RESUMEN
O-GlcNAcylation is the covalent addition of an O-linked ß-N-acetylglucosamine (O-GlcNAc) sugar moiety to hydroxyl groups of serine/threonine residues of cytosolic and nuclear proteins. O-GlcNAcylation, analogous to phosphorylation, plays critical roles in gene expression through direct modification of transcription factors, such as NF-κB. Aberrantly increased NF-κB O-GlcNAcylation has been linked to NF-κB constitutive activation and cancer development. Therefore, it is of a great biological and clinical significance to dissect the molecular mechanisms that tune NF-κB activity. Recently, we and others have shown that O-GlcNAcylation affects the phosphorylation and acetylation of NF-κB subunit p65/RelA. However, the mechanism of how O-GlcNAcylation activates NF-κB signaling through phosphorylation and acetylation is not fully understood. In this study, we mapped O-GlcNAcylation sites of p65 at Thr-305, Ser-319, Ser-337, Thr-352, and Ser-374. O-GlcNAcylation of p65 at Thr-305 and Ser-319 increased CREB-binding protein (CBP)/p300-dependent activating acetylation of p65 at Lys-310, contributing to NF-κB transcriptional activation. Moreover, elevation of O-GlcNAcylation by overexpression of OGT increased the expression of p300, IKKα, and IKKß and promoted IKK-mediated activating phosphorylation of p65 at Ser-536, contributing to NF-κB activation. In addition, we also identified phosphorylation of p65 at Thr-308, which might impair the O-GlcNAcylation of p65 at Thr-305. These results indicate mechanisms through which both non-pathological and oncogenic O-GlcNAcylation regulate NF-κB signaling through interplay with phosphorylation and acetylation.
Asunto(s)
Transducción de Señal/fisiología , Factor de Transcripción ReIA/metabolismo , Acetilación , Animales , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Ratones , Fosforilación/fisiología , Factor de Transcripción ReIA/genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
O-Linked ß-N-acetylglucosamine (O-GlcNAc) is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. Analogous to phosphorylation, O-GlcNAcylation plays crucial regulatory roles in cellular signaling. Recent work indicates that increased O-GlcNAcylation is a general feature of cancer and contributes to transformed phenotypes. In this minireview, we discuss how hyper-O-GlcNAcylation may be linked to various hallmarks of cancer, including cancer cell proliferation, survival, invasion, and metastasis; energy metabolism; and epigenetics. We also discuss potential therapeutic modulation of O-GlcNAc levels in cancer treatment.
Asunto(s)
Acetilglucosamina/metabolismo , Carcinogénesis , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Animales , Epigénesis Genética , Humanos , Neoplasias/irrigación sanguínea , Neoplasias/genética , Neovascularización Patológica/metabolismoRESUMEN
O-GlcNAc is a carbohydrate modification found on cytosolic and nuclear proteins. Our previous findings implicated O-GlcNAc in hippocampal presynaptic plasticity. An important mechanism in presynaptic plasticity is the establishment of the reserve pool of synaptic vesicles (RPSV). Dynamic association of synapsin I with synaptic vesicles (SVs) regulates the size and release of RPSV. Disruption of synapsin I function results in reduced size of the RPSV, increased synaptic depression, memory deficits, and epilepsy. Here, we investigate whether O-GlcNAc directly regulates synapsin I function in presynaptic plasticity. We found that synapsin I is modified by O-GlcNAc during hippocampal synaptogenesis in the rat. We identified three novel O-GlcNAc sites on synapsin I, two of which are known Ca(2+)/calmodulin-dependent protein kinase II phosphorylation sites. All O-GlcNAc sites mapped within the regulatory regions on synapsin I. Expression of synapsin I where a single O-GlcNAc site Thr-87 was mutated to alanine in primary hippocampal neurons dramatically increased localization of synapsin I to synapses, increased density of SV clusters along axons, and the size of the RPSV, suggesting that O-GlcNAcylation of synapsin I at Thr-87 may be a mechanism to modulate presynaptic plasticity. Thr-87 is located within an amphipathic lipid-packing sensor (ALPS) motif, which participates in targeting of synapsin I to synapses by contributing to the binding of synapsin I to SVs. We discuss the possibility that O-GlcNAcylation of Thr-87 interferes with folding of the ALPS motif, providing a means for regulating the association of synapsin I with SVs as a mechanism contributing to synapsin I localization and RPSV generation.
