Activation of intracellular proteases is an early event in TNF-induced apoptosis.
The serine protease inhibitor tosyl-argenine methyl ester inhibits TNF-induced apoptosis, suggesting that proteolysis is necessary for this response. To test this hypothesis, we asked whether protein fragmentation occurs during the death of C3HA fibroblasts, a 3T3-like cell that was rendered sensitive to TNF by cycloheximide. Our results show that the binding of fluorescamine, which binds primary amines, was increased in apoptotic cells by approximately 50%. We also found that 10-15% of the protein in apoptotic cells was no longer precipitable by TCA. Evidence for proteolysis was also revealed by SDS-PAGE analysis and from Western blots. We observed fragmentation and/or degradation of lamin B, topoisomerase I, histone H1, protein kinase C beta 1, and cPLA2, indicating that proteolysis during apoptosis is non-specific. We also found evidence of proteolysis in C3HA cells sensitized to TNF by the adenovirus dl758 (which lacks the E3 14.7-kDa resistance gene) suggesting that protease activation is common in TNF-induced apoptosis. In contrast, the adenovirus E3 14.7-kDa resistance gene prevented proteolysis suggesting that this protein acts at, or upstream of the proteases activated in this response. Finally, because tosyl-argenine methyl ester inhibits the release of [3H]arachidonic acid from apoptotic cells, we tested whether proteolysis of cPLA2 is necessary for enzyme activation. Our results failed, however, to reveal a common proteolytic fragment in different cell types, and when tested in vitro the cytosol from apoptotic cells had less cPLA2 activity. It is unlikely, therefore, that proteolysis is necessary for the activation of this enzyme during TNF-induced apoptosis.
Voelkel-Johnson
C
C
Department of Microbiology, North Carolina State University, Raleigh 27695.
Entingh
A J
AJ
Wold
W S
WS
Gooding
L R
LR
Laster
S M
SM
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000502:physiology; D000260:Adenoviruses, Human / Q000235:genetics / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D010450:Endopeptidases / Q000502:physiology*; D004789:Enzyme Activation; D005347:Fibroblasts; D051379:Mice; D008809:Mice, Inbred C3H; D010741:Phospholipases A / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014106:Tosylarginine Methyl Ester / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017500:Adenovirus E1A Proteins; D011506:Proteins; D014409:Tumor Necrosis Factor-alpha; D014106:Tosylarginine Methyl Ester; D003513:Cycloheximide; D010741:Phospholipases A; D010450:Endopeptidases