RESUMO
O-GlcNAcylation, which is a nutrient-sensitive sugar modification, participates in the epigenetic regulation of gene expression. The enzymes involved in O-linked ß-D-N-acetylglucosamine (O-GlcNAc) cycling - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - target key transcriptional and epigenetic regulators including RNA polymerase II, histones, histone deacetylase complexes and members of the Polycomb and Trithorax groups. Thus, O-GlcNAc cycling may serve as a homeostatic mechanism linking nutrient availability to higher-order chromatin organization. In response to nutrient availability, O-GlcNAcylation is poised to influence X chromosome inactivation and genetic imprinting, as well as embryonic development. The wide range of physiological functions regulated by O-GlcNAc cycling suggests an unexplored nexus between epigenetic regulation in disease and nutrient availability.
Assuntos
Acetilglucosamina/metabolismo , Epigênese Genética , Estado Nutricional/genética , Processamento de Proteína Pós-Traducional , Acetilglucosaminidase/química , Acetilglucosaminidase/metabolismo , Acetilglucosaminidase/fisiologia , Animais , Cromatina/genética , Cromatina/metabolismo , Glicosilação , Humanos , Redes e Vias Metabólicas , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/fisiologia , Conformação ProteicaRESUMO
Nutrient-driven O-GlcNAcylation is strikingly abundant in the brain and has been linked to development and neurodegenerative disease. We selectively targeted the O-GlcNAcase (Oga) gene in the mouse brain to define the role of O-GlcNAc cycling in the central nervous system. Brain knockout animals exhibited dramatically increased brain O-GlcNAc levels and pleiotropic phenotypes, including early-onset obesity, growth defects, and metabolic dysregulation. Anatomical defects in the Oga knockout included delayed brain differentiation and neurogenesis as well as abnormal proliferation accompanying a developmental delay. The molecular basis for these defects included transcriptional changes accompanying differentiating embryonic stem cells. In Oga KO mouse ES cells, we observed pronounced changes in expression of pluripotency markers, including Sox2, Nanog, and Otx2. These findings link the O-GlcNAc modification to mammalian neurogenesis and highlight the role of this nutrient-sensing pathway in developmental plasticity and metabolic homeostasis.
Assuntos
Acetilglucosamina/metabolismo , Encéfalo/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neurogênese/fisiologia , Acetilglucosamina/genética , Animais , Encéfalo/citologia , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , N-Acetilglucosaminiltransferases/genética , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Especificidade de Órgãos/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification. To pinpoint genomic regions that are regulated by O-GlcNAc levels, we performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in S2 cells. After OGA RNAi, we observed a genome-wide increase in the intensity of most O-GlcNAc-occupied regions including genes linked to cell cycle, ubiquitin, and steroid response. In contrast, O-GlcNAc levels were strikingly insensitive to OGA RNAi at sites of polycomb repression such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. We then produced a viable null allele of oga (oga(del.1)) in Drosophila allowing visualization of altered O-GlcNAc cycling on polytene chromosomes. We found that trithorax (TRX), absent small or homeotic discs 1 (ASH1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in oga(del.1) mutants. The oga(del.1) mutants displayed altered expression of a distinct set of cell cycle-related genes. Our results show that the loss of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1.
Assuntos
Acetilglucosamina/metabolismo , Cromatina/metabolismo , Drosophila/enzimologia , Epigênese Genética/genética , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Animais , Western Blotting , Células Cultivadas , Cromatina/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Imunoprecipitação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
Posttranslational modifications (PTM) including glycosylation, phosphorylation, acetylation, methylation and ubiquitination dynamically alter the proteome. The evolutionarily conserved enzymes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase are responsible for the addition and removal, respectively, of the nutrient-sensitive PTM of protein serine and threonine residues with O-GlcNAc. Indeed, the O-GlcNAc modification acts at every step in the "central dogma" of molecular biology and alters signaling pathways leading to amplified or blunted biological responses. The cellular roles of OGT and the dynamic PTM O-GlcNAc have been clarified with recently developed chemical tools including high-throughput assays, structural and mechanistic studies and potent enzyme inhibitors. These evolving chemical tools complement genetic and biochemical approaches for exposing the underlying biological information conferred by O-GlcNAc cycling.
