Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Summary

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Abstract

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC.

Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Introduction

One of the best-studied death receptors (DRs) is CD95 (Fas, APO-1). The extrinsic apoptotic pathway starts with the interaction of the DR with its cognate ligand, i.e., CD95L interacts with CD95 or TRAIL binds to TRAIL-Rs. This results in the formation of the DISC at the corresponding DR. DISC consists of CD95, FADD, procaspase-8/-10, and c-FLIP proteins^1,2. Furthermore, the DISC is assembled by interactions between death domain (DD)-containing proteins, such as CD95 and FADD, and DED-containing proteins such as FADD, procaspase-8/-10, and c-FLIP (Figure 1). Procaspase-8 undergoes oligomerization via association of its DEDs, resulting in the formation of DED filaments, followed by procaspase-8 activation and processing. This triggers a caspase cascade, which leads to cell death (Figure 1)^3,4. Thus, procaspase-8 is a central initiator caspase of the extrinsic apoptosis pathway mediated by CD95 or the TRAIL-Rs, activated at the corresponding macromolecular platform, DISC.

Two isoforms of procaspase-8, namely procaspase-8a (p55) and -8b (p53), are known to be recruited to the DISC⁵. Both isoforms comprise two DEDs. DED1 and DED2 are located at the N-terminal part of procaspase-8a/b followed by the catalytic p18 and p10 domains. Detailed cryo-electron microscopy (cryo-EM) analysis of procaspase-8 DEDs revealed the assembly of procaspase-8 proteins into filamentous structures called DED filaments^4,6. Remarkably, the linear procaspase-8 chains were initially suggested to be engaged in the dimerization followed by procaspase-8 activation at the DISC. Now, it is known that those chains are only a substructure of the procaspase-8 DED filament, the latter comprising three chains assembled into a triple helix^3,4,6,7.

Upon dimerization at the DED filament, conformational changes in procaspase-8a/b lead to the formation of the active center of procaspase-8 and its activation^3,8. This is followed by procaspase-8 processing, which is mediated via two pathways: the first one goes via the generation of a p43/p41 cleavage product and the second one via the initial generation of a p30 cleavage product. The p43/p41 pathway is initiated by the cleavage of procaspase-8a/b at Asp374, resulting in p43/p41 and p12 cleavage products (Figure 2). Further, these fragments are auto-catalytically cleaved at Asp384 and Asp210/216, giving rise to the formation of the active caspase-8 heterotetramer, p10₂/p18₂^9,10,11. In addition, it was shown that in parallel to the p43/p41 pathway of processing, procaspase-8a/b is also cleaved at Asp216, which leads to the formation of the C-terminal cleavage product p30, followed by its proteolysis to p10 and p18¹⁰ (Figure 2).

Procaspase-8a/b activation at the DED filament is strictly regulated by proteins named c-FLIPs¹². The c-FLIP proteins occur in three isoforms: c-FLIP_Long (c-FLIP_L), c-FLIP_Short (c-FLIP_S), and c-FLIP_Raji (c-FLIP_R). All three isoforms contain two DEDs in their N-terminal region. c-FLIP_L also has a C-terminal catalytically inactive caspase-like domain^12,13. Both short isoforms of c-FLIP-c-FLIP_S and c-FLIP_R-act in an anti-apoptotic manner by disrupting DED filament formation at the DISC^6,14,15. In addition, c-FLIP_L can regulate caspase-8 activation in a concentration-dependent manner. This can result in both pro- and anti-apoptotic effects^16,17,18. By forming the catalytically active procaspase-8/c-FLIP_L heterodimer, c-FLIP_L leads to the stabilization of the active center of procaspase-8 and its activation. The pro- or anti-apoptotic function of c-FLIP_L is directly dependent on its amount at the DED filaments and the subsequent amount of assembled procaspase-8/c-FLIP_L heterodimers¹⁹. Low or intermediate concentrations of c-FLIP_L at the DISC result in sufficient amounts of procaspase-8/c-FLIP_L heterodimers at the DED filament, which supports the activation of caspase-8. In contrast, increased amounts of c-FLIP_L directly lead to its anti-apoptotic effects at the DISC²⁰.

