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Diisopropylfluorophosphate (DFP) inhibits ricin-induced apoptosis of MDCK cells
T Oda1, N Komatsu, T Muramatsu
1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Japan.
Abstract:
Diisopropylfluorophosphate (DFP), a general serine protease inhibitor, inhibited the DNA fragmentation and cell death in MDCK cells treated with ricin, modeccin, Pseudomonas toxin, or diphtheria toxin. A trypsin-like serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK) also prevented ricin-induced DNA fragmentation and cell death, albeit less effectively than DFP. Microscopic observation showed that the morphological changes of MDCK cells induced by ricin were prevented by DFP. DFP did not affect the binding, internalization, or subsequent excretion of ricin, but reduced the degradation of ricin in MDCK cells, suggesting that DFP inhibits at least the cellular protease that may be involved in the degradation of internalized ricin. In addition, SDS-PAGE analysis of cytosolic proteins suggested that DFP-sensitive endogenous proteases are activated in the ricin-treated cells. In the cells treated with DFP, the protein synthesis inhibitory activity of ricin was increased rather than inhibited. The activities of modeccin and Pseudomonas toxin were also slightly increased by DFP, but no effect of DFP on the activity of diphtheria toxin was observed. Therefore, these results suggest that protein toxins have a DFP-sensitive common pathway leading to apoptosis that is distinct from the pathway leading to the inhibition of cellular protein synthesis.
Insights
Diisopropylfluorophosphate (DFP), a serine protease inhibitor, prevents cell death induced by protein toxins like ricin. DFP inhibits toxin degradation, suggesting a common apoptosis pathway distinct from protein synthesis inhibition.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Protein toxins like ricin, modeccin, Pseudomonas toxin, and diphtheria toxin induce cell death.
- Serine proteases are implicated in cellular processes and toxin-induced damage.
Purpose of the Study:
- To investigate the role of serine proteases in protein toxin-induced cell death.
- To determine the mechanism by which Diisopropylfluorophosphate (DFP) affects toxin activity and cellular response.
Main Methods:
- Treatment of Madin-Darby Canine Kidney (MDCK) cells with various protein toxins and serine protease inhibitors (DFP, TLCK).
- Assessment of DNA fragmentation, cell death, and morphological changes.
- Analysis of toxin binding, internalization, excretion, and degradation using SDS-PAGE.
- Measurement of protein synthesis inhibition.
Main Results:
- DFP inhibited DNA fragmentation and cell death induced by ricin, modeccin, Pseudomonas toxin, and diphtheria toxin.
- DFP reduced ricin degradation and prevented ricin-induced morphological changes in MDCK cells.
- DFP increased the protein synthesis inhibitory activity of ricin, modeccin, and Pseudomonas toxin, but not diphtheria toxin.
Conclusions:
- A Diisopropylfluorophosphate (DFP)-sensitive common pathway mediates apoptosis induced by protein toxins, separate from the pathway inhibiting protein synthesis.
- Cellular proteases inhibited by DFP play a role in the degradation of internalized protein toxins.
- Serine proteases are critical in the cellular response to protein toxin intoxication.