The molecular mechanism of apoptosis upon caspase-8 activation: Quantitative experimental validation of a mathematical model

• Published in: Biochimica et biophysica acta 2012-10, Vol.1823 (10), p.1825-1840
• Main Authors: Kominami, Katsuya, Nakabayashi, Jun, Nagai, Takeharu, Tsujimura, Yuki, Chiba, Kumiko, Kimura, Haruna, Miyawaki, Atsushi, Sawasaki, Tatsuya, Yokota, Hideo, Manabe, Noboru, Sakamaki, Kazuhiro
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - drug effects; BH3 Interacting Domain Death Agonist Protein - metabolism; Biosensing Techniques; Caspase 3 - metabolism; Caspase 6 - metabolism; Caspase 8 - metabolism; Caspase cascade; Caspase Inhibitors; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Cell Survival - drug effects; Down-Regulation - drug effects; Enzyme Activation - drug effects; Feedback, Physiological - drug effects; Fluorescence Resonance Energy Transfer; FRET; HeLa Cells; Humans; Mathematical model; Models, Biological; Peptides - pharmacology; Positive-feedback loop; Receptors, Death Domain - metabolism; Reproducibility of Results; Signal Transduction - drug effects
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2012.07.003

Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway. ► A monitoring system with high sensitivity FRET-based biosensors has been developed. ► The established system enabled us to detect the most minute dynamics of caspase activation in single cells. ► The kinetics, dynamics, and selectivity in apoptotic signaling pathways with activation of caspase-8 were redefined. ► The actual number of pro-apoptotic molecules required for extrinsic apoptotic signal transduction was evaluated. ► A mathematical model predicted the minimal concentration of caspase-8 required to act as an initiator.