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Activation of intracellular proteases is an early event in TNF-induced apoptosis
C Voelkel-Johnson1, A J Entingh, W S Wold
1Department of Microbiology, North Carolina State University, Raleigh 27695.
Abstract:
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.
Insights
Proteolysis, or protein breakdown, occurs during tumor necrosis factor (TNF)-induced apoptosis. This process is non-specific and involves the degradation of multiple proteins, but not the activation of cPLA2.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Tumor necrosis factor (TNF)-induced apoptosis involves complex cellular signaling pathways.
- The role of proteolysis in TNF-induced apoptosis has been suggested but not fully elucidated.
- C3HA fibroblasts are sensitive to TNF-induced apoptosis when treated with cycloheximide.
Purpose of the Study:
- To investigate whether protein fragmentation occurs during TNF-induced apoptosis in C3HA fibroblasts.
- To identify specific proteins undergoing degradation during apoptosis.
- To determine the role of proteolysis in the activation of cPLA2 during TNF-induced apoptosis.
Main Methods:
- Measurement of primary amine binding using fluorescamine.
- Trichloroacetic acid (TCA) precipitation to assess protein integrity.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to detect protein fragmentation.
- Analysis of cPLA2 activity in apoptotic cells.
Main Results:
- Increased primary amine binding (approx. 50%) and loss of TCA-precipitable protein (10-15%) in apoptotic cells indicate proteolysis.
- Fragmentation and degradation of lamin B, topoisomerase I, histone H1, protein kinase C beta 1, and cPLA2 were observed, suggesting non-specific proteolysis.
- Adenovirus dl758 lacking the E3 14.7-kDa gene promoted proteolysis, while the E3 14.7-kDa gene inhibited it.
- Proteolysis of cPLA2 was not found to be necessary for its activation during TNF-induced apoptosis.
Conclusions:
- Proteolysis is a significant event during TNF-induced apoptosis, affecting multiple cellular proteins non-specifically.
- The adenovirus E3 14.7-kDa protein likely acts upstream of protease activation in this process.
- Proteolysis of cPLA2 is unlikely to be a prerequisite for its activation in TNF-induced apoptosis.