Mutations to bid cleavage sites protect hepatocytes from apoptosis after ischemia/reperfusion injury.
BACKGROUND
Apoptosis of hepatocytes contributes to many forms of liver pathology and can compromise liver function. Hepatocytes have been shown to require mitochondrial disruption to execute apoptosis, a process that is controlled by members of the Bcl-2 family. Bid is a proapoptotic Bcl-2 family member that is cleaved to its active form, tBid, by caspase 8 and granzyme B. Studies in the Bid-deficient mouse have established that hepatocytes require Bid to undergo apoptosis.


METHODS
We generated aspartic acid to glutamic acid mutations in the rat Bid protein, at the caspase 8 and granzyme B cleavage sites, and utilized recombinant adenoviruses to express this protein in hepatoma cells and in the livers of rats.


RESULTS
Cells transduced with recombinant adenoviruses encoding Bid containing mutations to the caspase 8 and granzyme B cleavage sites are significantly protected from both tumor necrosis factor-alpha-induced and cell-mediated apoptosis. Protection occurs through a mechanism that includes decreased Bid cleavage, caspase activation, and mitochondrial membrane damage. Further, after warm ischemia/reperfusion injury, we show that rats expressing cleavage-resistant Bid in the liver display significantly less hepatocyte apoptosis as compared to control rat livers and this results in improved liver function and survival.


CONCLUSIONS
Our results suggest that reagents that prevent the cleavage of Bid would be an effective strategy to inhibit hepatocyte apoptosis and decrease liver injury.
Riddle-Taylor
Erica
E
Department of Surgery, Division of Transplantation, Stanford University School of Medicine, Stanford, CA, USA.
Nagasaki
Kazuhito
K
Lopez
Joseph
J
Esquivel
Carlos O
CO
Martinez
Olivia M
OM
Krams
Sheri M
SM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D053804:Granzymes / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000235:genetics* / Q000473:pathology / Q000517:prevention & control*
D051023:BH3 Interacting Domain Death Agonist Protein; C494816:Bid protein, rat; D001224:Aspartic Acid; D018698:Glutamic Acid; D053804:Granzymes; D053181:Caspase 8