Asunto(s)
Acetilglucosamina/metabolismo , Hipocampo/metabolismo , Pliegue de Proteína , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Acetilglucosamina/genética , Secuencias de Aminoácidos , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Glicosilación , Humanos , Ratones , Transporte de Proteínas/fisiología , Ratas , Sinapsinas/genética , Vesículas Sinápticas/genéticaRESUMEN
Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-κB transcriptional activity. The NF-κB p65 subunit and upstream kinases IKKα/IKKß were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-κB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKα and p65 O-GlcNAcylation, accompanied by activation of NF-κB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB oncogenic activation in PDAC.
Asunto(s)
Apoptosis , Carcinoma Ductal Pancreático/metabolismo , Núcleo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Factor de Transcripción ReIA/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Transporte Activo de Núcleo Celular/genética , Acilación/genética , Sustitución de Aminoácidos , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Transformada , Núcleo Celular/genética , Núcleo Celular/patología , Proliferación Celular , Humanos , Ratones , Mutación Missense , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Factor de Transcripción ReIA/genética , Transcripción Genética/genética , Uridina Difosfato N-Acetilgalactosamina/genéticaRESUMEN
Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. We therefore speculated that increasing tau O-GlcNAc could be a strategy to hinder pathological tau-induced neurodegeneration. Here we found that treatment of hemizygous JNPL3 tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAc, hindered formation of tau aggregates and decreased neuronal cell loss. Notably, increases in tau O-GlcNAc did not alter tau phosphorylation in vivo. Using in vitro biochemical aggregation studies, we found that O-GlcNAc modification, on its own, hinders tau oligomerization. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease.
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas tau/metabolismo , Acetilglucosamina/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Conformación de Carbohidratos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ratones , Ratones Transgénicos , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Piranos/farmacología , Tiazoles/farmacología , Proteínas tau/genéticaRESUMEN
Cancer cells universally increase glucose and glutamine consumption, leading to the altered metabolic state known as the Warburg effect; one metabolic pathway, highly dependent on glucose and glutamine, is the hexosamine biosynthetic pathway. Increased flux through the hexosamine biosynthetic pathway leads to increases in the post-translational addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to various nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer, and recently, O-GlcNAcylation was shown to play a role in breast cancer; however, O-GlcNAcylation in other cancers remains poorly defined. Here, we show that O-GlcNAc transferase (OGT) is overexpressed in prostate cancer compared with normal prostate epithelium and that OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines. Reducing O-GlcNAcylation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF, resulting in inhibition of invasion and angiogenesis. OGT-mediated regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, a key regulator of invasion and angiogenesis, as reducing OGT expression led to increased FoxM1 protein degradation. Conversely, overexpression of a degradation-resistant FoxM1 mutant abrogated OGT RNAi-mediated effects on invasion, MMP levels, angiogenesis, and VEGF expression. Using a mouse model of metastasis, we found that reduction of OGT expression blocked bone metastasis. Altogether, these data suggest that as prostate cancer cells alter glucose and glutamine levels, O-GlcNAc modifications and OGT levels become elevated and are required for regulation of malignant properties, implicating OGT as a novel therapeutic target in the treatment of cancer.
Asunto(s)
N-Acetilglucosaminiltransferasas/metabolismo , Neovascularización Patológica/enzimología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Acetilglucosamina/metabolismo , Animales , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Mutación , N-Acetilglucosaminiltransferasas/deficiencia , N-Acetilglucosaminiltransferasas/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteolisis , Interferencia de ARN , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
O-linked ß-N-actylglucosamine (O-GlcNAc) is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. Analogous to phosphorylation, O-GlcNAcylation plays crucial regulatory roles in a variety of cellular processes. O-GlcNAc was termed a nutritional sensor, as global levels of the modification are elevated in response to increased glucose and glutamine flux into the hexosamine biosynthetic pathway. A unique feature of cancer cell energy metabolism is a shift from oxidative phosphorylation to the less efficient glycolytic pathway (Warburg effect), necessitating greatly increased glucose uptake. Additionally, to help meet increased biosynthetic demands, cancer cells also up-regulate glutamine uptake. This led us to hypothesize that the universal feature of increased glucose and glutamine uptake by cancer cells might be linked to increased O-GlcNAc levels. Indeed, recent work in many different cancer types now indicates that hyper-O-GlcNAcylation is a general feature of cancer and contributes to transformed phenotypes. In this review, we describe known/potential links between hyper-O-GlcNAcylation and specific hallmarks of cancer, including cancer cell proliferation, survival, cell stresses, invasion and metastasis, aneuploidy, and energy metabolism. We also discuss inhibition of hyper-O-GlcNAcylation as a potential novel therapeutic target for cancer treatment.