Taken together, the activation and processing of procaspase-8a/b at the DISC is a highly regulated process involving several steps. This paper discusses the measurement of procaspase-8 processing directly at the DISC as well as the analysis of the composition of this complex. This will be presented using CD95 DISC as the exemplary DR complex.

Protocol

​T cell experiments were performed according to the ethical agreement 42502-2-1273 Uni MD.

1. Preparing cells for the experiment

NOTE: The average number of cells for this immunoprecipitation is 1 × 10⁷_. Adherent cells have to be seeded one day before the experiment so that there are 1 × 10⁷ cells on the day of the experiment.

1. Preparing adherent cells for the experiment
   1. Seed 5-8 × 10⁶ adherent cells in 20 mL of medium (see the Table of Materials for the composition) for each condition in 14.5 cm dishes one day before the experiment starts.
   2. On the day of the experiment, ensure that the cells are 80-90% confluent and adherent to the dish. Discard the medium and add fresh medium to the adherent cells.
2. Preparing suspension cells for the experiment
   1. Carefully place 1 × 10⁷ suspension cells in 10 mL of culture medium (see the Table of Materials for the composition) per condition in 14.5 cm dishes immediately before the experiment starts.
   2. If using primary cells, isolate primary T cells according to the previously described procedure²¹. Treat primary T cells with 1 µg/mL phytohemagglutinin for 24 h, followed by 25 U/mL IL2 treatment for 6 days.
   3. Carefully place 1 × 10⁸ primary T cells in 10 mL of culture medium (see the Table of Materials for the composition) per condition in 14.5 cm dishes immediately before the experiment starts.
      ​NOTE: This higher number of primary T cells is recommended, as these cells are smaller.

2. CD95L stimulation

1. Stimulate the cells with CD95L (produced as described previously²⁰ or commercially available (see the Table of Materials)).
   ​NOTE: The concentration of the CD95L and the time of stimulation are cell-type dependent^{13,15,22,23,24,25}. Prepare one stimulation condition twice to generate a 'bead control' sample in parallel.
   1. Stimulate adherent cells with the selected concentration of CD95L. Hold the plate at an angle and pipet the ligand into the medium without touching the adherent cells.
   2. Stimulate suspension cells with CD95L by pipetting the ligand solution into the cell suspension.

3. Cell harvest and lysis

1. Place the cell dishes on ice.
   NOTE: Do not discard the medium. Dying cells float in the medium and are important for the analysis.
2. Add 10 mL of cold phosphate-buffered saline (PBS) to the cell suspension and scrape the attached cells off the plate. Collect the cell suspension in a 50 mL tube.
3. Wash the cell dish with 10 mL of cold PBS twice and place the wash solution into the same 50 mL tube. Centrifuge the cell suspension at 500 × g for 5 min, 4 °C.
4. Discard the supernatant and resuspend the cell pellet with 1 mL of cold PBS. Transfer the cell suspension into a 1.5 mL tube.
5. Centrifuge the cell suspension at 500 × g for 5 min, 4 °C. Discard the supernatant and resuspend the cell pellet with 1 mL of cold PBS.
6. Centrifuge the cell suspension at 500 × g for 5 min, 4 °C. Discard the supernatant and resuspend the cell pellet with 1 mL of lysis buffer (containing 4% protease inhibitor cocktail). Incubate it for 30 min on ice.
7. Centrifuge the lysate at maximal speed (~17,000 × g) for 15 min, 4 °C.
8. Transfer the supernatant (lysate) to a clean tube. Discard the pellet. Take 50 µL of the lysate in another tube. Analyze the protein concentration by Bradford assay and take the amount of lysate corresponding to 25 µg of protein in a vial. Add loading buffer (see the Table of Materials for the composition) to the vial. Store it at -20 °C as lysate control.

4. Immunoprecipitation (IP)

1. Add 2 µL of anti-APO-1 antibodies and 10 µL of protein A sepharose beads (prepared as recommended by the manufacturer) to the lysate. Add only 10 µL of the beads to a separate tube containing lysate (stimulated sample) to generate a 'bead control.'
   NOTE: Use pipet tips with wide orifices either by cutting the tips or buying special tips for IP while handling the protein A sepharose beads.
2. Incubate the mixture of lysate with antibodies/protein A sepharose beads with gentle mixing overnight at 4 °C. Centrifuge the lysates with antibodies/protein A sepharose beads at 500 × g for 4 min, 4 °C. Discard the supernatant, add 1 mL of cold PBS to the beads, and repeat this step at least three times.
3. Discard the supernatant. Aspirate the beads preferably with a 50 µL Hamilton syringe.

