Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1.
Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome.
Steel
Rohan
R
Peter MacCallum Cancer Centre, A'Beckett St., Locked Bag #1, Melbourne, Victoria 8006, Australia.
Doherty
Judith P
JP
Buzzard
Katherine
K
Clemons
Nicholas
N
Hawkins
Christine J
CJ
Anderson
Robin L
RL
D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012235:Rhodamines / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014466:Ultraviolet Rays
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D005456:Fluorescent Dyes; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D011994:Recombinant Proteins; D012235:Rhodamines; D012492:Salts; D014409:Tumor Necrosis Factor-alpha; D005047:Etoposide; D045304:Cytochromes c; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; C029773:rhodamine B