Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins.
Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system, we show that selective lysosome disruption with L-leucyl-L-leucine methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases to the cytosol, could be prevented by 15 microM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 microM N-benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg(65) or Arg(71). No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Tina
T
Cirman
Kristina
K
Oresić
Gabriela Droga
GD
Mazovec
Vito
V
Turk
John C
JC
Reed
Richard M
RM
Myers
Guy S
GS
Salvesen
Boris
B
Turk
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002403:Cathepsins / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009206:Myocardium / Q000378:metabolism; D010206:Papain / Q000737:chemistry*; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014162:Transfection
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C494815:Bid protein, mouse; D002352:Carrier Proteins; D011994:Recombinant Proteins; D045304:Cytochromes c; D002403:Cathepsins; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases; D010206:Papain