title: 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/Bak-deficient (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 (α6 and α7) 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.
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.
authors.forename: Kai
authors.initials: K
authors.lastname: Huang
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Xiangya Hospital, Central South University, Changsha 410008, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases.
authors.forename: Katelyn L
authors.initials: KL
authors.lastname: O'Neill
authors.affiliation: Department of Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, Nebraska 68198-7696.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
authors.affiliation: Department of Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and.
authors.forename: Rolen M
authors.initials: RM
authors.lastname: Quadros
authors.affiliation: Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178, and.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Tu
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, xuluo@unmc.edu.
authors.forename: Xu
authors.initials: X
authors.lastname: Luo
mesh_terms: D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D053181:Caspase 8 / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051336:Mitochondrial Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C474007:Bax protein (53-86); D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D053181:Caspase 8
keywords: B-cell lymphoma 2 (Bcl-2) family; CRISPR/Cas; apoptosis; mitochondria; transcription activator-like effector nuclease (TALEN)