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FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens
M Muzio1, G S Salvesen, V M Dixit
1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Abstract:
Engagement of CD95 or tumor necrosis factor 1 receptor (TNFR-1) by ligand or agonist antibodies is capable of activating the cell death program, the effector arm of which is composed of mammalian interleukin-1beta converting enzyme (ICE)-like cysteine proteases (designated caspases) that are related to the Caenorhabditis elegans death gene, CED-3. Caspases, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Furthermore, proteases belonging to this family exist as zymogens that in turn require cleavage at internal aspartate residues to generate the two-subunit active enzyme. As such, family members are capable of activating each other. Remarkably, both CD95 and TNFR-1 death receptors initiate apoptosis by recruiting a novel ICE/CED-3 family member, designated FLICE/MACH, to the receptor signaling complex. Therefore, FLICE/MACH represents the apical triggering protease in the cascade. Consistent with this, recombinant FLICE was found capable of proteolytically activating downstream caspases. Furthermore, CrmA, a pox virus-encoded serpin that inhibits Fas and tumor necrosis factor-induced cell death attenuates the ability of FLICE to activate downstream caspases.
Insights
CD95 and tumor necrosis factor receptor-1 (TNFR-1) trigger apoptosis by activating caspases, a family of proteases. FLICE/MACH is identified as the apical protease initiating this cell death cascade.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- CD95 and tumor necrosis factor receptor-1 (TNFR-1) are key mediators of programmed cell death (apoptosis).
- The effector arm of apoptosis involves mammalian interleukin-1beta converting enzyme (ICE)-like cysteine proteases, known as caspases, which are evolutionarily related to Caenorhabditis elegans CED-3.
- Caspases are characterized by their ability to cleave substrates after aspartate residues and exist as zymogens requiring internal cleavage for activation.
Purpose of the Study:
- To identify the apical protease responsible for initiating the caspase cascade triggered by CD95 and TNFR-1.
- To investigate the role of FLICE/MACH in the receptor-mediated apoptosis pathway.
Main Methods:
- Utilized receptor engagement assays with CD95 and TNFR-1.
- Investigated the recruitment of ICE/CED-3 family members to receptor signaling complexes.
- Employed recombinant FLICE to assess its proteolytic activity on downstream caspases.
- Examined the effect of CrmA, a viral serpin, on FLICE-mediated caspase activation.
Main Results:
- Both CD95 and TNFR-1 initiate apoptosis by recruiting a novel ICE/CED-3 family member, FLICE/MACH, to the receptor signaling complex.
- Recombinant FLICE demonstrated the ability to proteolytically activate downstream caspases.
- The viral serpin CrmA was found to inhibit FLICE's capacity to activate downstream caspases, thereby attenuating cell death.
Conclusions:
- FLICE/MACH acts as the apical triggering protease in the CD95 and TNFR-1-mediated apoptosis cascade.
- FLICE/MACH's activation of downstream caspases is a critical step in initiating programmed cell death.
- Inhibition of FLICE by viral factors like CrmA can modulate apoptotic signaling pathways.