Caspase-8 cleaves its substrates from the plasma membrane upon CD95-induced apoptosis

Abstract

Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells. Apoptosis is coordinated by the activity of initiator and effector caspases.1-4 While effector caspases are dimeric zymogens that become activated by cleavage, initiator caspases are normally expressed as monomeric zymogens and their activity is initiated by dimerization in a multimeric complex. 5,6 The initiator caspase procaspase-8 dimerizes in the death-inducing signaling complex (DISC) formed around activated death receptors. 7 In the context of CD95, the DISC contains clustered receptors bound to the adaptor protein FADD. FADD can recruit several proteins, 8,9 including procaspase-8 and -10 through their prodomain. In type I cells, active caspase-8/10 directly cleaves and activates effector caspase-3/-7, thus inducing apoptosis. In type II cells, this activation is blocked by XIAP, but cleavage of BID by active caspase-8/10 induces mitochondrial outer membrane permeabilization (MOMP), followed by initiator caspase-9 activation and release of XIAP-inhibitor SMAC, leading to massive caspase-3/7 activity. 10,11 Biochemical and structural studies showed that dimerization but also cleavage in the catalytic subunit of caspase-8 is required for efficient cleavage of caspase-3 and BID and for apoptosis.12-14 Supporting this, the non-cleavable D387A caspase-8 mutant compromises apoptosis in mice. 15Although caspase-8 dimerization generates some activity, cleavage in the catalytic units likely stabilizes the active form and increases activity. 13,16 Caspase-8 is also cleaved between the prodomain and the catalytic unit of the enzyme, at D210 and D216, 17,18 and subsequently released from the DISC. Active caspase-8 can be detected on the plasma membrane, before release, with fluorescent inhibitors. 19 In contrast, by designing a procaspase-8 artificially dimerized on the plasma membrane, Martin et al.18 suggested that the cytosolic release is necessary to cleave caspase-3 and BID. Other studies proposed that fully processed ...

Intra- and Interdimeric Caspase-8 Self-Cleavage Controls Strength and Timing of CD95-Induced Apoptosis

“…Upon caspase-8 activation, the linker is cleaved and the fluorescent protein is released from its localization domain, allowing the fluorescent proteins to equilibrate over the entire cell. Dynamic changes in nuclear intensity, measured by time-resolved confocal microscopy imaging, served as the readout for enzymatic activity (19). …”

“…Caspase-8 activation is enhanced by caspase-3–mediated feedback cleavage, which is activated after MOMP in the last 6 to 8 min before apoptosis (23). Before MOMP, caspase-8 and not caspase-3 contributes to probe cleavage (19). Because we here focused on the initial dynamics of caspase-8 autoprocessing, we excluded the last 10 min before bleb appearance from analysis.…”

“…BID was fused to mGFP C-terminally and mCherry N-terminally with the linkers GSIAT and RSRG, respectively, and cloned into the pEGFP N1 vector. Myristoylation-palmitoylation (MyrPalm) probes were constructed as described in (19). The MyrPalm domain was fused to a spacer of snap tags, alternative cleavage sequences, and either mCherry or mGFP.…”

Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway

“…Caspase 8 also plays an important role in apoptosis. As the first among the caspase family of proteins to be activated, it then activates caspase 3 so that apoptosis can proceed (Beaudouin et al, 2013). Our results indicate that both caspase 3 and caspase 8 were activated after LAMPs treatment.…”

Applications in Cancer Research: Mathematical Models of Apoptosis

“…This problem is overcome by fluorescence-based microscopic methods that were developed to obtain quantitative data of single cells with high temporal resolution: The activity of caspases can be monitored with FRET reporters or smart probes that harbor caspase cleavage sites (Tyas et al 2000;Rehm et al 2002;Beaudouin et al 2013). However, understanding its mechanistic basis is challenging owing to complex interactions of a large number of signaling proteins and emergent behavior at the systems level.…”