Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation.
Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.
Li
C Y
CY
Department of Biochemistry and Ilchun Molecular Medicine Institute Medical Research Center, Seoul National University College of Medicine, Korea.
Lee
J S
JS
Ko
Y G
YG
Kim
J I
JI
Seo
J S
JS
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005434:Flow Cytometry; D017353:Gene Deletion; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020298:U937 Cells
D003574:Cytochrome c Group; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases