Caspase 8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase 8 activation involves dimerization and subsequent interdomain autoprocessing of caspase 8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase 8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase 8-deficient mice are shared by mice lacking the dimerization adapter FADD (Fas-associated death domain) or the caspase paralogue FLIP_L [FLICE (FADD-like interleukin 1β-converting enzyme)-inhibitory protein, long form] has led to the hypothesis that FADD-dependent formation of heterodimers between caspase 8 and FLIP_L could mediate the developmental role of caspase 8. In the present study, using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase 8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIP_L as a partner for caspase 8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase 8 in complex with FLIP_L does not generate a fully active enzyme, but an attenuated species able to process only selected natural substrates. Based on these results we propose a mechanism of caspase 8 activation by dimerization in the presence of FLIP_L, as well as a mechanism of caspase 8 functional divergence in apoptotic and non-apoptotic pathways.

Significance: The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Using this approach, we have designed an extremely active substrate of NE and subsequently converted it into an ultrasensitive activity-based probe for imaging active elastase during the process of neutrophil extracellular trap formation. Our study could have a substantial effect on the design of substrates, inhibitors, and probes for any endopeptidase.

This review aims to highlight the proteases required for regulated cell death mechanisms in animals and plants. The aim is to be incisive, and not inclusive of all the animal proteases that have been implicated in various publications. The review also aims to focus on instances when several publications from disparate groups have demonstrated the involvement of an animal protease, and also when there is substantial biochemical, mechanistic and genetic evidence. In doing so, the literature can be culled to a handful of proteases, covering most of the known regulated cell death mechanisms: apoptosis, regulated necrosis, necroptosis, pyroptosis and NETosis in animals. In plants, the literature is younger and not as extensive as for mammals, although the molecular drivers of vacuolar death, necrosis and the hypersensitive response in plants are becoming clearer. Each of these death mechanisms has at least one proteolytic component that plays a major role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes. Some similarities are found among animal and plant cell death proteases but, overall, the pathways that they govern are kingdom‐specific with very little overlap.