Assuntos
Acetilglucosamina/metabolismo , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Animais , Humanos , N-Acetilglucosaminiltransferases/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
O-GlcNAc cycling is maintained by the reciprocal activities of the O-GlcNAc transferase and the O-GlcNAcase (OGA) enzymes. O-GlcNAc transferase is responsible for O-GlcNAc addition to serine and threonine (Ser/Thr) residues and OGA for its removal. Although the Oga gene (MGEA5) is a documented human diabetes susceptibility locus, its role in maintaining insulin-glucose homeostasis is unclear. Here, we report a conditional disruption of the Oga gene in the mouse. The resulting homozygous Oga null (KO) animals lack OGA enzymatic activity and exhibit elevated levels of the O-GlcNAc modification. The Oga KO animals showed nearly complete perinatal lethality associated with low circulating glucose and low liver glycogen stores. Defective insulin-responsive GSK3ß phosphorylation was observed in both heterozygous (HET) and KO Oga animals. Although Oga HET animals were viable, they exhibited alterations in both transcription and metabolism. Transcriptome analysis using mouse embryonic fibroblasts revealed deregulation in the transcripts of both HET and KO animals specifically in genes associated with metabolism and growth. Additionally, metabolic profiling showed increased fat accumulation in HET and KO animals compared with WT, which was increased by a high fat diet. Reduced insulin sensitivity, glucose tolerance, and hyperleptinemia were also observed in HET and KO female mice. Notably, the respiratory exchange ratio of the HET animals was higher than that observed in WT animals, indicating the preferential utilization of glucose as an energy source. These results suggest that the loss of mouse OGA leads to defects in metabolic homeostasis culminating in obesity and insulin resistance.
Assuntos
Técnicas de Inativação de Genes , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Teste de Tolerância a Glucose , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Homeostase , Humanos , Insulina/metabolismo , Camundongos , Camundongos Knockout , Morte Perinatal/etiologia , Período Pós-Parto , GravidezRESUMO
Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide ß-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase. As many cytoplasmic and nuclear proteins can be glycosylated by O-GlcNAc, we studied whether the expression of the HA synthases that synthesize HA could be controlled by O-GlcNAcylation in human aortic smooth muscle cells. Among the three HAS isoenzymes, only HAS2 mRNA increased after O-GlcNAcylation induced by glucosamine treatments or by inhibiting O-GlcNAc transferase with PUGNAC (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate). We found that the natural antisense transcript of HAS2 (HAS2-AS1) was absolutely necessary to induce the transcription of the HAS2 gene. Moreover, we found that O-GlcNAcylation modulated HAS2-AS1 promoter activation by recruiting the NF-κB subunit p65, but not the HAS2 promoter, whereas HAS2-AS1 natural antisense transcript, working in cis, regulated HAS2 transcription by altering the chromatin structure around the HAS2 proximal promoter via O-GlcNAcylation and acetylation. These results indicate that HAS2 transcription can be finely regulated not only by recruiting transcription factors to the promoter as previously described but also by modulating chromatin accessibility by epigenetic modifications.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/genética , Acetilglucosamina/química , Animais , Aorta/enzimologia , Sequência de Bases , Núcleo Celular/enzimologia , Cromatina/química , Citoplasma/enzimologia , Epigênese Genética , Inativação Gênica , Glucuronosiltransferase/fisiologia , Humanos , Hialuronan Sintases , Masculino , Camundongos , Camundongos Knockout , Modelos Genéticos , Dados de Sequência Molecular , Monossacarídeos/química , Miócitos de Músculo Liso/enzimologia , N-Acetilglucosaminiltransferases/química , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
O-GlcNAcylation is an abundant posttranslational modification in the brain implicated in human neurodegenerative diseases. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized Caenorhabditis elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the phenotype in transgenic models of tauopathy, amyloid ß-peptide, and polyglutamine expansion. Intriguingly, loss of function of O-GlcNAc transferase alleviated, whereas loss of O-GlcNAcase enhanced, the phenotype of multiple neurodegenerative disease models. The O-GlcNAc cycling mutants act in part by altering DAF-16-dependent transcription and modulating the protein degradation machinery. These findings suggest that O-GlcNAc levels may directly influence neurodegenerative disease progression, thus making the enzymes of O-GlcNAc cycling attractive targets for neurodegenerative disease therapies.
Assuntos
Acetilglucosamina/metabolismo , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Mutação , Doenças Neurodegenerativas/patologia , Alelos , Animais , Caenorhabditis elegans/genética , Humanos , Doenças Neurodegenerativas/metabolismo , ProteóliseRESUMO
The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.
