Determining the contributions of caspase-2 and caspase-8 to intracellular protease activities during apoptosis

Apoptosis is the main mechanism of programmed cell death involved in maintaining cellular homeostasis. Dysregulation in apoptosis has been implicated in diseases including cancer, chronic inflammation and neurodegenerative disorders. The understanding of the mechanism of apoptosis regulation in the last decades has revealed a number of highly promising therapeutic targets against these disorders. In this regard, apoptosis signalling crucially depends on caspase activity. This thesis provides novel insights into the more controversial and less established activation scenarios of caspase-2 and caspase-8. So far, approaches towards measuring caspase-2 activity were restricted to analysis in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. In addition, recent publications reported the interplay of caspase-8-dependent apoptosis signalling, autophagy and proteasomal protein degradation to be involved in cell fate decisions. Although the function of caspase-8 in death receptor signalling is well documented, less knowledge exists on its activation in these alternative scenarios. For that purpose, broadly accepted biochemical techniques and highly sensitive Forster resonance energy transfer (FRET)-based flow cytometry were used to monitor caspase activity in different scenarios at single cell and population levels.

In this study, caspase-2 activity during apoptosis execution was negligible and no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), death receptor stimulation (TRAIL, Fas), microtubule destabilization (vincristine), or heat shock. In addition, when limited proteolysis of a VDVAD substrate during apoptosis initiation was observed, this activity was attributable to caspase-8 rather than caspase-2. Therefore, these findings indicate that, in contrast to several previous studies, caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signalling.

Furthermore, in this thesis bortezomib was used to inhibit proteasomes in highly apoptosis resistant cells. Proteasome inhibition induced autophagy and apoptotic cell death but did not induce necroptosis or ROS generation. Caspase-8 was identified as the apical caspase activated after bortezomib and its activation depended on autophagy induction. In addition, high glucose concentration in

medium was able to modulate the cell death response and caspase-8 activation induced after proteasome inhibition by preventing bortezomib-induced rapid inhibition of proteasomes and delaying the induction of autophagy. While further research is needed in order to decipher this novel molecular mechanism, this study provides solid evidence for an autophagy-dependent mechanism that promotes caspase-8 activation when proteasomes are inhibited.