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, 433 (3), 447-457

FLIP(L) Induces Caspase 8 Activity in the Absence of Interdomain Caspase 8 Cleavage and Alters Substrate Specificity


FLIP(L) Induces Caspase 8 Activity in the Absence of Interdomain Caspase 8 Cleavage and Alters Substrate Specificity

Cristina Pop et al. Biochem J.


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.


Figure 1
Figure 1. Caspase-8 mutants and activation scheme
(A) Cleavage mutants designed in the context of full-length caspase-8 used for cellular expression, or in the context of chimeric caspase-8 used for in vitro activation studies. Mutations are shown in bold. Chimeric caspase-8 consists of the catalytic domain fused with the artificial dimerization domain FKBP (~14 kDa) [34]. Note that upon expression in E. coli, chimeric caspase-8 underwent proteolysis at Site-1 producing a two-chain species that we refer to as “Mature” throughout the manuscript. We refer to caspase-8 as wild-type (WT) only in the case of caspase-8 expressed in mammalian cells. (B) Diagram of homo- and hetero-dimerization between caspase-8 and FLIPL in the presence of the specified dimerization compounds (AP20187 and AP21967, respectively). FRB is fused to FLIPL to provide a specific heterodimerization with FKBP of capase-8 fusions [34]. (C) Coomassie stained SDS-PAGE gel showing purified FKBP-caspase-8 and FRB-FLIPL mutants expressed in vitro. Two-chain species underwent autoprocessing during expression, whereas one-chain species displayed no autoproteolytic activity.
Figure 2
Figure 2. Caspase-8 uncleavable at Site-1 can only be activated by hetero-dimerization with FLIPL
(A) Homo- and hetero-dimerization activity assays of FKBP-caspase-8. For homodimerization, FKBP-caspase-8 mutants (25 nM) were dissolved in the assay buffer containing either the homodimerizer compound AP20187 (25 nM) or the activator kosmotrope Na-citrate. For heterodimerization, FKBP -caspase-8 mutants (25 nM) were incubated with FRB-FLIP (125 nM) and heterodimerization compound AP21967 (125 nM). Samples were incubated at 25°C for 30 min to permit complete activation. Relative activity was determined at 30°C with the fluorescent substrate Ac-IETF-afc. Data represent the mean of three independent experiments (±SEM); (B) Size exclusion separation of FKBP-Site-1 mutant (3 μM) in the presence and absence of homo-dimerizer (3 μM). The caspase-8 mutant (100 μL) was pre-activated in the assay buffer as described above and applied to a Superdex200 size exclusion column. Calculation of apparent molecular weight was based on column pre-calibration with protein standards; (C) FKBP-Site-1 mutant caspase-8 (50 nM) activation in the presence of Na-citrate and homodimerizer (500 nM). Caspase-8 was added to a mixture of homodimerizer and Na-citrate dissolved in assay buffer, to reach the indicated concentration of kosmotrope. The mixture was incubated for 30 min at 25°C and activity was monitored using Ac-IETD-afc as described above; (D) Cleavage of FRB-FLIPL at LEVD/G is not required for FKBP-Site-1 mutant caspase-8 activation during heterodimerization. FKBP-casp8(Site-1 mutant) (0.7μM) was activated in the presence of the heterodimerizer AP21967 and FRB-FLIPL(wt) or FRB-FLIPL(D/A) as detailed above. The mixture was split and subjected to IETD-ase activity testing or electrophoresis separation in 4-20% SDS-PAGE gels, followed by Coomassie staining; (E) FRB-FLIPL rescues the inactive FKBP-Site-1 mutant caspase-8 during co-expression in HeLa cells, inducing cell death. The depicted caspase mutants were transiently transfected in HeLa cells with pcDNA3 vectors encoding the FKBP and FRB hybrids, as indicated. Transfection was followed, after 24 hours, by treatment with 500 nM heterodimerization compound. Apoptotic cell death was quantified after an additional 24 hours by Annexin V staining (average ±SEM of three independent experiments). In all panels, IETD-ase is expressed as RFU/min.
Figure 3
Figure 3. Substrate specificity of caspase-8 dimers
Specificity was tested for P2-P4 positions of a tetra-peptide on a substrate library with fixed P1 position (Asp) and an ACC group in P1’. Mutants of caspase-8 dimers used in the analysis are specified on the side. FKBP-caspase-8 mutants (100-400 nM) were activated either by homodimerization (in the case of Mature/Mature) or heterodimerization (in the case of Mature/FLIPL and Site-1 mutant/FLIPL) prior to library screening. Full-length caspase-8 containing uncleavable pro-domain and mutations for increased solubility (D210A,D216A,D223A,L122S,F123Y) (0.5 μM), as well as the ΔDED-caspase-8 mutant (50 nM) did not require artificial-induced activation, as they displayed spontaneous enzymatic activity in the assay buffer under the same conditions. The concentration of assembled dimers was chosen based on similar amounts of activity on Ac-IETD-afc prior to library testing.
Figure 4
Figure 4. Cleavage of natural substrates by caspase-8 homo/heterodimers
Caspase-8 dimers were pre-activated for 45 min following addition of the appropriate dimerization compound and/or FRB-FLIPL. Protein substrates (~0.1-3 μM final concentration) were added to serially diluted caspase-8. Final caspase concentrations were 100 nM (Mature/Mature), 100-200 nM (Mature/FLIPL) or 400 nM (Site-1 mutant/FLIPL), as indicated. The protein mix was incubated at 25°C for 3 h and reactions were stopped by the addition of 3× SDS buffer. Cleavage was analyzed by either staining the gels with Coomassie or by Western blotting (HDAC-7). Caspase-3 used as a caspase-8 substrate harbored the catalytically inactive mutation C285A (caspase-1 nomenclature). The lower panels compare IETD-ase activity (RFU/min) generated by the same caspase-8 dilutions as in the protein cleavage assays. Arrows point to the enzyme concentration producing 50% substrate cleavage. * FKBP-Caspase-8(Mature) runs at the same size as caspase-3 zymogen.
Figure 5
Figure 5. Hypothetical model for caspase-8 dimerization
Homodimerization primes the active state of caspase-8, but additional stabilization or interaction, such as cleavage or perhaps C-terminal ubiquitination, is required. In contrast, heterodimerization with FLIPL primes the active state via interface recruitment, and activates without a requirement of further stabilization. On the other hand, kosmotropes promote the activation by uniformly priming the entire caspase surface for association.

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