Asunto(s)
Acetilglucosamina/metabolismo , Neoplasias/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/antagonistas & inhibidores , Animales , Humanos , Neoplasias/patología , Neoplasias/terapiaRESUMEN
The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-D: -glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.
Asunto(s)
Acetilglucosamina/metabolismo , Anticuerpos/análisis , Mapeo Peptídico/métodos , Proteínas tau/química , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Glicosilación , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico/instrumentación , Conejos , Ratas , Proteínas tau/genética , Proteínas tau/inmunologíaRESUMEN
Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer's disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.
Asunto(s)
Acetilglucosamina/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Sinapsis/química , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Corteza Cerebral/química , Corteza Cerebral/metabolismo , Glicosilación , Humanos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Plasticidad Neuronal , Mapeo Peptídico , Fosforilación , Proteínas/química , Proteínas/genética , Alineación de SecuenciaRESUMEN
N-Acetylglucosamine O-linked to serines and threonines of cytosolic and nuclear proteins (O-GlcNAc) is an abundant reversible post-translational modification found in all higher eukaryotes. Evidence for functional regulation of proteins by this dynamic saccharide is rapidly accumulating. Deletion of the gene encoding the enzyme that attaches O-GlcNAc (OGT) is lethal at the single cell level, indicating the fundamental requirement for this modification. Recent studies demonstrate a role for O-GlcNAcylation in processes as diverse as transcription in the nucleus and signaling in the cytoplasm, suggesting that O-GlcNAc has both protein and site-specific influences on biochemistry and metabolism throughout the cell.
Asunto(s)
Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Glucosa/metabolismo , Humanos , N-Acetilglucosaminiltransferasas/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
In this chapter we describe the application of lectin weak affinity chromatography (LWAC) for the enrichment of peptides modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a single carbohydrate moiety post-translational modification of intracellular proteins. The stoichiometry of the modification is low and identification of the sites of O-GlcNAc attachment is challenging. To map O-GlcNAc sites we use the approach where a protein sample of interest is digested with trypsin and subjected to LWAC, which employs weak interaction between lectin wheat germ agglutinin and O-GlcNAc. Obtained sample is enriched with O-GlcNAc-modified peptides, which can be identified by means of mass spectrometry.
Asunto(s)
Cromatografía de Afinidad/métodos , Glicoproteínas/aislamiento & purificación , Aglutininas del Germen de Trigo/metabolismo , Acetilglucosamina/metabolismo , Cromatografía de Afinidad/instrumentación , Cromatografía Líquida de Alta Presión , Equipo Reutilizado , Glicoproteínas/metabolismo , Espectrometría de Masas , Politetrafluoroetileno/química , Proteolisis , Sefarosa/química , Tripsina/metabolismoRESUMEN
Administration of carbon monoxide derived from carbon monoxide-releasing molecules (CORMs) have been demonstrated to enhance coagulation and diminish fibrinolysis in vitro at small concentrations (100-200 µmol/l) in human and rabbit plasma, whereas in vivo administration of large concentrations (>1400 µmol/l) of carbon monoxide has mildly increased bleeding time in vivo in rats. We sought to determine whether CORM-2 [tricarbonyldichlororuthenium (II) dimer] would improve coagulation and attenuate tissue-type plasminogen activator (tPA)-mediated fibrinolysis in rabbit whole blood as determined in vitro by thrombelastography and in an in vivo preclinical rabbit model of ear bleeding time administered intravenous tPA (1 mg/kg). Addition of 200, 400 and 600 µmol/l CORM-2 to whole blood significantly improved coagulation and attenuated fibrinolysis compared with blood without CORM-2. Rabbits administered CORM-2 (10 mg/kg, 279 µmol/l) had a small but significant decrease in bleeding time before tPA administration. Administration of tPA resulted in bleeding times more than six-fold greater than baseline in animals not exposed to CORM-2, whereas rabbits administered CORM-2 had significantly smaller (more than five-fold less) bleeding time values after tPA administration. CORM-2 administration significantly decreases fibrinolytic bleeding in the rabbit in vivo. Additional preclinical investigation of the effects of CORM-2 on coagulopathy (e.g. heparin-mediated or clopidogrel-mediated) utilizing this rabbit model are planned.
