Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation

J Exp Med. 2000 Nov 20;192(10):1403-14. doi: 10.1084/jem.192.10.1403.

Abstract

The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Animals
  • Apoptosis*
  • BH3 Interacting Domain Death Agonist Protein
  • Bone Marrow Cells
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Caspases / metabolism
  • Cells, Cultured
  • Enzyme Activation
  • Granzymes
  • Humans
  • Jurkat Cells
  • Mice
  • Mitochondria / metabolism
  • Models, Biological
  • Protein Processing, Post-Translational
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • Serine Endopeptidases / metabolism*
  • Signal Transduction
  • fas Receptor / metabolism

Substances

  • Amino Acid Chloromethyl Ketones
  • BH3 Interacting Domain Death Agonist Protein
  • BID protein, human
  • Bid protein, mouse
  • Carrier Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • fas Receptor
  • GZMB protein, human
  • Granzymes
  • Gzmb protein, mouse
  • Serine Endopeptidases
  • Caspases