beta-cells. Not coincidentally, STZ has also been shown to directly inhibit the O-GlcNAcase activity of the enzyme NCOAT in vitro, which is the only enzyme that possesses the ability to remove O-GlcNAc modifications on proteins in the nucleus and cytosol. Since O-GlcNAc modification plays a role on a number of proteins in a vast amount of cellular processes, this shift in whole-cell protein O-GlcNAcylation state affords another source of cell death. We set about to find the exact mechanism by which STZ inhibits O-GlcNAcase activity. Inhibition is achievable because the GlcNAc analog STZ targets the active site of the enzyme whereby it is catalyzed. During this process, the enzyme converts STZ to a compound that closely resembles the natural ligand transition state, but is distinctly more stable energetically. As a result, this analog is catalyzed to completion at a much slower rate, thereby out-competing GlcNAc substrate for the active site, and inhibiting the enzyme.
Asunto(s)
Acetilglucosamina/química , Acetilglucosaminidasa/antagonistas & inhibidores , Acetilglucosaminidasa/química , Inhibidores Enzimáticos/química , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/química , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Estreptozocina/análogos & derivados , Estreptozocina/química , Estreptozocina/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosaminidasa/biosíntesis , Conformación de Carbohidratos , Catálisis , Inhibidores Enzimáticos/metabolismo , Histona Acetiltransferasas/biosíntesis , Cinética , Espectrometría de Masas , Complejos Multienzimáticos/biosíntesis , Resonancia Magnética Nuclear Biomolecular , Especificidad por Sustrato , beta-N-AcetilhexosaminidasasRESUMEN
Nuclear cytoplasmic O-GlcNAcase and acetyltransferase (NCOAT) is a bifunctional enzyme with both glycoside hydrolase and alkyltransferase activity. Its O-GlcNAcase active site lies in the N terminus of the enzyme and its histone acetyltransferase (HAT) domain lies in the C terminus. Whereas the HAT domain of the enzyme is catalytically and structurally similar to other acetyltransferases across subfamilies, NCOAT has a motif resembling a zinc finger-like domain unique to the MYST family of HATs. Among the MYST family, this zinc finger, or zinc finger-like domain, is responsible for making contacts with the histone tails within nucleosomes for the HAT to catalyze its respective reaction. Here, we show that NCOAT has the ability to directly associate with both an acetylated and unacetylated histone H4 tail in vitro, and a potential zinc finger-like motif found in NCOAT is implicated in this nucleosomal contact, and is necessary for fully efficient enzymatic activity. Subsequent to the catalysis of acetyltransfer to lysine 8 of histone H4 for the enzyme, however, the substrate is released and NCOAT can no longer bind H4 in our assays. Furthermore, this finger domain by itself is sufficient to bind histone H4.
Asunto(s)
Acetilglucosaminidasa/química , Histona Acetiltransferasas/química , Complejos Multienzimáticos/química , Dedos de Zinc , Acetilglucosaminidasa/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Ditiotreitol/farmacología , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Ratones , Complejos Multienzimáticos/metabolismo , beta-N-AcetilhexosaminidasasRESUMEN
Histones and transcription factors are regulated by a number of post-translational modifications that in turn regulate the transcriptional activity of genes. These modifications occur in large, multisubunit complexes. We have reported previously that mSin3A can recruit O-GlcNAc transferase (OGT) along with histone deacetylase into such a corepressor complex. This physical association allows OGT to act cooperatively with histone deacetylation in gene repression by catalyzing the O-GlcNAc modification on specific transcription factors to inhibit their activity. For rapid, reversible gene regulation, the enzymes responsible for the converse reactions must be present. Here, we report that O-GlcNAcase, which is responsible for the removal of O-GlcNAc additions on nuclear and cytosolic proteins, possesses intrinsic histone acetyltransferase (HAT) activity in vitro. Free as well as reconstituted nucleosomal histones are substrates of this bifunctional enzyme. This protein, now termed NCOAT (nuclear cytoplasmic O-GlcNAcase and acetyltransferase) has a typical HAT domain that has both active and inactive states. This finding demonstrates that NCOAT may be regulated to reduce the state of glycosylation of transcriptional activators while increasing the acetylation of histones to allow for the concerted activation of eukaryotic gene transcription.