Asunto(s)
Monóxido de Carbono/administración & dosificación , Fibrinólisis/efectos de los fármacos , Compuestos Organometálicos/farmacología , Animales , Coagulación Sanguínea/efectos de los fármacos , Masculino , ConejosRESUMEN
Cigarette smoking is associated with plasmatic hypercoagulability, and carbon monoxide has been demonstrated to enhance coagulation by binding to a fibrinogen-bound heme. Our objective was to design and test a redox-based method to detect carboxyhemefibrinogen. Normal, pooled, citrated plasma was exposed to 0-100â µmol/l carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2) before or after exposure to the organic reductant phenylhydroxylamine (PHA, 0-30â mmol/l), a compound that rapidly converts Fe(+2) to Fe(+3) in heme. Addition of calcium and tissue factor activation in disposable thrombelastographic cups was performed, followed by data collection at 37°C for 15â min. Elastic modulus (G, dynes/cm(2)) was the primary endpoint. CORM-2 significantly increased G values by 67.8% compared to unexposed plasma; pretreatment with 10â mmol/l PHA significantly decreased G values in CORM-2-exposed plasma by 77.1%, whereas 30â mmol/l PHA was required to significantly decrease G values by 64.0% in plasma following CORM-2 pre-exposure. G values were not significantly different between unexposed plasma and plasma exposed to CORM-2 followed by 30â mmol/l PHA addition. Conversion of fibrinogen-bound to the metheme state alone decreased G by 34.3-38.9% following exposure to 10-30â mmol/l PHA. Conversion of fibrinogen-bound heme Fe(+2) to Fe(+3) with PHA abrogated carbon monoxide-mediated increases in clot strength. Clinical trials are planned to investigate smoking individuals to mechanistically link carboxyhemefibrinogen formation with in-vitro hypercoagulability.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Módulo de Elasticidad/efectos de los fármacos , Plasma/química , Fumar/sangre , Tromboelastografía/métodos , Trombofilia/sangre , Calcio/farmacología , Monóxido de Carbono/metabolismo , Relación Dosis-Respuesta a Droga , Fibrinógeno/metabolismo , Hemo/metabolismo , Humanos , Hidroxilaminas/farmacología , Compuestos Organometálicos/efectos adversos , Oxidación-Reducción , Plasma/metabolismo , Unión Proteica/efectos de los fármacos , Fumar/efectos adversos , Tromboplastina/análisis , Tromboplastina/biosíntesisRESUMEN
O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that is implicated in the etiology of type II diabetes and Alzheimer's disease, as well as cardioprotection. This unit covers simple and comprehensive techniques for identifying proteins modified by O-GlcNAc, studying the enzymes that add and remove O-GlcNAc, and mapping O-GlcNAc modification sites.
Asunto(s)
Acetilglucosamina/análisis , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Enfermedad de Alzheimer/metabolismo , Anticuerpos/inmunología , Núcleo Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Glicosilación , HumanosRESUMEN
O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that is implicated in the etiology of type II diabetes and Alzheimer's disease, as well as cardioprotection. This unit covers simple and comprehensive techniques for identifying proteins modified by O-GlcNAc, studying the enzymes that add and remove O-GlcNAc, and mapping O-GlcNAc modification sites.
