Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis

Abstract

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo. In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid D60E and a BH3 defective mutant Bid G94E failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bakdeficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (␣6 and ␣7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis.Apoptosis, an efficient cell death program, is primarily mediated through the intrinsic or the extrinsic pathway in response to different stimuli in various cell types. Both pathways lead to the activation of a common set of effector caspases, caspase 3, 6, or 7 (1). In the intrinsic pathway, stress signals, e.g. UV irradiation, serum starvation, DNA damage, etc., elicit discrete intracellular pathways that converge on the mitochondria, causing the release of cytochrome c and other apoptogenic proteins. Once in the cytoplasm, cytochrome c triggers formation of the apoptosome, consisting of cytochrome c, Apaf-1, and procaspase 9, and leads to the auto-activation of caspase 9 (2). In the extrinsic pathway, the death ligands, Fas/Apo1/CD95 ligand (FasL) 3 or Apo2L/TRAIL, bind to their cell surface death receptors Fas/Apo-1/CD95 receptor or death receptors 4/5 (DR4/5), respectively, and trigger their trimerization (3). The trimerized receptors then recruit pro-caspase 8 through the adaptor molecule FADD, and form a death-induced signaling complex (DISC), in which caspase 8 becomes auto-activated (4). Once activated, either caspase 8 or 9, in turn, cleaves and activates the more abundant downstream effector caspases 3, 6, and 7, initiating the execution stage of apoptosis (5).The mitochondrial event responsible for the release of apoptogenic factors in the intrinsic pathway, which is termed mitochondrial outer me...

miR-128 Targets the SIRT1/ROS/DR5 Pathway to Sensitize Colorectal Cancer to TRAIL-Induced Apoptosis

“…7A, the combination of miR-128 with TRAIL clearly increased the cleavage of caspase-8. However, the SIRT1 plasmid or NAC inhibited caspase-8 activation in TRAIL [20]. Our results showed that miR-128 significantly enhanced the translocation of tBid to mitochondria in TRAIL-treated SW480 cells (Fig.…”

Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5

“…Primary antibodies against DR5 (#AB16942, Milllipore), caspase-8 (#9746, Cell Signaling Technology), FLIP (#3210, Cell Signaling Technology), FADD (#610399, BD Transduction Laboratories™), CHOP (#2895, Cell Signaling Technology), β-actin (Santa Cruz Biotechnology) and Bid (kindly provided by Dr. Xu Luo) [20] were used to detect relevant proteins of interest.…”

Gold nanoparticles enhance TRAIL sensitivity through Drp1-mediated apoptotic and autophagic mitochondrial fission in NSCLC cells

“…37 Briefly, Calu-1 cells were seeded (1×10 5 per well) on to 12-well plates 24 h prior to exposure to 50 µg/mL AuNPs with various sizes for 3, 6, and 12 h. Cells were washed three times with phosphate-buffered saline (PBS) to remove free AuNPs. Samples were then trypsinized and suspended in medium, and the uptake amount of AuNPs was analyzed with FACScan flow cytometry (BD Biosciences).…”

“…3,4 TRAIL binding to its receptors leads to the assembly of death-inducing signaling complex by recruiting Fas-associated death domain and caspase-8, which in turn initiates a cascade of caspase activation events mediating apoptosis. 5 Preclinical trials reported that recombinant TRAIL and its receptor agonists have been shown to preferentially eliminate cancer cells while leaving normal cells Recently, gold nanoparticles (AuNPs) have been shown to hold great promise for future applications because of their distinctive properties, such as small size, unique photophysical features, easy to surface modify, and favorable biocompatibility. 9,10 All these properties render AuNPs as a versatile nanoplatform for emerging biomedical applications in the design of biosensors, targeted drug delivery vehicles, photothermal therapy, and diagnostic bioimaging.…”