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1.
Cell Rep ; 5(2): 546-52, 2013 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-24120863

RESUMEN

Protein modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2(-/-) cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts.


Asunto(s)
Mitocondrias/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Acetilglucosamina/metabolismo , Animales , Línea Celular , Electroforesis en Gel de Campo Pulsado , Colorantes Fluorescentes/química , Glicoproteínas/metabolismo , Glicosilación , Células HEK293 , Humanos , Células Jurkat , Ratones , Proteómica , Especificidad por Sustrato , Canal Aniónico 2 Dependiente del Voltaje/deficiencia , Canal Aniónico 2 Dependiente del Voltaje/genética
2.
ACS Chem Biol ; 6(8): 829-36, 2011 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-21604797

RESUMEN

Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide's sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.


Asunto(s)
Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Péptidos/química , Proteínas/química , Proteómica/métodos , Algoritmos , Animales , Bovinos , Halogenación , Humanos , Células Jurkat , Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Sensibilidad y Especificidad
3.
J Biol Chem ; 286(4): 2492-503, 2011 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-21068383

RESUMEN

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is an important regulator of cytokine-mediated inflammatory responses in both in vitro and in vivo models of rheumatoid arthritis (RA). However, treatment of RA patients with sPLA(2)-IIA inhibitors shows only transient benefit. Using an activity-impaired sPLA(2)-IIA mutant protein (H48Q), we show that up-regulation of TNF-dependent PGE(2) production and cyclooxygenase-2 (COX-2) induction by exogenous sPLA(2)-IIA in RA fibroblast-like synoviocytes (FLSs) is independent of its enzyme function. Selective cytosolic phospholipase A(2)-α (cPLA(2)-α) inhibitors abrogate TNF/sPLA(2)-IIA-mediated PGE(2) production without affecting COX-2 levels, indicating arachidonic acid (AA) flux to COX-2 occurs exclusively through TNF-mediated activation of cPLA(2)-α. Nonetheless, exogenous sPLA(2)-IIA, but not H48Q, stimulates both AA mobilization from FLSs and microparticle-derived AA release that is not used for COX-2-dependent PGE(2) production. sPLA(2)-IIA-mediated AA production is inhibited by pharmacological blockade of sPLA(2)-IIA but not cPLA(2)-α. Exogenous H48Q alone, like sPLA(2)-IIA, increases COX-2 protein levels without inducing PGE(2) production. Unlike TNF, sPLA(2)-IIA alone does not rapidly mobilize NF-κB or activate phosphorylation of p38 MAPK, two key regulators of COX-2 protein expression, but does activate the ERK1/2 pathway. Thus, sPLA(2)-IIA regulates AA flux through the cPLA(2)-α/COX-2 pathway in RA FLSs by up-regulating steady state levels of these biosynthetic enzymes through an indirect mechanism, rather than direct provision of substrate to the pathway. Inhibitors that have been optimized for their potency in enzyme activity inhibition alone may not adequately block the activity-independent function of sPLA(2)-IIA.


Asunto(s)
Ácido Araquidónico/metabolismo , Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Fosfolipasas A2 Grupo II/metabolismo , Líquido Sinovial/metabolismo , Sustitución de Aminoácidos , Animales , Ácido Araquidónico/genética , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Línea Celular , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Dinoprostona/biosíntesis , Dinoprostona/genética , Perros , Fibroblastos/patología , Fosfolipasas A2 Grupo II/genética , Humanos , Mutación Missense , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
5.
Proc Natl Acad Sci U S A ; 107(9): 3988-93, 2010 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-20142501

RESUMEN

Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Delta strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Polisacáridos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Glicoproteínas/química , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Polisacáridos/química
6.
Antimicrob Agents Chemother ; 51(10): 3659-71, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17606674

RESUMEN

New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.


Asunto(s)
Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Quinolinas/farmacología , Sulfonamidas/farmacología , Animales , Antimaláricos/síntesis química , Antimaláricos/farmacocinética , Conductos Biliares/metabolismo , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Remoción de Radical Alquila , Femenino , Humanos , Malaria/tratamiento farmacológico , Malaria/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Pruebas de Mutagenicidad , Pruebas de Sensibilidad Parasitaria , Plasmodium berghei/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Quinolinas/síntesis química , Quinolinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética
7.
J Med Chem ; 49(10): 2858-60, 2006 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-16686528

RESUMEN

Using the X-ray structure of human group X secreted phospholipase A(2) (hGX), we carried out structure-based design of indole-based inhibitors and prepared the compounds using a new synthetic route. The most potent compound inhibited hGX and the mouse orthologue with an IC(50) of 75 nM. This compound is the most potent hGX inhibitor reported to date and was also found to inhibit a subset of the other mouse and human sPLA(2)s.