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas/análisis , Proteínas/aislamiento & purificación , Anticuerpos/metabolismo , Núcleo Celular/metabolismo , Cromatografía de Afinidad , Galactosiltransferasas/metabolismo , Glicosilación , Hexosaminidasas/metabolismo , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismoRESUMEN
The objective of this study was to determine how carbon monoxide directly modifies fibrinogen utilizing liquid chromatography-mass spectrometry (LC-MS/MS) by examining fibrinogen exposed to carbon monoxide releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer; CORM-2]. Purified fibrinogen was exposed to 0, 25, 50 or 100 µmol/l CORM-2 for 5 min at 37°C and then stored at -80°C before analyses with LC-MS/MS. In a second series of experiments, normal plasma was exposed to 0 or 100 µmol/l CORM-2 in the absence or presence of the nitric oxide donor sodium nitroprusside and hydroquinone (an organic reductant) to compete with carbon monoxide binding to a putative heme group found on fibrinogen. Coagulation was activated with tissue factor (n=8 per condition). Thrombus growth was monitored with thrombelastography for 15 min. LC-MS/MS did not detect any direct modifications of amino acids in fibrinogen, but detection of small regions of both the alpha and gamma chains was lost following exposure to CORM-2 and endoproteinase digestion with trypsin and Glu-C. An ion with the same m/z and expected retention time as heme was found in the purified fibrinogen. Exposure of plasma to nitric oxide/hydroquinone significantly decreased CORM-2-mediated enhancement of coagulation without affecting the coagulation kinetics of plasma not exposed to CORM-2. Carbon monoxide derived from CORM-2 likely modifies fibrinogen via modulation of a fibrinogen-associated heme group(s). Whereas the precise molecular location of heme attachment and three-dimensional conformational change secondary to carbon monoxide exposure remain to be determined, fibrinogen appears to be a carbon monoxide sensing molecule.
Asunto(s)
Monóxido de Carbono/metabolismo , Fibrinógeno/metabolismo , Hemo/metabolismo , Coagulación Sanguínea , Humanos , Cinética , Compuestos Organometálicos/farmacología , Plasma , Espectrometría de Masas en Tándem , Tromboelastografía , TrombosisRESUMEN
Cigarette smoke and carbon monoxide (CO) released from tricarbonyldichlororuthenium (II) dimer (CORM-2) attenuate fibrinolysis. The purpose of the present study was to determine whether CO diminished fibrinolysis by enhancement of α2-antiplasmin via a putative heme group. Plasma, isolated α2-antiplasmin and isolated plasmin were exposed to CO released from CORM-2 and nitric oxide (NO) via a NO donor to induce carboxyheme and metheme states, respectively. Exposed, isolated enzymes were placed in either α2-antiplasmin-deficient or normal plasma. Effects of CO and NO on tissue-type plasminogen activator initiated fibrinolysis were determined by thrombelastography. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify heme released from α2-antiplasmin and plasmin. CO significantly enhanced α2-antiplasmin activity, but decreased plasmin activity. NO decreased both α2-antiplasmin and plasmin activity. Although inadequate LC-MS/MS data were obtained with α2-antiplasmin (secondary to glycosylation), a putative plasmin-associated heme was identified. CO elicits hypofibrinolysis by enhancing α2-antiplasmin activity and decreasing plasmin activity. On the basis of the responses to NO and LC-MS/MS data, it is highly likely that both enzymes are modulated by attached heme groups. Efforts to develop methods to detect CO-mediated hypercoagulability are ongoing, with the goal of identifying populations at risk of thrombotic morbidity secondary to cigarette smoking.
Asunto(s)
Monóxido de Carbono/metabolismo , Fibrinolisina/farmacología , Fibrinólisis/efectos de los fármacos , Hemo/metabolismo , Óxido Nítrico/metabolismo , Fumar/sangre , Tromboelastografía/métodos , alfa 2-Antiplasmina/farmacología , Antifibrinolíticos/farmacología , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Humanos , Hidrazinas/farmacología , Hidroxilaminas/farmacología , Compuestos Organometálicos/farmacología , Plasma/química , Plasma/metabolismo , Unión Proteica , Fumar/efectos adversos , Espectrometría de Masas en Tándem , Trombosis/sangre , Trombosis/patología , Activador de Tejido Plasminógeno/sangreRESUMEN
Administration of carbon monoxide derived from carbon monoxide-releasing molecules has been demonstrated to enhance coagulation in vitro at small concentrations (100-200âµmol/l) in human and rabbit plasma. We sought to determine if carbon monoxide-releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer, CORM-2] would improve coagulation in rabbit plasma in vitro via thrombelastography and in an in vivo preclinical rabbit model of ear bleeding time following administration of clopidogrel (20âmg/kg) with aspirin (10âmg/kg) via gavage. Addition of 100âµmol/l CORM-2 to rabbit plasma significantly improved coagulation. This procoagulant effect was blocked by pre-exposure of plasma to an agent that converts hemefibrinogen to methemefibrinogen in human plasma, preventing carbon monoxide binding and enhancement of coagulation. Rabbit ear bleeding time was 5.8â±â1.1âmin 2-3âh after clopidogrel/aspirin administration. Bleeding time significantly decreased to 2.6â±â0.6âmin, 5âmin after administration of CORM-2 (10âmg/kg; 279âµmol/l 'best-case' instantaneous concentration) intravenously. CORM-2 enhances plasmatic coagulation in a manner similar to that of human plasma in vitro, and plasmatic coagulation is enhanced in vivo by CORM-2 as well. Additional preclinical investigation of the effects of CORM-2 on coagulopathy (e.g. heparin or hemodilution mediated) utilizing this rabbit model is planned.