title: 20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated.
abstract: The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.
authors.affiliation: Departmento de Biologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277 São Paulo, São Paulo 05508-900, Brazil. mari_mais@hotmail.com
authors.forename: Marilene
authors.initials: M
authors.lastname: Demasi
authors.forename: Gustavo Monteiro
authors.initials: GM
authors.lastname: Silva
authors.forename: Luis Eduardo Soares
authors.initials: LE
authors.lastname: Netto
mesh_terms: D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002470:Cell Survival; D002614:Chelating Agents / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D004369:Pentetic Acid / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism
chemical_list: D001643:Bridged Bicyclo Compounds; D002614:Chelating Agents; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D016877:Oxidants; D029701:Saccharomyces cerevisiae Proteins; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D004369:Pentetic Acid; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; C028381:monobromobimane