Related Experiment Videos
Fas-induced DNA fragmentation and proteolysis of nuclear proteins
A Kawahara1, M Enari, R V Talanian
1Department of Genetics, Osaka University Medical School, Suita, Japan.
Background:
Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti-Fas antibody causes apoptosis in Fas-bearing cells, by activating various members of the caspase family.
Results:
Specific fluorogenic substrates (MCA-DEVDAPK[dnp] and MCA-VEVDAPK[dnp]) for caspases 3 and 6 were prepared. Using these substrates, a gradual increase of the caspase 3-and 6-like proteases were detected during the Fas engagement in human Jurkat. This activation of caspases correlated well with the cleavage of poly(ADP-ribose) polymerase and lamin B1, as well as with DNA fragmentation. When the recombinant caspases were added to the extracts from Jurkat cells, caspase 3 produced active caspase 6-like protease, while caspase 6 activated the caspase 3 protease, suggesting that these proteases can activate each other. The caspase-treated cell extracts, as well as the extracts from the Fas-activated cells, caused the proteolysis of nuclear proteins and DNA degradation. The cleavage of nuclear proteins was inhibited by caspase inhibitors, while the same inhibitors had no effect on DNA degradation.
Conclusions:
At one stage of the caspase cascade, caspases activate each other, and amplify the apoptotic signal. Caspases downstream of the cascade then cause the proteolysis of nuclear proteins and DNA degradation.
Insights
Fas receptor activation initiates a caspase cascade where caspases 3 and 6 activate each other, amplifying the apoptotic signal. This leads to nuclear protein breakdown and DNA fragmentation during Fas-mediated apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Apoptosis Research
Background:
- Fas is a tumor necrosis factor (TNF) receptor family member.
- Fas activation triggers apoptosis via caspase cascades.
Purpose of the Study:
- Investigate the role of caspases 3 and 6 in Fas-induced apoptosis.
- Elucidate the auto-activation mechanism within the caspase cascade.
Main Methods:
- Synthesis of specific fluorogenic caspase substrates (MCA-DEVDAPK[dnp], MCA-VEVDAPK[dnp]).
- Assay of caspase activity in Fas-engaged human Jurkat cells.
- Analysis of poly(ADP-ribose) polymerase and lamin B1 cleavage.
- Assessment of DNA fragmentation and nuclear protein proteolysis.
Main Results:
- Detected increased caspase 3- and 6-like protease activity during Fas engagement.
- Observed auto-activation between caspase 3 and caspase 6.
- Correlated caspase activation with poly(ADP-ribose) polymerase cleavage, lamin B1 degradation, and DNA fragmentation.
- Demonstrated that downstream caspases degrade nuclear proteins and DNA.
Conclusions:
- Caspase auto-activation amplifies the apoptotic signal in the Fas pathway.
- Downstream caspases are responsible for nuclear protein proteolysis and DNA degradation.
- Caspase inhibitors block nuclear protein cleavage but not DNA degradation.