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Monóxido de Carbono/metabolismo , Hemorragia/sangre , Compuestos Organometálicos/farmacología , Plasma/metabolismo , Administración Oral , Animales , Aspirina/farmacología , Tiempo de Sangría , Coagulación Sanguínea/fisiología , Clopidogrel , Fibrinolíticos/farmacología , Hemorragia/tratamiento farmacológico , Inyecciones Intravenosas , Masculino , Compuestos Organometálicos/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Conejos , Tromboelastografía , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
Many neuronal cytosolic and nuclear proteins are post-translationally modified by the reversible addition of O-linked N-acetylglucosamine (O-GlcNAc) on serines and threonines. The cellular functions of O-GlcNAc modifications in neuronal development are not known. We report that O-GlcNAc-modified proteins are distributed nonuniformly throughout cultured primary chicken forebrain neurons, with intense immunostaining of the cell body, punctuate immunostaining in axons and all processes, and localization in filopodia/lamellipodia. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, increased the percentage of neurons exhibiting axon branching without altering the frequency of axon branches on a per neuron basis and increased the numbers of axonal filopodia. Conversely, pharmacologically increasing O-GlcNAc levels on proteins through specific inhibition of O-GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching. Treatment with an alternative O-GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia. Furthermore, axon branching induced by the adenylyl cyclase activator forskolin was suppressed by pharmacological inhibition of O-GlcNAcase. Western analysis revealed that O-GlcNAc levels regulate the phosphorylation of some PKA substrates in response to forskolin. These data provide the first evidence of O-GlcNAc modification-specific influences in neuronal development in primary culture, and indicate specific roles for O-GlcNAc in the regulation of axon morphology.
Asunto(s)
Acetilglucosamina/metabolismo , Axones/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , Neuronas/citología , Procesamiento Proteico-Postraduccional , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacología , Animales , Axones/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , N-Acetilglucosaminiltransferasas/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oximas/farmacología , Faloidina/metabolismo , Fenilcarbamatos/farmacología , Fosforilación/efectos de los fármacos , Prosencéfalo/citología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factores de Tiempo , Transfección/métodosRESUMEN
O-Linked N-acetylglucosamine (O-GlcNAc) is a cytosolic and nuclear carbohydrate post-translational modification most abundant in brain. We recently reported uniquely extensive O-GlcNAc modification of proteins that function in synaptic vesicle release and post-synaptic signal transduction. Here we examined potential roles for O-GlcNAc in mouse hippocampal synaptic transmission and plasticity. O-GlcNAc modifications and the enzyme catalyzing their addition (O-GlcNAc transferase) were enriched in hippocampal synaptosomes. Pharmacological elevation or reduction of O-GlcNAc levels had no effect on Schaffer collateral CA1 basal hippocampal synaptic transmission. However, in vivo elevation of O-GlcNAc levels enhanced long term potentiation (LTP), an electrophysiological correlate to some forms of learning/memory. Reciprocally, pharmacological reduction of O-GlcNAc levels blocked LTP. Additionally, elevated O-GlcNAc led to reduced paired-pulse facilitation, a form of short term plasticity attributed to presynaptic mechanisms. Synapsin I and II are presynaptic proteins that increase synaptic vesicle availability for release when phosphorylated, thus contributing to hippocampal synaptic plasticity. Synapsins are among the most extensively O-GlcNAc-modified proteins known. Elevating O-GlcNAc levels increased phosphorylation of Synapsin I/II at serine 9 (cAMP-dependent protein kinase substrate site), serine 62/67 (Erk 1/2 (MAPK 1/2) substrate site), and serine 603 (calmodulin kinase II site). Activation-specific phosphorylation events on Erk 1/2 and calmodulin kinase II, two proteins required for CA1 hippocampal LTP establishment, were increased in response to elevation of O-GlcNAc levels. Thus, O-GlcNAc is a novel regulatory signaling component of excitatory synapses, with specific roles in synaptic plasticity that involve interplay with phosphorylation.