Generation of aberrant forms of DFF40 concurrent with caspase-3 activation during acute and chronic liver injury in rats.
DNA fragmentation factors (DFF) form protein complexes consisting of nuclease DFF40/CAD and inhibitory chaperon DFF45/ICAD. Although activated caspase-3 has been shown to cleave DFF complexes with the release of active DFF40 and DNA fragmentation, the organ-specific mechanisms of DFF turnover during liver injury accompanied by massive apoptosis are unclear. In this study, we investigated hepatic profile of DFF40-immunopositive proteins in two models of liver injury in rats: acute ischemia/reperfusion (I/R) and chronic alcohol administration. We show that DFF40-like proteins occur in intact rat liver mainly as a 52kDa protein. Hepatic I/R-induced caspase-3 activation and a time-dependent accumulation of DFF40-positive protein fragments (40 and 20kDa), most likely via specific caspase-3 cleavage as evidenced by in vitro digestion of intact liver tissue with recombinant caspase-3. In addition, immunoprecipitation with DFF40 followed by Western blot with active caspase-3 antibody revealed the presence of active caspase-3 in DFF40-immunopositive 20kDa proteins. Chronic alcohol administration in rats also resulted in a dose-dependent fragmentation of DFF40 proteins similar to I/R injury. Collectively, these data demonstrate that DFF40 immunopositive proteins exist in the liver as distinct, tissue-specific molecular forms that may be processed by caspase-3 during both acute and chronic liver injury.
Xiang
Yiwen
Y
Laboratory of Molecular and Cellular Mechanisms of Injury, Center for Neuroproteomics and Multiple Organ Injury, Department of Neuroscience, University of Florida, P.O. Box 100244, Gainesville, FL 32610, USA.
Johnson
Erik A
EA
Zhang
Chun
C
Huang
Guangling
G
Hayes
Ronald L
RL
Wang
Kevin K W
KK
Svetlov
Stanislav I
SI
D000208:Acute Disease; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D002908:Chronic Disease; D003851:Deoxyribonucleases / Q000032:analysis / Q000378:metabolism*; D004789:Enzyme Activation; D008099:Liver / Q000098:blood supply / Q000201:enzymology*; D008107:Liver Diseases / Q000201:enzymology*; D008108:Liver Diseases, Alcoholic / Q000201:enzymology; D000075223:Poly-ADP-Ribose Binding Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology
D000075223:Poly-ADP-Ribose Binding Proteins; D003851:Deoxyribonucleases; C491882:Dffb protein, rat; D053148:Caspase 3