Assuntos
Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Compostos Organometálicos/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Níquel/química , Compostos Organometálicos/química , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Protein-O-linked N-Acetyl-ß-D-glucosaminidase (O-GlcNAcase, OGA; also known as hexosaminidase C) participates in a nutrient-sensing, hexosamine signaling pathway by removing O-linked N-acetylglucosamine (O-GlcNAc) from key target proteins. Perturbations in O-GlcNAc signaling have been linked to Alzheimer's disease, diabetes and cancer. Mammalian O-GlcNAcase exists as two major spliced isoforms differing only by the presence (OGA-L) or absence (OGA-S) of a histone-acetyltransferase domain. Here we demonstrate that OGA-S accumulates on the surface of nascent lipid droplets with perilipin-2; both of these proteins are stabilized by proteasome inhibition. We show that selective downregulation of OGA-S results in global proteasome inhibition and the striking accumulation of ubiquitinylated proteins. OGA-S knockdown increased levels of perilipin-2 and perilipin-3 suggesting that O-GlcNAc-dependent regulation of proteasomes might occur on the surface of lipid droplets. By locally activating proteasomes during maturation of the nascent lipid droplet, OGA-S could participate in an O-GlcNAc-dependent feedback loop regulating lipid droplet surface remodeling. Our findings therefore suggest a mechanistic link between hexosamine signaling and lipid droplet assembly and mobilization.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Inibidores de Cisteína Proteinase/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , Células HeLa , Humanos , Leupeptinas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Proteínas de Membrana/genética , Camundongos , Perilipina-2 , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Inibidores de Proteassoma , Isoformas de Proteínas/genética , Transporte Proteico , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/genética , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the O-GlcNAc cycling enzymes are viable and fertile. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where O-GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, O-GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. These marked genes are linked to insulin-like signaling, metabolism, aging, stress, and pathogen-response pathways in C. elegans. Whole-genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that O-GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans, including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes and neurodegeneration.
Assuntos
Acetilglucosamina/genética , Caenorhabditis elegans/genética , Longevidade/genética , Animais , Caenorhabditis elegans/metabolismo , Carboidratos/química , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Sistema Imunitário , Insulina/metabolismo , Lipídeos/química , Óperon , Fosforilação , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
The Student Spaceflight Experiments Program (SSEP) is a United States national science, technology, engineering, and mathematics initiative that aims to increase student interest in science by offering opportunities to perform spaceflight experiments. The experiment detailed here was selected and flown aboard the third SSEP mission and the first SSEP mission to the International Space Station (ISS). Caenorhabditis elegans is a small, transparent, self-fertilizing hermaphroditic roundworm that is commonly used in biological experiments both on Earth and in Low Earth Orbit. Past experiments have found decreased expression of mRNA for several genes whose expression can be controlled by the FOXO transcription factor DAF-16. We flew a daf-16 mutant and control worms to determine if the effects of spaceflight on C. elegans are mediated by DAF-16. The experiment used a Type Two Fluids Mixing Enclosure (FME), developed by Nanoracks LLC, and was delivered to the ISS aboard the SpaceX Dragon and returned aboard the Russian Soyuz. The short time interval between experiment selection and the flight rendered preflight experiment verification tests impossible. In addition, published research regarding the viability of the FME in life science experiments was not available. The experiment was therefore structured in such a way as to gather the needed data. Here we report that C. elegans can survive relatively short storage and activation in the FME but cannot produce viable populations for post-flight analysis on extended missions. The FME appears to support short-duration life science experiments, potentially on supply or crew exchange missions, but not on longer ISS expeditions. Additionally, the flown FME was not properly activated, reportedly due to a flaw in training procedures. We suggest that a modified transparent FME could prevent similar failures in future flight experiments.
RESUMO
The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These 'evo-devo' relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the 'vicious cycle' observed in children of mothers with type-2 diabetes and metabolic disease.
Assuntos
Acetilglucosamina/metabolismo , Epigênese Genética , Morfogênese , Animais , Evolução Biológica , Hexosaminas/biossíntese , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologiaRESUMO
A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic ß-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.
Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Homeostase/fisiologia , Insulina , Acetilglucosamina/genética , Animais , Drosophila melanogaster , Técnicas de Silenciamento de Genes , Glucose/genéticaRESUMO
The enzymes of O-GlcNAc cycling couple the nutrient-dependent synthesis of UDP-GlcNAc to O-GlcNAc modification of Ser/Thr residues of key nuclear and cytoplasmic targets. This series of reactions culminating in O-GlcNAcylation of targets has been termed the hexosamine signaling pathway (HSP). The evolutionarily ancient enzymes of O-GlcNAc cycling have co-evolved with other signaling effecter molecules; they are recruited to their targets by many of the same mechanisms used to organize canonic kinase-dependent signaling pathways. This co-recruitment of the enzymes of O-GlcNAc cycling drives a binary switch impacting pathways of anabolism and growth (nutrient uptake) and catabolic pathways (nutrient sparing and salvage). The hexosamine signaling pathway (HSP) has thus emerged as a versatile cellular regulator modulating numerous cellular signaling cascades influencing growth, metabolism, cellular stress, circadian rhythm, and host-pathogen interactions. In mammals, the nutrient-sensing HSP has been harnessed to regulate such cell-specific functions as neutrophil migration, and activation of B-cells and T-cells. This review summarizes the diverse approaches being used to examine O-GlcNAc cycling. It will emphasize the impact O-GlcNAcylation has upon signaling pathways that may be become deregulated in diseases of the immune system, diabetes mellitus, cancer, cardiovascular disease, and neurodegenerative diseases.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/fisiologia , Acetilglucosaminidase/metabolismo , Animais , Caenorhabditis elegans , Domínio Catalítico , Diabetes Mellitus Tipo 2/fisiopatologia , Evolução Molecular , Alimentos , Regulação da Expressão Gênica/fisiologia , Humanos , Resistência à Insulina/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Animais , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Sirtuínas/fisiologia , InaniçãoRESUMO
O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP-OGT. Elevated GFP-OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.