5. Western blot

1. Add 20 µL of 4x loading buffer (see the Table of Materials for the composition) to the beads and heat at 95 °C for 10 min. Heat the lysate controls at 95 °C for 5 min.
2. Load the lysates, IPs, and a protein standard onto a 12.5% sodium dodecyl sulfate (SDS) gel (see the Table of Materials for the gel preparation) and run with a constant voltage of 80 V.
3. Transfer the proteins from the SDS gel to a nitrocellulose membrane.
   NOTE: Here, the semi-dry technique, optimized for the proteins of interest, was used for the transfer over 12 min (25 V; 2.5 A= constant). Soak the nitrocellulose membrane and two mini-size transfer stacks in electrophoresis buffer (see the Table of Materials for the composition, prepare according to the manufacturer's instructions) for a few minutes before western blotting.
4. Place the blotted membrane in a box and block it for 1 h in blocking solution (0.1% Tween-20 in PBS (PBST) + 5% milk). Incubate the membrane with the blocking solution under gentle agitation.
5. Wash the membrane three times with PBST for 5 min each wash.

6. Western blot detection

1. Add the first primary antibody at the indicated dilution (see the Table of Materials) to the membrane and incubate it overnight at 4 °C with gentle agitation.
2. Wash the membrane three times with PBST for 5 min each wash.
3. Incubate the membrane with 20 mL of secondary antibody (diluted 1:10,000 in PBST + 5% milk) with gentle shaking for 1 h at room temperature.
4. Wash the membrane three times with PBST for 5 min each wash.
5. Discard PBST and add approximately 1 mL of horseradish peroxidase substrate to the membrane.
6. Detect the chemoluminescent signal (see the Table of Materials).
   NOTE: The exposure time and the number of captured images depend on the amount of protein in the cell and the specificity of the antibodies used. It must be established empirically for each antibody used for the detection.

Results

To analyze caspase-8 recruitment to the DISC and its processing at the CD95 DISC, this paper describes a classical workflow, which combines IP of the CD95 DISC with western blot analysis. This allows the detection of several key features of caspase-8 activation at the DISC: the assembly of the caspase-8-activating macromolecular platform, recruitment of procaspase-8 to the DISC, and the processing of this initiator caspase (Figure 1 and Figure 2). This workflow ...

Discussion

This approach was first described by Kischkel et al.²⁷ and has successfully been developed since then by several groups. Several important issues have to be considered for efficient DISC-immunoprecipitation and monitoring caspase-8 processing in this complex.

First, it is essential to follow all washing steps during immunoprecipitation. Especially important are the final washing steps of the sepharose beads and the drying of the sepharose beads. This must be done correc...

