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Intracellular Refolding Assay
Published on: January 24, 2012
CD95-mediated alteration in Hsp70 levels is dependent on protein stabilization
Caoimhín G Concannon1, Una FitzGerald, Carina I Holmberg
1Cell Stress and Apoptosis Research Group, Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
Cell Stress & Chaperones
|April 19, 2005
Summary
CD95 signaling increases heat shock protein 70 (Hsp70) levels by enhancing protein stability, not mRNA accumulation. This novel mechanism involves reduced Hsp70 turnover during CD95-mediated apoptosis.
Area of Science:
- Cellular Biology
- Molecular Biology
- Immunology
Background:
- Engagement of death receptors triggers caspase activation and apoptosis.
- Previous studies indicated elevated heat shock protein 70 (Hsp70) levels during CD95-mediated apoptosis.
Purpose of the Study:
- To investigate the mechanism behind increased Hsp70 levels during CD95 receptor-mediated apoptosis.
- To determine if Hsp70 accumulation is regulated at the mRNA or posttranslational level.
Main Methods:
- Challenging cells with a monoclonal antibody against the CD95 receptor.
- Assessing Hsp70 levels, heat shock factor 1 activation, and Hsp70 mRNA accumulation.
- Utilizing cycloheximide to inhibit protein synthesis and evaluating Hsp70 stability.
- Comparing CD95 signaling with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment.
Main Results:
- Hsp70 levels increased in a dose-dependent manner upon CD95 receptor engagement.
- Hsp70 accumulation occurred independently of heat shock factor 1 activation or increased Hsp70 mRNA levels.
- CD95 signaling maintained Hsp70 protein levels despite inhibited de novo protein synthesis, unlike control cells.
- TRAIL-induced apoptosis did not affect Hsp70 levels, indicating receptor-specific effects.
Conclusions:
- CD95 receptor signaling enhances Hsp70 protein stability, leading to its accumulation.
- This mechanism involves increased protein stability and reduced turnover, a novel pathway for heat shock protein accumulation during apoptosis.
- The findings highlight a posttranslational regulation of Hsp70 during CD95-mediated cell death.
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