Assuntos
Acetilglucosamina/metabolismo , Apoptose , Mitocôndrias/enzimologia , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Transdução de Sinais , Regulação para Cima , Animais , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , N-Acetilglucosaminiltransferases/química , Estrutura Terciária de Proteína , Proteínas/metabolismo , RatosRESUMO
The title compound, which differs from the powerful O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline only at the chalcogen atom (Se for S), is a much weaker inhibitor in a direct OGA assay. In human cells, however, the selenazoline shows comparable ability to induce hyper-O-GlcNAc-ylation, and the two show similar reduction of insulin-stimulated translocation of glucose transporter 4 in differentiated 3T3 adipocytes.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucosamina/análogos & derivados , Tiazóis/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Células 3T3-L1 , Acetilglucosamina/análise , Acetilglucosamina/metabolismo , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Clostridium perfringens/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Glicosilação , Células HeLa , Humanos , Insulina/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Alinhamento de Sequência , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Nutrient-driven O-GlcNAcylation has been linked to epigenetic regulation of gene expression in metazoans. In C. elegans, O-GlcNAc marks the promoters of over 800 developmental, metabolic, and stress-related genes; these O-GlcNAc marked genes show a strong 5', promoter-proximal bias in the distribution of RNA Polymerase II (Pol II). In response to starvation or feeding, the steady state distribution of O-GlcNAc at promoters remain nearly constant presumably due to dynamic cycling mediated by the transferase OGT-1 and the O-GlcNAcase OGA-1. However, in viable mutants lacking either of these enzymes of O-GlcNAc metabolism, the nutrient-responsive GlcNAcylation of promoters is dramatically altered. Blocked O-GlcNAc cycling leads to a striking nutrient-dependent accumulation of O-GlcNAc on RNA Pol II. O-GlcNAc cycling mutants also show an exaggerated, nutrient-responsive redistribution of promoter-proximal RNA Pol II isoforms and extensive transcriptional deregulation. Our findings suggest a complex interplay between the O-GlcNAc modification at promoters, the kinase-dependent "CTD-code," and co-factors regulating RNA Pol II dynamics. Nutrient-responsive O-GlcNAc cycling may buffer the transcriptional apparatus from dramatic swings in nutrient availability by modulating promoter activity to meet metabolic and developmental needs.
RESUMO
O-GlcNAcase (OGA) promotes O-GlcNAc removal, and thereby plays a key role in O-GlcNAc metabolism, a feature of a variety of vital cellular processes. Two splice transcripts of human OGA encode "long OGA", which contains a distinct N-terminal O-GlcNAcase domain and a C-terminal histoneacetylferase (HAT) domain, and "short OGA", which lacks the HAT domain. The functional roles of long OGA are only beginning to be unraveled, and the characteristics of short OGA remain almost unknown. We find that short OGA, which possesses O-GlcNAcase catalysis machinery like that of long OGA, exhibits comparative resistance to previously described potent inhibitors of long OGA and lysosomal hexosaminidases, including PUGNAc and NAG-thiazoline, suggesting a role for the HAT domain in O-GlcNAcase catalysis. We also find that alpha-GlcNAc thiolsulfonate (2) is the most potent inhibitor of short OGA yet described (Ki = 10 microM), and exhibits some degree of selectivity versus long OGA and lysosomal hexosaminidases. In contrast to its mode of inhibition of short OGA, 2 acts as a irreversible inhibitor of long OGA by covalently modifying the enzyme as an S-GlcNAc derivative. Covalent attachment of GlcNAc to the HAT domain of long OGA dramatically changes its properties with respect to enzymatic activity and caspase-3 cleavage.
Assuntos
Inibidores Enzimáticos/farmacologia , Tioglucosídeos/farmacologia , Compostos de Tosil/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Inibidores Enzimáticos/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Estrutura Molecular , Tioglucosídeos/química , Compostos de Tosil/químicaRESUMO
The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Genes Reporter , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.