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Doctoral Thesis
DOI
10.11606/T.46.2016.tde-10082016-171123
Document
Author
Full name
Maísa Ribeiro Pereira Lima Brigagão
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2004
Supervisor
Committee
Colepicolo Neto, Pio (President)
Augusto, Ohara
Junqueira, Virginia Berlanga Campos
Laurindo, Francisco Rafael Martins
Netto, Luis Eduardo Soares
Title in Portuguese
Estudo do processo de S-glutationação protéica no "BURST" respiratório de leucócitos: modulação pela lactona sesquiterpênica licnofolido
Keywords in Portuguese
Ânion superóxido
Bioquímica
Glutationa transferase
Inflamação
Lactona sesquiterpênica
Leucócitos
NADPH-oxidase
Neutrófilos
S-glutationação
Abstract in Portuguese
Foi estudado o efeito da lactona sesquiterpênica licnofolido sobre o "burst" respiratório de leucócitos polimorfonucleares inflamatórios (PMN) estimulados por forbol (PMA), pelo peptídeo quimiotático fMLP ou zimozan opsonizado (OZ). O licnofolido inibiu de forma dose-dependente a liberação de O2•- pelos PMN, sem alteração do período "Iag" do complexo NADPH. oxidase. O efeito foi mais acentuado quando os PMN foram estimulados diretamente pela via de proteína quinase C. A adição de ditiotreitol ou glutationa reduzida (GSH) às suspensões celulares antes da incubação com licnofolido preveniu parcialmente o efeito inibitório. O tratamento dos PMN com a lactona determinou uma queda drástica dos níveis celulares de GSH livre, sem incremento de glutationa oxidada (GSSG). A reação direta entre GSH e licnofolido foi confirmada com a detecção de um aduto glutationil-licnofolido através de identificação por espectrometria de massa (ESI-MS/MS). A S-tiolação protéica induzida pelo PMA foi reduzida em PMN tratados com Iicnofo/ido, como detectado através de determinação de incorporação de [35S], sendo que 80% desses tióis foram identificados como GSH. Uma série de proteínas S-glutationadas foi detectada através de autoradiografias, sendo que aquelas correspondentes a 38 e 24 kDa tiveram essa modificação póstraducional suprimida pelo tratamento com dose de licnofolido capaz de suprimir o "burst" respiratório dos PMN. Estes resultados indicam que a depleção celular de GSH causada pelo licnofolido impede a sustentação do "burst" respiratório pelos PMN, em correlação direta com a diminuição de S-glutationação protéica.
Title in English
Study process S-glutationação protein in "Burst" respiratory leukocyte: modulation by sesquiterpene lactone licnofolido
Keywords in English
Biochemistry
Glutathione transferase
Inflammation
Leukocyte
NADPH oxidase
Neutrophils
S-glutathione action
Sesquiterpene lactone
Superoxide
Abstract in English
An investigation was made into the action of the sesquiterpene lactone lychnopholide on the respiratory burst of inflammatory polymorphonuclear leukocytes. Lychnopholide determined concentration-related inhibition of the generation of phorbol 12-myristate 13-acetate-, chemotatic peptide-, and opsonized zymozan-induced superoxide anion with no effect on the lag time of the assembly of the NADPH oxidase complex, such action was greater on the protein kinase C pathway that on both membrane receptor dependent stimuli via. Subsequent additions of D-glucose, Ca2+, Mg2+, dithiothreitol ar reduced glutathione (GSH) did not reverse the inhibitory action. The addition of both thiols prior to the lychnopholide treatment partially hindered the inhibition rate. The endogenous level of GSH in leukocytes was drastically depleted under the lychnopholide treatment, without corresponding increases occurring in the oxidized form (GSSG). A direct reaction between glutathione and lychnopholide was confirmed from a glutathionyl-lychnopholide adduct detected by electrospray mass spectrometry analysis and identified by tandem mass analysis in cellular extracts. Protein S-thiolation induced by PMA stimulation was decreased in lactone-treated PMN as detected by [35S] scintillation count, which indicated that about 80% of the thiols were glutathione. A subset of S-glutathionylated proteins was identified through gel electrophoresis, which revealed that the modification of the phorbol-triggered protein sulfhydryl in the protein bands corresponding to 38 and 24 kDa was precluded by the lychnopholide treatment correlated with respiratory burst inhibition. These results show that GSH depletion determined by lychnopholide treatment renders PMN to sustain respiratory burst, whose action is proportional to protein S-glutahionylation decrease.
 
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Publishing Date
2016-08-10
 
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