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1.
mBio ; 8(3)2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28512097

RESUMO

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione.IMPORTANCEYersinia pestis, the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Glutationa/metabolismo , Peste/microbiologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidade , Animais , Antígenos de Bactérias/genética , Apoptose , Linhagem Celular , Cisteína/química , Citocinas/metabolismo , Modelos Animais de Doenças , Dissulfetos/metabolismo , Feminino , Glutationa Sintase/deficiência , Glutationa Sintase/genética , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Macrófagos/microbiologia , Macrófagos/patologia , Espectrometria de Massas , Camundongos , Peste/imunologia , Proteínas Citotóxicas Formadoras de Poros/genética , Ratos , Virulência , Yersinia pestis/genética
2.
Cell Metab ; 22(3): 508-15, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26190651

RESUMO

We discovered recently that the central metabolite α-ketoglutarate (α-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.


Assuntos
Adenosina Trifosfatases/metabolismo , Caenorhabditis elegans/fisiologia , Glioblastoma/metabolismo , Glutaratos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Proliferação de Células , Glioblastoma/genética , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Longevidade , Mutação
3.
Int. arch. otorhinolaryngol. (Impr.) ; 19(2): 141-150, Apr-Jun/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-747151

RESUMO

Introduction Chronic otomastoiditis causes pain, otorrhea, and hearing loss resulting from the growth of tissue within the normally hollow mastoid cavity. Objectives In this report, we used a lipidomics approach to profile major mastoid bone and tissue lipids from patients with and without otomastoiditis. Methods The bone dust created during mastoidectomy, as well as the mastoid tissue, was analyzed from seven patients. Bone dust was also collected and analyzed in an additional four otologic cases (parotidectomy requiring mastoidectomy). Samples were subjected to a modified Bligh/Dyer lipid extraction, then high-performance thin-layer chromatography (HPTLC), combined gas chromatography/electron impact-mass spectrometry (GC/EI-MS), and flow-injection/electrospray ionization-tandem mass spectrometry (FI/ESI-MSMS). Data were analyzed for identification and profiling of major lipid components. Results HPTLC revealed the presence of various lipid classes, including phosphatidylcholines, cholesterol, and triacylglycerols. GC/EI-MS analysis revealed the presence of cholesterol and several fatty acids. FI/ESI-MSMS analysis revealed a host of phosphatidylcholines, phosphatidylethanolamines, and cholesteryl esters. Conclusion We used a lipidomics approach to develop an efficient (both in time and tissue amount) methodology for analysis of these tissues, identify the most abundant and common lipid species, and create a base of knowledge from which more focused endeavors in biomarker discovery can emerge. In an effort toward improved patient categorization and individualized intervention, the ultimate goal of this work is to correlate these lipid molecules to disease state and progression. This is the first reported study of its kind on these tissues. .


Assuntos
Humanos , Síndrome de Linfonodos Mucocutâneos/diagnóstico , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Aneurisma Coronário/etiologia , Glucocorticoides/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Síndrome de Linfonodos Mucocutâneos/etiologia , Síndrome de Linfonodos Mucocutâneos/fisiopatologia , Fatores de Risco , Fator de Necrose Tumoral alfa/antagonistas & inibidores
4.
Clin Transl Sci ; 8(2): 107-15, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25588779

RESUMO

Sinusitis is a cause of significant morbidity, substantial healthcare costs, and negative effects on quality of life. The primary objective of this study is to characterize the previously unknown lipid profile of sinonasal mucosa from patients with chronic rhinosinusitis (CRS) and from controls. Sinus mucosa samples were analyzed from 9 CRS patients with concomitant nasal polyps, 11 CRS patients without polyps, and 12 controls. Ten lone polyp samples were also analyzed. Samples were subjected to a modified Bligh/Dyer lipid extraction, then high performance thin layer chromatography (HPTLC), combined gas chromatography/electron impact-mass spectrometry (GC/EI-MS), and flow-injection/electrospray ionization-tandem mass spectrometry (FI/ESI-MS/MS). Data was analyzed for identification and profiling of major components. HPTLC revealed an array of species reflecting the lipid complexity of the samples. GC/EI-MS revealed cholesterol and several fatty acids. FI/ESI-MSMS revealed numerous lipid species, namely a host of phosphatidylcholines, phosphatidylethanolamines, ceramides and cholesteryl esters, but no detectable amounts of phosphatidyinositols or sulfated lipids. These results are a first step to uncover unique molecular biomarkers in CRS.


Assuntos
Lipídeos/química , Pólipos Nasais/química , Sinusite/fisiopatologia , Biomarcadores/química , Estudos de Casos e Controles , Ceramidas/química , Ésteres do Colesterol/química , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Doença Crônica , Humanos , Mucosa Nasal/patologia , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Qualidade de Vida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
5.
Elife ; 32014 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-24891238

RESUMO

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.


Assuntos
Aminoácidos/química , Aminoacil-tRNA Sintetases/química , Código Genético , Oxigênio/química , Trifosfato de Adenosina/química , Escherichia coli/genética , Hidrólise , Estresse Oxidativo , Fenilalanina-tRNA Ligase/genética , Plasmídeos , Biossíntese de Proteínas , Proteínas/química , Proteoma , Aminoacil-RNA de Transferência/genética , Saccharomyces cerevisiae/genética
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