Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation.

Abstract

In the original version of this manuscript, we inadvertently included the incorrect blot for HSP90, a control for equal protein loading, in supplemental Fig. S3. The HSP90 signal shown in supplemental Fig. S3 was taken from the blot shown in Fig. 3B. In both of these experiments, primary antibodies against caspase-8 and HSP90 were used concurrently, so signals for both caspase-8 and the loading control HSP90 are visible on each blot. Below is the corrected version of Fig. S3 with the correct location of the HSP90 loading control shown. These changes do not affect the interpretation or conclusions of this work.

Type 1 inositol‐1,4,5‐trisphosphate receptor is a late substrate of caspases during apoptosis

“…Actually, because there is no cleavage site for this protease in the human proteome, this approach is ideal to post‐translationally modify a protein including decoupling a cleavage event occurring during apoptosis from full‐blown apoptosis. For example, we previously used this approach to demonstrate that caspase‐8 activation does not lead to apoptosis unless it is cleaved [Oberst et al, 2010].…”

Caspase-8 as a therapeutic target in cancer

“…Like all caspases, the caspase-8 protein is translated as a monomeric, procaspase zymogen that is later activated by dimerization [3], typically accompanied by the maturation cleavage of the caspase itself [4; 5]. In the case of caspase-8 this yields large and small ‘subunits’ of approximately 18 and 12 kilodaltons.…”

“…However, these two fragments are not actually disparate domains, but rather non-covalently associated parts of the same protease domain, consisting of a twisted β-sheet (6 stranded, one anti-parallel) bordered by 5 α helices [6]. Rather, the purpose of the observed cleavage is to accommodate the conformational rearrangements required for stable formation of the dimer and the catalytic sites [5], which are otherwise absent in the solved structure of the monomeric procaspase [7]. Nonetheless, the lack of cleavage of the caspase catalytic subunit, at least in the case of caspase-8, should not serve as an indication of an absolute absence of caspase-8 catalytic activity.…”

“…The mechanism by which the procaspase may transiently form a catalytic site is unclear, but given the requirement for dimerization to form a stable enzyme, it appears that this may represent an unfavored, but semi-stable conformer of the procaspase, or alternatively may reflect rearrangements of capsase-8 with other binding partners, such as c-FLIP [10]. Indeed, while dimerization of caspase-8 can be achieved in vitro with chaotropic salts, dimerization of caspase-8 in vivo appears to require maturation cleavage as an essential event [5]. …”

Inflammatory cell death in intestinal pathologies

“…Caspase‐8 (p55/53) has a prodomain that interacts with FADD, and two catalytic subdomains that undergo sequential cleavage during activation. The linker between prodomain and catalytic subunits can be cleaved between Asp 374 and Asp 384 by caspase‐8 itself to generate procaspase‐8 (43/41) that is further processed into (p18) 2 (p10) 2 heterodimer that induces apoptosis . Processing of caspase‐8 changes the range of downstream substrates and potency of its activity …”