Materials

Name	Company	Catalog Number	Comments
12.5% SDS gel	self made		for two separating gels: 3.28 mL distilled H2O 2.5 mL Tris; pH 8.8; 1.5 M 4.06 mL acrylamide 100 µL 10% SDS 100 µL 10% APS 7.5 µL TEMED for two collecting gels: 3.1 mL distilled H2O 1.25 mL Tris; pH 6.8; 1.5 M 0.5 mL acrylamide 50 µL 10% SDS 25 µL 10% APS 7.5 µL TEMED
14.5 cm cell dishes	Greiner	639160
acrylamide	Carl Roth	A124.1
anti-actin Ab	Sigma Aldrich	A2103	dilution: 1:4000 in PBST + 1:100 NaN3
anti-APO-1 Ab	provided in these experiments by Prof. P. Krammer or can be purchased by Enzo	ALX-805-038-C100	used only for immunoprecipitation
anti-caspase-10 Ab	Biozol	MBL-M059-3	dilution: 1:1000 in PBST + 1:100 NaN3
anti-caspase-3 Ab	cell signaling	9662 S	dilution: 1:2000 in PBST + 1:100 NaN3
anti-caspase-8 Ab C15	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ALX-804-242-C100	dilution: 1:20 in PBST + 1:100 NaN3
anti-CD95 Ab	Santa Cruz	sc-715	dilution: 1:2000 in PBST + 1:100 NaN3
anti-c-FLIP NF6 Ab	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ALX-804-961-0100	dilution: 1:10 in PBST + 1:100 NaN3
anti-FADD 1C4 Ab	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ADI-AAM-212-E	dilution: 1:10 in PBST + 1:100 NaN3
anti-PARP Ab	cell signaling	9542	dilution: 1:1000 in PBST + 1:100 NaN3
APS	Carl Roth	9592.3
β-mercaptoethanol	Carl Roth	4227.2
Bradford solution Protein Assay Dye Reagent Concentrate 450ml	Bio Rad	500-0006	used according to manufacturer's instructions
CD95L	provided in these experiments by Prof. P. Krammer or can be purchased by ENZO	ALX-522-020-C005
chemoluminescence detector Chem Doc XRS+	Bio Rad
cOmplete Protese Inhibitor Cocktail (PIC)	Sigma Aldrich	11 836 145 001	prepared according to manufacturer's instructions
DPBS (10x) w/o Ca, Mg	PAN Biotech	P04-53500	dilution 1:10 with H2O, storage in the fridge
eletrophoresis buffer	self made		10x electrophoresis buffer: 60.6 g Tris 288 g glycine 20 g SDS ad 2 L H2O 1:10 dilution before usage
glycine	Carl Roth	3908.3
Goat Anti-Mouse IgG1 HRP	SouthernBiotech	1070-05	dilution 1:10.000 in PBST + 5% milk
Goat Anti-Mouse IgG2b	SouthernBiotech	1090-05	dilution 1:10.000 in PBST + 5% milk
Goat Anti-Rabbit IgG-HRP	SouthernBiotech	4030-05	dilution 1:10.000 in PBST + 5% milk
Interleukin-2 Human(hIL-2)	Merckgroup/ Roche	11011456001	for activation of T cells
KCl	Carl Roth	6781.2
KH2PO4	Carl Roth	3904.1
loading buffer 4x Laemmli Sample Buffer,10 mL	Bio Rad	161-0747	prepared according to manufacturer's instructions
Luminata Forte Western HRP substrate	Millipore	WBLUFO500
lysis buffer	self made		13.3 mL Tris-HCl; pH 7.4; 1.5 M 27.5 mL NaCl; 5 M 10 mL EDTA; 2 mM 100 mL Triton X-100 add 960 mL H2O
medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine,  2,438 g/L	PAN Biotech	P04-41154	adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium
medium for primary T cells	gibco by Life Technologie	21875034	adding 10% FCS and 1% Penicillin-Streptomycin to the medium
milk powder	Carl Roth	T145.4
Na2HPO4	Carl Roth	P030.3
NaCl	Carl Roth	3957.2
PBST	self made		20x PBST: 230 g NaCl 8 g KCl 56.8 g Na2HPO4 8 g KH2PO4 20 mL Tween-20 ad 2 L H2O dilution 1:20 before usage
PBST + 5% milk	self made		50 g milk powder + 1 L PBST
PHA	Thermo Fisher Scientific	R30852801	for actavation of T ells
Power Pac HC	Bio Rad
Precision Plus Protein Standard All Blue	Bio Rad	161-0373	use between 3-5 µL
Protein A Sepharose CL-4B beads	Novodirect/ Th.Geyer	GE 17-0780-01	affinity resin beads prepared according to manufacturer's instructions
scraper	VWR	734-2602
SDS	Carl Roth	4360.2
shaker	Heidolph
sodium azide	Carl Roth	K305.1
TEMED	Carl Roth	2367.3
Trans Blot Turbo mini-size transfer stacks	Bio Rad	170-4270	used according to manufacturer's instructions
TransBlot Turbo 5x Transfer Buffer	Bio Rad	10026938	prepared according to manufacturer's instructions
TransBlot Turbo Mini-size nictrocellulose membrane	Bio Rad	170-4270	used according to manufacturer's instructions
Trans-Blot-Turbo	Bio Rad
Tris	Chem Solute	8,08,51,000
Triton X-100	Carl Roth	3051.4
Tween-20	Pan Reac Appli Chem	A4974,1000