Mechanism of procaspase-8 activation by c-FLIP _L

Abstract

Cellular FLICE-inhibitory protein (c-FLIPL) is a key regulator of the extrinsic cell death pathway. Although widely regarded as an inhibitor of initiator caspase activation and cell death, c-FLIPL is also capable of enhancing procaspase-8 activation through heterodimerization of their respective protease domains. However, the underlying mechanism of this activation process remains enigmatic. Here, we demonstrate that cleavage of the intersubunit linker of c-FLIPL by procaspase-8 potentiates the activation process by enhancing heterodimerization between the two proteins and vastly improving the proteolytic activity of unprocessed caspase-(C)8. The crystal structures of the protease-like domain of c-FLIPL alone and in complex with zymogen C8 identify the unique determinants that favor heterodimerization over procaspase-8 homodimerization, and induce the latent active site of zymogen C8 into a productive conformation. Together, these findings provide molecular insights into a key aspect of c-FLIPL function that modulates procaspase-8 activation to elicit diverse responses in different cellular contexts.apoptosis ͉ caspase-8 ͉ extrinsic pathway ͉ cellular FLICE-inhibitory protein A poptosis is a cellular suicide program essential for early development, tissue homeostasis, and immune system maintenance in all metazoans (1, 2). This program is executed by a family of cysteine proteases known as caspases, which are synthesized as latent zymogens, and are activated in a hierarchical manner as initiators and effectors of the cell death process (3). In the extrinsic cell death pathway, ligands such as FasL/CD95L directly engage their cognate death receptors at the cell surface to recruit and activate the initiator caspases (4). This process occurs through homotypic death domain (DD) interactions between the intracellular portion of the Fas receptor and the adaptor protein FADD, and homotypic death effector domain (DED) interactions between FADD and the N terminus of procaspase-8 or procaspase-10. The resulting activation platform constitutes the death-inducing signaling complex (DISC) (5), effectively homo-oligomerizing and activating the protease domains of procaspase-8 and procaspase-10. Once activated and processed to the mature form, these initiator caspases then cleave and activate the effector caspases, which ultimately proteolyze and dismantle the contents of the cell.During the early stages of death receptor signaling, the key regulatory proteins c-FLIP L , c-FLIP S , and c-FLIP R are also recruited to the DISC, where they modulate activation of procaspase-8 and procaspase-10 (6, 7). The long form of FLIP (c-FLIP L ) is highly homologous to procaspase-8 and procaspase-10, possessing the same architecture with tandem DEDs at its N terminus (the prodomain) and a protease-like domain at its C terminus that is catalytically inactive. The short forms (c-FLIP S and c-FLIP R ) by contrast primarily contain the N-terminal DEDs. All forms of FLIP are widely accepted as antiapoptotic proteins that compete with procaspase-8 ...

DR5‐Cbl‐b/c‐Cbl‐TRAF2 complex inhibits TRAIL‐induced apoptosis by promoting TRAF2‐mediated polyubiquitination of caspase‐8 in gastric cancer cells

“…1D) was found to be decreased after TRAIL treatment. The FLIP/L is an endogenous inhibitor of caspase‐8 that negatively interferes with DISC formation (Yu et al ., 2009). In the present study, the cleavage of caspase‐8 and the translocation of FLIP/L were not detected in BGC823 and MGC803 cells in response to TRAIL.…”

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

“…This assumption was motivated by structural considerations. Namely, procaspase-8 homodimers or procaspase-8/c-FLIP L heterodimers form hydrogen bonds between the catalytic subunits, 28,33 which are lacking in the short isoforms of c-FLIP as well as in the procaspase-8 prodomain.…”

“…27,28 Recently, we studied the stoichiometry of the CD95 DISC by AQUA peptide-based mass spectrometry. 29 In combination with western blot analysis supported by mathematical modeling, we revealed an unexpected stoichiometry in which procaspase-8 is more abundant at the DISC compared with FADD.…”

Redesigning the procaspase‐8 dimer interface for improved dimerization

“…In cellular signaling, formation of the procaspase-8 dimer controls the fate of the cell because it also forms heterodimers with the structurally related protein, FLIP. [18][19][20] It is currently thought that the procaspase-8:FLIP heterodimer represents the default pathway in the cell because the heterodimer is more stable than the homodimer in vitro, 25,32 and it prevents programmed necrosis by inactivating the RIPK1-RIPK3 signaling pathway. 21 High stress loads or activation of death receptors shifts the procaspase-8 oligomer toward the homodimeric state, which then activates procaspase-3 to initiate apoptosis.…”

“…17 In addition to homodimerization, procaspase-8 also forms heterodimers with the structurally similar protein, FLIP. [18][19][20] In the cell, the heterodimer inactivates RIPK1, preventing necroptosis and promoting cell survival, and it is thought that heterodimerization represents the default cellular pathway. 21 Under strong apoptotic stimuli, procaspase-8 forms homodimers and promotes apoptosis.…”