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.
Cirman
Tina
T
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Oresić
Kristina
K
Mazovec
Gabriela Droga
GD
Turk
Vito
V
Reed
John C
JC
Myers
Richard M
RM
Salvesen
Guy S
GS
Turk
Boris
B
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