Death-effector Filaments: Novel Cytoplasmic Structures that Recruit Caspases and Trigger Apoptosis

Abstract

The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

“…3c) showed that HSV-2 R1 co-immunoprecipitated with casp-8 DED-AB GFP (lane 3), but not with casp-8 CD GFP (lane 4) or with FADD YFP (lane 5). Consistent with these results, we also observed that HSV-2 R1 could block the formation of previously-described [30] cytoplasmic filaments in casp-8 DED-AB GFP-expressing cells but not those seen in FADD YFP-expressing cells (Fig. S3).…”

“…3b). In these adherent cells, fluorescence appeared diffuse throughout the cytoplasm, as reported in conditions where apoptosis induced by casp-8 GFP was inhibited by z-VADfmk [30]. Immunoblotting with anti-GFP antibody showed that casp-8 GFP (*85 kDa) was nearly completely absent in control cells, which accumulated the processed casp-8 p10 GFP product (*42 kDa) (Fig.…”

“…The calcium phosphate technique [7] was used to transfect 10 lg per well and 50 lg per dish of the plasmids pAdCMV5 (empty), pAdCMV5-R1 (HSV-2 R1); pLBPF1-GST (GST), pLBPF1-GST-R1 (GST-HSV-1 R1) (kindly provided by A. Pearson); pcDNA3 (empty), pcDNA3-HA-EBV R1 (HA-EBV R1) [29]; pEGFP C1 (GFP) and plasmids encoding for caspase-8 GFP (casp-8 GFP), caspase-8 C360S GFP (casp-8 C360S GFP), caspase-8 DED-AB (1-209) GFP (casp-8 DED-AB GFP), caspase-8 CD (210-479) GFP (casp-8 CD GFP) and FADD YFP [30]. Immunofluorescence was performed on cells fixed with paraformaldehyde as previously described [31].…”

“…To determine whether HSV-2 R1 could prevent apoptosis induced by caspase-8 over-expression, we transfected into HeLa cells a GFPtagged version of caspase-8 (casp-8 GFP depicted in Fig. 3a) known to potently induce apoptosis in these cells [30]. Observations by fluorescence microscopy at 24 h following casp-8 GFP transfection showed that all the GFP-positive cells in control dishes (mock-infected or preinfected with an empty control Ad recombinant) were rounded up and floating in the medium (Fig.…”

Requirement of cooperative functions of two repeated death effector domains in caspase‐8 and in MC159 for induction and inhibition of apoptosis, respectively

“…Similarly, caspase-10, c-FLIP (Irmler et al 1997) and v-FLIP (Bertin et al 1997;Thome et al 1997) have two DEDs, while caspase-1, -2, -4 and -5 have only one CARD. Interestingly, two repeated DEDs of caspase-8 (Casp8NC-DED) were involved in induction of apoptosis (Siegel et al 1998), while two repeated DEDs of v-FLIP inhibit apoptosis (Bertin et al 1997;Thome et al 1997).…”