Background

Caspases are proteases that are produced as inactive zymogens and are activated by proteolytic cleavage (Degterev et al., 2003). The activation of the caspase cascade is the most important event during apoptotic cell death, which induces the typical biochemical and morphological changes of the apoptotic cell. In contrast to inactive executioner procaspases, the initiator procaspases 8/9/10 have restricted proteolytic activity and become fully activated in high molecular weight complexes (Lavrik et al., 2005). Procaspase-9 is activated at the platform termed apoptosome during intrinsic apoptosis, while stimulation of the death receptors TNF-R1 (Tumor Necrosis Factor Receptor 1), CD95/Fas (Cluster of Differentiation 95), TRAIL-R1 (Tumor Necrosis Factor Related Apoptosis Inducing Ligand Receptor 1), TRAIL-R2 (Tumor Necrosis Factor Related Apoptosis Inducing Ligand Receptor 2) leads to the recruitment of procaspases-8 and -10 with adapter proteins to form the Death-Inducing Signalling Complex (DISC) during extrinsic apoptosis (Schleich et al., 2012). Here, caspases-8 and -10 enter close proximity and perform several intra- and inter-molecular cleavages. This processing results in the release of a small and a large subunit from the prodomain. These form the active heterotetramers p18₂p10₂ and p17₂p12₂ that triggers the caspase cascade leading to the demolition of the cell (see Figures 1A and 1B).

To verify apoptosis induction, it is important to show the activation of caspases. Checking the activation of the initiator procaspases 8 and 10, accompanied by their cleavage, gives main evidence for the induction of the extrinsic apoptosis via death receptors. The kinetics of procaspases-8 and -10 processing can be analysed by monitoring its cleavage steps by Western blot (see Figure 1C) and give information about the induction of apoptotic cell death after death receptor stimulation (Schleich et al., 2016). Depending on the part of the caspase that is recognized by the antibody, the intermediate products of the caspase containing this particular part can be analysed by western blot. Healthy, unstimulated cells only contain the unprocessed procaspases-8 and -10 (p55/53 and p59/55, respectively), but after stimulation the amount of procaspase decreases and the intermediate cleavage products (caspase-8 p43/41 or caspase-10 p47/43) and active subunits (caspase-8 p18) are enriched and can be detected by Western blot (Figure 1C). By performing time-dependent analysis, it is possible to follow the course of caspase cleavage including the enrichment of the cleaved forms (Schleich et al., 2016). This also allows to compare the time-dependent cleavage of caspases between several conditions.

To ensure getting the complete information on procaspase-8/-10 processing we have developed the protocol presented below. In contrast to a number of other protocols for Western blot analysis of procaspase-8/-10 processing, here we do not follow only a part of the Western blot of a specific molecular weight, e.g., by cutting the membrane, or use antibodies specific only to the active forms of the caspases. In this way, we can follow simultaneously several cleavage products of procaspases-8 and -10: proform, intermediate and final cleavage products. Only this way of measuring procaspase-8/-10 processing allows to escape from misjudgement on the efficiency of procaspase cleavage, e.g., in some studies only the proform of procaspases is followed and in case of a low stimulation strength and a weak caspase processing, the small differences in the decrease of the proform are not detected and might be misleading. Furthermore, another very important feature of our protocol is performing the measurement over different time intervals, which is also often neglected, and thus, the results might be misleading due to missing the key time points of processing, for example the appearance of active caspases, that are degraded fast. To the later point, there is a cell type and stimulation strength specificity with respect to the timing of procaspase-8/-10 processing and the corresponding time intervals have to be carefully selected in each particular case.


Figure 1. Kinetics of procaspase-8 and procaspase-10 processing. A. Scheme of caspase-8 processing. The cleavage at D216, D374 and D384 results in the release of the active subunits (p18 and p10). The point marks the region of the caspase that is recognized by the antibody (epitope). B. Scheme of caspase-10 processing. The cleavage at D219, D372 results in the release of the active subunits (p23/p17 and p12). The point marks the region of the caspase that is recognized by the antibody (epitope). C. Western blot: Hela-CD95 cells (Neumann et al., 2010) were stimulated with 250 ng/ml CD95L for the indicated periods of time. Samples were lysed, subjected to SDS-PAGE and analysed by Western blot for caspase-8 and caspase-10. Western blot for actin was used as loading control.