Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.
While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca2(+)-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response.
McDuffee
A T
AT
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Senisterra
G
G
Huntley
S
S
Lepock
J R
JR
Sekhar
K R
KR
Meredith
M J
MJ
Borrelli
M J
MJ
Morrow
J D
JD
Freeman
M L
ML
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D006528:Carcinoma, Hepatocellular; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000378:metabolism*; D005456:Fluorescent Dyes; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008168:Lung / Q000166:cytology; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D015398:Signal Transduction / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D014812:Vitamin K / Q000494:pharmacology
D004220:Disulfides; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D006490:Hemostatics; D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D014812:Vitamin K; D000252:Calcium-Transporting ATPases; D005978:Glutathione