Abstract
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
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📋 Methods
Casp-8 tDED Filament, FADD DED Filament, and cFLIP tDED Filament Preparation Casp-8 tDED and FADD DED were expressed as His-GFP fusions and purified using Ni-NTA affinity and gel filtration chromatography. The void fractions contained the respective filament samples. cFLIP tDED was expressed as a His-MBP fusion and similarly purified. cFLIP tDED filaments were generated upon removal of His-MBP by the TEV protease.
Cryo-EM Data Collection and Image Processing
Cryo-EM data collection was performed on a Tecnai F20 electron microscope (FEI) operated at an acceleration voltage of 200 kV using a K2 Summit direct electron detection camera (Gatan) operated in super-resolution mode with dose-fractionation. A total of 388 images were used for analysis and processing. Helical averaging was performed using the iterative helical real space reconstruction (IHRSR) algorithm ( Egelman, 2010 ).
Fluorescence Polarization Assay His-MBP-Casp-8 tDED -Sumo
(Y8A) or His-MBP-cFLIP-Sumo was purified as a monomer and labeled with TAMRA through a sortase reaction. FP readings were taken on a SpectraMax M5e (Molecular Devices) using excitation and emission wavelengths of 561 nm and 585 nm.
Cell Death Assay Induced by Fas Ligand Casp-8 deficient
Jurkat cells were transfected with wild type and mutant Casp-8 constructs in the pcDNA3.1-YFP vector. YFP + cells were treated with FasL fused to an isoleucine zipper (FasL-LZ) and analyzed for cell death via Annexin V and Live/Dead staining (Invitrogen). Recruitment of Caspase-8 to ASC Specks Co-expressed Casp-8 and ASC were immunostained and speck formation was assessed using changes in the ratio of fluorescence peak height to area.
Supplementary Material supplement
📊 Figures
Figure 1
The Fas/FADD Complex, FADD DED and ASC PYD Promote Casp-8 tDED Filament Formation
(A) Domain composition and interaction hierarchy of Casp-8 activation in the death receptor pathway and the inflammasome pathway. (B) A gel filtration profile of GFP-Casp-8 tDED purification. An SDS-P...
Figure 2
Crystal Structure of MBP-Casp-8 tDED -F122G/L123G and Cryo-EM Reconstruction of the GFP-Casp-8 tDED Filament
(A) A ribbon diagram of the Casp-8 tDED (F122G/L123G) crystal structure labeled with secondary structures, and locations of the mutations. (B) Sequence alignment among Casp-8, cFLIP, MC159 and FADD. S...
Figure 3
Quasi-Equivalent Interactions in the Casp-8 tDED Filament
(A) Ribbon and schematic diagram of Casp-8 tDED helical assembly. Each DED is shown as a hexagon and the three tDED molecules in the asymmetric unit are encircled in blue lines. (B) The type I interfa...
Figure 4
Structure-Based Mutants Disrupt Casp-8 tDED Filament Formation in Vitro and in Cells
(A) Gel filtration profiles of WT and mutant Casp-8 tDED showing the filamentous, void fraction and the monomeric fraction from a Superdex 200 column. (B) Morphology of transfected mCherry-fused WT an...
Figure 5
Filament Formation-Defective Mutants of Casp-8 Compromised Death Receptor- and Inflammasome-Mediated Signaling
(A) Cell death in Casp-8 deficient Jurkat cell line I9.2C reconstituted with WT and mutant Casp-8 induced by isoleucine zipper-fused FasL (FasL-LZ). (B) Histogram of cell death in I9.2C reconstituted ...
Figure 6
Insights into DED/DED interactions among FADD DED , Casp-8 tDED and cFLIP tDED
(A) An electron micrograph of GFP-FADD DED filament. (B) Schematic diagram of FADD DED -nucleated Casp-8 tDED filament formation. (C) Predicted type I interface of the FADD DED /Casp-8 tDED interactio...
Figure 7
Capping as the Inhibition Mechanism of Casp-8 tDED Filament Formation by MC159
(A) Inhibition of Fas DD /FADD-mediated Casp-8 tDED polymerization by MC159. The apparent inhibitory constant (K i ), with fitting error, is shown. (B) Comparison of Casp-8 tDED surface with MC159. Ty...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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