Selective Knockdown of the Long Variant of Cellular FLICE Inhibitory Protein Augments Death Receptor-mediated Caspase-8 Activation and Apoptosis

Abstract

Apoptosis is essential for development, tissue homeostasis, and immune function (1). The key mediators of apoptosis are caspases, a family of cysteine proteases that cleave a critical set of cellular proteins near specific aspartic acid residues (2). Apoptosis-inducing members of the tumor necrosis factor superfamily, such as Fas ligand (FasL) 1 and Apo2L/TRAIL, activate caspases through the cell extrinsic apoptosis signaling pathway by engaging their respective "death" receptors: Fas and DR4 or DR5, leading to assembly of the DISC (3). Upon ligand stimulation, the adaptor protein FADD (Fas-associated death domain) binds to the receptor through homophilic death domain interactions. FADD then recruits the apoptosis initiator proteases caspase-8 and -10, through homophilic death effector domain (DED) interactions. The proximity of caspase molecules in the DISC facilitates their dimerization and thereby stimulates their proteolytic activity (4 -6). Activation of caspase-8 is followed by two self-processing events; the first cleaves a 10-kDa fragment (p10) from the full-length caspase, leaving an intermediate fragment (p43/41) at the DISC, and the second cleaves an 18-kDa fragment (p18) from the p43/41 intermediate, allowing formation of an active caspase-8 (p18/ p10) 2 heterotetramer, which is released into the cytosol. In turn, cytosolic caspase-8 catalyzes the cleavage and activation of downstream effector caspases, such as caspase-3 and -7, which execute the apoptotic death program.c-FLIP (cellular FLICE inhibitory protein) is structurally related to procaspase-8 and -10 but lacks enzymatic activity (7,8). At least 10 splice variants of c-FLIP exist on the mRNA level, but often only two c-FLIP proteins are detected: a 26-kDa short form (c-FLIP S ) and a 55-kDa long form (c-FLIP L ) (9, 10). c-FLIP S resembles its viral counterpart, v-FLIP, consisting of two DEDs and a short C-terminal tail but entirely lacking a caspase-like domain (11,12). In contrast, c-FLIP L contains two N-terminal DEDs and a C-terminal caspase-like domain, similar to caspase-8 and -10. However, c-FLIP L is proteolytically inactive, because it lacks a specific cysteine residue that is critical for caspase activity.Both c-FLIP variants are capable of binding to the Fas DISC (7,8). The presence of c-FLIP S or c-FLIP L in the Fas DISC does not preclude caspase-8 recruitment; rather, DISC-associated caspase-8/c-FLIP complexes are formed (9,(13)(14)(15). Because c-FLIP S lacks caspase cleavage sites, it is not processed by apical caspases in the DISC. In contrast, c-FLIP L is cleaved between its large (p20) and small (p12) subunits by apical caspases in the DISC; however, a second cleavage between the large subunit and the N-terminal DEDs has not been detected, probably because of the lack of a conserved cleavage site (9). Interaction of caspase-8 with c-FLIP S does not support caspase-8 activation; therefore, binding of c-FLIP S to the Fas DISC inhibits Fas-mediated apoptosis (7,8). Indeed, ectopic overexpression of c-FLIP S inhibits apoptosis i...

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

“…In agreement with recent observations, 44 our studies demonstrate that FLIP L is a key determinant in mediating TRAIL resistance in malignant urothelial cells. Sensitization to TRAIL-induced apoptosis after pretreatment with CHX, CAPE or SB202190 correlated with decreases in FLIP L and FLIP S , and siRNA knockdown experiments showed a marked increase in apoptotic susceptibility that correlated with the degree of loss of FLIP L protein expression.…”

Dual role of TMS1/ASC in death receptor signaling

“…One possibility is that the upregulation of TMS1 by TNFa, while not required for initial activation of caspase-8, serves to amplify the apoptotic signal induced by death receptor ligation. Current models indicate that death receptors elicit an apoptotic response through ligand-induced recruitment of the adaptor protein FADD and procaspase-8 to the receptors' cytoplasmic surface to form the DISC (Ashkenazi and Dixit, 1998;Sharp et al, 2005), allowing for activation of the procaspase through induced proximity (Salvesen and Dixit, 1999;Shi, 2004). However, there is mounting evidence that caspase-8 can also be activated independently of DISC formation.…”

New insights into apoptosis signaling by Apo2L/TRAIL

“…The Apo2L/TRAIL DISC has been well characterized. As the FasL DISC, it is composed of oligomerized receptors, the adaptor molecule FADD, the initiator proteases caspase-8 and/ or caspase-10, and, depending on the context, the cellular FLICE-inhibitory protein (c-FLIP) Kischkel et al, 2000Kischkel et al, , 2001Sprick et al, 2000Sprick et al, , 2002Sharp et al, 2005). The stoichiometry of the different DISC components has not been fully defined.…”