Activation of caspases-8 and -10 by FLIP(L)

Abstract

The first step in caspase activation is transition of the latent zymogen to an active form. For the initiator caspases, this occurs through dimerization of monomeric zymogens at an activating complex. Recent studies have suggested that FLIP(L) [FLICE-like inhibitory protein, long form; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1beta-converting enzyme], previously thought to act solely as an inhibitor of caspase-8 activation, can under certain circumstances function to enhance caspase activation. Using an in vitro induced-proximity assay, we demonstrate that activation of caspases-8 and -10 occurs independently of cleavage of either the caspase or FLIP(L). FLIP(L) activates caspase-8 by forming heterodimeric enzyme molecules with substrate specificity and catalytic activity indistinguishable from those of caspase-8 homodimers. Significantly, the barrier for heterodimer formation is lower than that for homodimer formation, suggesting that FLIP(L) is a more potent activator of caspase-8 than is caspase-8 itself.

Figure 1. Schematic representation of conserved caspase residues

Conserved residues of the caspase family are shown. As the caspase-1 numbering system is used throughout, this Figure allows translation of conserved residues across the various members of the caspase family. For caspases-8 and -10, the numbering system describing isoform a is used.

Figure 2. FLIP_L activates caspase-8 by heterodimerization

(A) Non-cleavable caspase-8 (50 nM) was incubated with either 1 μM FLIP_L or 1 μM caspase-8 C285A at the indicated concentrations of sodium citrate at 37 °C, followed 20 min later by the addition of Ac-IETD-AFC. Substrate hydrolysis was followed at 37 °C, and is expressed as relative fluorescence units (RFU)·min⁻¹. (B) Both non-cleavable caspase-8 and FLIP_L (each at 7.5 μM) were incubated for 45 min at 37 °C in the presence of 10 mM dithiothreitol and 10 μM Z-VAD-FMK. Following incubation, samples were analysed by size exclusion chromatography on a Superdex 200 column. The broken line indicates samples prepared in the absence of Z-VAD-FMK. (C) Same as (B) with the exclusion of FLIP_L; (D) same as (B) with the exclusion of caspase.

Figure 3. Non-cleavable caspase-8 is activated most efficiently by FLIP_L and closely approximates endogenous procaspase-8

(A) Caspase-8 cleaved monomer, cleaved dimer or non-cleavable monomer at 75 nM was incubated with an equimolar amount of FLIP_L in the presence of 20 mM Tris, pH 7.4, 100 mM NaCl (low-salt buffer) or kosmotrope buffer (containing 0.7 M sodium citrate). After 20 min at 37 °C, Ac-IETD-AFC substrate was added at 100 μM and substrate hydrolysis monitored at 37 °C. Results are expressed as fold increase over caspase alone under the respective buffer conditions. (B, C) Endogenous procaspase-8 from a Jurkat cytosolic extract or non-cleavable caspase-8 prepared in hypotonic buffer at a comparable concentration (15 nM) was immunoprecipitated using polyclonal antisera raised against recombinant caspase-8. (B) Immunoprecipitates were assayed for their ability to hydrolyse Ac-IETD-AFC substrate in the presence or absence of 1 μM FLIP_L in either low-salt buffer or kosmotrope buffer as indicated. (C) Western blot of immunoprecipitates using monoclonal anti-caspase-8 antiserum raised against an epitope of the large subunit. (D) Non-cleavable caspase-8, -9 or -10 (500 nM) was added to buffer control, 1 μM of their respective C285A mutant or 1 μM FLIP_L in either low-salt buffer or kosmotrope buffer for 20 min at 37 °C before the addition of substrate (Ac-IETD-AFC for caspase-8, Ac-LEHD-AFC for caspase-9, and Ac-DEVD-AFC for caspase-10).

Figure 4. Cleavage of FLIP_L is not required for activation of caspase-8, but an ability to form heterodimers is

(A) Non-cleavable caspase-8 (50 nM) was incubated with increasing concentrations of the indicated protein in the presence of 0.7 M sodium citrate for 20 min at 37 °C. Following the activation period, activity was monitored by Ac-IETD-AFC (AFC) hydrolysis. (B) ΔDED caspase-10 (50 nM) was treated as in (A). Following the incubation, activity was monitored by hydrolysis of Ac-DEVD-AFC. Symbols represent FLIP (♦), non-cleavable FLIP D287A (▪), dimer blocking FLIP Q390D (▴), and a catalytic mutant of caspase-8 or -10 (•).

Figure 5. Substrate specificity of the caspase-8 homodimer and the caspase-8/FLIP_L heterodimer

Caspase-8 was incubated in 0.7 M sodium citrate in the presence (A) or absence (B) of FLIP_L and assayed with a positional scanning substrate library with P1 fixed as aspartic acid. The y-axis is the rate of hydrolysis presented as a percentage of the maximal rate observed. The x-axis provides the positionally defined L-amino acid (single-letter code).