The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model.
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.
Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Katsuya
K
Kominami
Jun
J
Nakabayashi
Takeharu
T
Nagai
Yuki
Y
Tsujimura
Kumiko
K
Chiba
Haruna
H
Kimura
Atsushi
A
Miyawaki
Tatsuya
T
Sawasaki
Hideo
H
Yokota
Noboru
N
Manabe
Kazuhiro
K
Sakamaki
D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D015374:Biosensing Techniques; D053148:Caspase 3 / Q000378:metabolism; D053178:Caspase 6 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism*; D061945:Caspase Inhibitors; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D025461:Feedback, Physiological / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D010455:Peptides / Q000494:pharmacology; D053218:Receptors, Death Domain / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects
D051023:BH3 Interacting Domain Death Agonist Protein; D061945:Caspase Inhibitors; D010455:Peptides; D053218:Receptors, Death Domain; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8