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Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
Published on: August 30, 2012
Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment
1Institute of Molecular Medicine, University of Düsseldorf, Universitätsstrasse 1, Düsseldorf D-40225, Germany.
Abstract:
Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.
Insights
Cellular FLICE-inhibitory protein (c-FLIP) isoform R in mice is characterized, revealing its anti-apoptotic function requires both death effector domains and C-terminal tail for stability and DISC binding. Viral and cellular FLIPs utilize distinct mechanisms for apoptosis inhibition.
Area of Science:
- Cellular and Molecular Biology
- Immunology
- Virology
Background:
- Cellular FLICE-inhibitory protein (c-FLIP) inhibits apoptosis by blocking caspase-8 activation at the death-inducing signaling complex (DISC).
- Three human c-FLIP isoforms exist: c-FLIP(long), c-FLIP(short), and c-FLIP(R), with c-FLIP(R) being poorly understood.
- Murine c-FLIP(R) is identified as the sole short c-FLIP isoform in mice.
Purpose of the Study:
- To characterize murine c-FLIP(R) and elucidate its role in apoptosis regulation.
- To investigate the structural requirements for c-FLIP(R) binding to the DISC and its anti-apoptotic activity.
- To compare the mechanism of action of c-FLIP(R) with viral FLIPs.
Main Methods:
- Generation and analysis of c-FLIP(R) mutants.
- Assessment of DISC binding and anti-apoptotic function.
- Three-dimensional structural modeling of c-FLIP(R).
- Comparison of structural motifs with viral FLIP MC159.
Main Results:
- Both death effector domains (DEDs) of c-FLIP(R) are essential for DISC binding and anti-apoptotic function.
- The C-terminal tail of c-FLIP(R) plays a critical role in protein stability and DISC recruitment.
- Murine c-FLIP(R) shares structural similarities with viral FLIP MC159 but employs distinct surface motifs for DISC association.
- A hydrophobic patch on DED2 of c-FLIP(R) is crucial for DISC binding, differing from MC159's mechanism.
Conclusions:
- Murine c-FLIP(R) is a functional inhibitor of apoptosis, requiring specific structural features for DISC interaction.
- Despite structural similarities, cellular and viral FLIPs exhibit divergent mechanisms for apoptosis inhibition.
- The findings provide new insights into the regulation of death receptor-mediated apoptosis by c-FLIP isoforms.
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