Asunto(s)
Indoles/síntesis química , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Fosfolipasas A2 Grupo X , Humanos , Indoles/química , Indoles/farmacología , Ratones , Modelos Moleculares , Fosfolipasas A2 , Relación Estructura-Actividad
8.
J Biol Chem ; 281(24): 16245-55, 2006 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-16603549

RESUMEN

Gastric epithelial cells liberate prostaglandin E(2) in response to cytokines as part of the process of healing of gastric lesions. Treatment of the rat gastric epithelial cell line RGM1 with transforming growth factor-alpha and interleukin-1beta leads to synergistic release of arachidonate and production of prostaglandin E(2). Results with highly specific and potent phospholipase A(2) inhibitors and with small interfering RNA show that cytosolic phospholipase A(2)-alpha and group IIA secreted phospholipase A(2) contribute to arachidonate release from cytokine-stimulated RGM1 cells. In the late phase of arachidonate release, group IIA secreted phospholipase A(2) is induced (detected at the mRNA and protein levels), and the action of cytosolic phospholipase A(2)-alpha is required for this induction. Results with RGM1 cells and group IIA secreted phospholipase A(2)-transfected HEK293 cells show that the group IIA phospholipase acts prior to externalization from the cells. RGM1 cells also express group XIIA secreted phospholipase A(2), but this enzyme is not regulated by cytokines nor does it contribute to arachidonate release. The other eight secreted phospholipases A(2) were not detected in RGM1 cells at the mRNA level. These results clearly show that cytosolic and group IIA secreted phospholipases A(2) work together to liberate arachidonate from RGM1 cell phospholipids in response to cytokines.


Asunto(s)
Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Riñón/citología , Fosfolipasas A/fisiología , Animales , Ácido Araquidónico/metabolismo , Células CACO-2 , Dinoprostona/metabolismo , Fosfolipasas A2 Grupo II , Fosfolipasas A2 Grupo IV , Humanos , Interleucina-1/metabolismo , Riñón/embriología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Prostaglandinas/metabolismo , Interferencia de ARN , Ratas , Transfección
9.
J Biol Chem ; 280(9): 7519-29, 2005 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-15475363

RESUMEN

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.


Asunto(s)
NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Fosfolipasas A/fisiología , Factor de Activación Plaquetaria/biosíntesis , Animales , Ácido Araquidónico/metabolismo , Líquido del Lavado Bronquioalveolar , Antígeno CD11b/biosíntesis , Citosol/enzimología , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Fosfolipasas A2 Grupo IV , Humanos , Inflamación , Ionomicina/farmacología , Leucotrieno B4/metabolismo , Ratones , Ratones Transgénicos , Neutrófilos/citología , Neutrófilos/microbiología , Oxígeno/metabolismo , Fagocitosis , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Neumonía/metabolismo , Pirrolidinas/farmacología , Receptores de IgG/biosíntesis , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo
10.
Bioorg Med Chem ; 12(7): 1737-49, 2004 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-15028265

RESUMEN

Structure-guided design was employed in a search for potent and selective inhibitors of mammalian secreted phospholipases A(2) (sPLA(2)s). Using the X-ray structures of human groups IIA and X sPLA(2)s (hGIIA and hGX) as templates, homology structural models were made for the other human and mouse sPLA(2)s (hGIB, mGIB, mGIIA, mGIIC, hGIID, mGIID, hGIIE, mGIIE, hGIIF, mGIIF, hGV, mGV, and mGX). Me-Indoxam is a previously discovered indole analogue that binds tightly to many sPLA(2)s, and the X-ray structure of the hGX-Me-Indoxam complex was determined at a resolution of 2.0 A. Modeling suggests that the residues near the N(1)-substituent of Me-Indoxam vary significantly among the mammalian sPLA(2)s, and therefore a library of 83N(1)-variants was prepared by parallel synthesis. Several Me-Indoxam analogues bearing a 4-(2-oxy-ethanoic acid) side chain were potent inhibitors (IC(50) <0.05 microM) of hGIIA, mGIIA, mGIIC, hGIIE, mGIIE, hGV, and mGV, while they displayed intermediate potency (0.05-5 microM) against hGIB, mGIB, hGX, and mGX, and poorly inhibited (>5 microM) hGIID, mGIID, hGIIF, and mGIIF. Me-Indoxam analogues bearing a 5-(4-oxy-butanoic acid) side chain were generally less potent inhibitors. Although no compounds were found to be highly specific for a single human or mouse sPLA(2), combinations of Me-Indoxam analogues were discovered that could be used to distinguish the action of various sPLA(2)s in cellular events. For example, Me-Indoxam and compound 5 are approximately 5-fold more potent on hGIIA than on hGV, and compound 21 is 10-fold more potent on hGV versus hGIIA.


Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Indoles/química , Indoles/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/química , Animales , Carbamatos/química , Carbamatos/farmacología , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Fosfolipasas A2 Grupo II , Fosfolipasas A2 Grupo X , Humanos , Indoles/síntesis química , Indolizinas/química , Indolizinas/farmacología , Ratones , Estructura Molecular , Relación Estructura-Actividad
11.
J Biol Chem ; 277(7): 5061-73, 2002 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-11741884

RESUMEN

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable. GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2). GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.


Asunto(s)
Neutrófilos/enzimología , Fosfolipasas A/biosíntesis , Fosfolipasas/clasificación , Fosfolipasas/fisiología , Western Blotting , Carbamatos/farmacología , Separación Celular , Citosol/enzimología , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Eicosanoides/biosíntesis , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Citometría de Flujo , Fosfolipasas A2 Grupo IV , Fosfolipasas A2 Grupo V , Fosfolipasas A2 Grupo X , Humanos , Hidrólisis , Indolizinas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Fosfolipasas A/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética
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