Synthesis of heat shock proteins following oxidative challenge: role of glutathione.
The relationship between glutathione metabolism, menadione sodium bisulphite oxidation of protein thiols, and the synthesis of hsc70 was investigated using CHO cells. A 30-min/37 degrees C exposure to menadione, a compound which redox cycles to produce superoxide anion radicals and hydrogen peroxide, resulted in rapid accumulation of hsc70 mRNA. PAGE and Western blot analysis indicated increased synthesis such that accumulation of hsc70 occurred. These changes were preceded by rapid oxidation of GSH to GSSG, followed by GSH depletion, and subsequent protein thiol oxidation. As a test of whether a correlation existed between GSH oxidation and depletion, protein thiol oxidation and hsp synthesis, cells were exposed to menadione in the absence and presence of glucose. Synthesis of hsc70 was increased in cells exposed to menadione in the absence of glucose compared with its presence. As a further test, cells were exposed to BSO/DEM in order to deplete GSH and then exposed to menadione. The synthesis of hsc70 following exposure to menadione was greatly increased in GSH-depleted cells compared with GSH-replete cells. Experiments were conducted to determine if electroporation of cells in GSSG containing buffer affected hsp synthesis. Electroporation in glucose-free buffer containing 3 mM GSSG did not affect hsp synthesis. We interpret these results to indicate that the inability to maintain glutathione in a reduced form during menadione redox cycling resulted in protein thiol oxidation. This, in turn, resulted in accumulation of hsc70 mRNA with a subsequent increase in the synthesis of hsc70.
Sierra-Rivera
E
E
Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Meredith
M J
MJ
Voorhees
G J
GJ
Oberley
L W
LW
Eisert
D R
DR
Freeman
M L
ML
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis; D006224:Cricetinae; D018274:Electroporation; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D019803:Glutathione Disulfide; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D014812:Vitamin K; D009249:NADP; D005978:Glutathione; D019803:Glutathione Disulfide