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Analysis of Caspase Activation During Apoptosis

Scott H. Kaufmann1,  Timothy J. Kottke1,  L. Miguel Martins2,  Alexander J. Henzing3,  William C. Earnshaw3

1Mayo Clinic, Rochester, Minnesota
2Imperial Cancer Research Fund, London, United Kingdom
3University of Edinburgh, Edinburgh, Scotland, United Kingdom



Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 18.2
DOI: 
10.1002/0471143030.cb1802s11
Online Posting Date: 
August, 2001
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Abstract

This unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low-molecular-weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole-cell lysates or subcellular fractions.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Enzymatic Assays for Caspase Activity
  • Basic Protocol 2: Detection of Caspase Activation by Immunoblotting
  • Alternate Protocol 1: Cell Lysis with Guanidine Hydrochloride for Immunoblotting
  • Support Protocol 1: Removing (Stripping) Primary and Secondary Antibodies from Blots
  • Basic Protocol 3: Labeling and Detecting Active Caspases Using Biotinylated Substrate Analogs
  • Alternate Protocol 2: In Vitro Activation of Caspases in Naive Lysates Followed by Affinity Labeling
  • Support Protocol 2: Controls for Specificity of Affinity-Labeled Active Caspases
  • Support Protocol 3: Stripping Membrane in the Presence of d-Biotin for Reprobing with Antibody
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enzymatic Assays for Caspase Activity

 Materials
  • Cells of interest and appropriate medium
  • Apoptosis-inducing stimulus
  • CMF-DPBS (appendix 2A), ice cold
  • Lysis buffer (see recipe), 4°C
  • 0.5 M EDTA, pH 7.4 (see recipe)
  • 1 M dithiothreitol (DTT; appendix 2A)
  • 5 mM EDTA (pH 7.4)/1 mM DTT in lysis buffer, ice cold
  • 20 mM (14.6 mg/ml) acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (acetyl-DEVD-AFC; Biomol), or other fluorogenic or chromogenic caspase substrate, in dimethyl sulfoxide (DMSO)
  • HEPES/CHAPS buffer (see recipe), room temperature and ice cold
  • 10 mM (4.6 mg/ml) 7-amino-4-trifluoromethylcoumarin (free AFC; Sigma), or appropriate standard for other substrate, in DMSO
  • Cell scraper (optional)
  • 2-ml (total volume) tight-fitting Dounce homogenizers
  • Beckman TL100 ultracentrifuge and TL100.2 rotor, or equivalent, 4°C, and appropriate ultracentrifuge tubes
  • Fluorometer
  • Additional reagents and equipment for Ficoll-Hypaque density sedimentation (unit 2.2), cell trypsinization (for adherent cell lines only; unit 1.1), trypan blue staining to detect lysed cells (unit 1.1), and determination of protein concentration (appendix 3B)

NOTE: Fluorogenic and chromogenic caspase substrates are available from a number of suppliers, including Bachem Bioscience, Biomol Research Laboratories, Calbiochem-Novabiochem, Molecular Probes, and Osaka Peptide Institute. Stock solutions for substrates and standards are prepared as 20 mM solutions in DMSO and stored in aliquots for up to 1 year at –20°C.

Basic Protocol 2: Detection of Caspase Activation by Immunoblotting

 Materials
  • CMF-DPBS (appendix 2A), ice cold
  • Serum-free tissue culture medium (appropriate for cells of interest), ice cold, optional
  • SDS sample buffer (see recipe)
  • Fast green dye solution: 0.1% (w/v) fast green FCF/20% (v/v) methanol/5% (v/v) acetic acid
  • Fast green destain: 20% (v/v) methanol/5% (v/v) acetic acid
  • Blocking buffer (see recipe)
  • Anti-caspase primary antibody (Table 18.2.1)
  • PBS-T (see recipe)
  • Appropriate secondary antibody conjugated to horseradish peroxidase (HRP), alkaline phosphatase (AP), or a radiolabel
  • 3% (w/v) nonfat dry milk in CMF-DPBS
  • Enhanced chemiluminescence reagents (e.g., ECL from Amersham Pharmacia Biotech), for HRP-conjugated secondary antibodies
  • X-ray film
  • Cell scraper (optional)
  • Sonicator equipped with microprobe (e.g., Branson)
  • 70° or 100°C water bath or heating block
  • Additional reagents and equipment for inducing apoptosis (see Basic Protocol 1), trypsinizing and counting cells (optional; unit 1.1), SDS-polyacrylamide gel electrophoresis (unit 6.1), and electrophoretic transfer of polypeptides to a solid support (unit 6.2)
     
    Table 18.2.1 Selected Properties of Human Caspasesa
    New nameOld name(s)Molecular weight (kDa)bPreferred small substratescAntibody suppliersd
    PrLgSm
    Caspase-1ICE452414YEVD/XWEHD/XBP, ORP, UB
    2010
    Caspase-2Ich-1, NEDD2L483214VDVAD/XDEHD/XBP, BTL, ORP, UB
    1812
    Caspase-3CPP32, YAMA, Apopain322012DMQD/XDEVD/XBP, BTL, CI, ORP, UB
    17
    Caspase-4Tx, Ich-2, ICErelIILEVD/X(W/L)EHD/XBP, ORP
    Caspase-5ICErelIII, TyUnknown(W/L)EHD/XCN
    Caspase-6Mch2342113VEID/XVEHD/XBP, CI, CN, NEB, UB
    1811
    Caspase-7Mch3, CMH-1, ICE-LAP3342012DEVD/XDEVD/XBP, BTL, NEB, ORP
    Caspase-8Mch5, FLICE,MACH534312IETD/XLETD/XBP, CI, CN, NEB, ORP
    551811
    Caspase-9ICE-LAP6, Mch6503712UnknownLEDH/XBP, CI, CN, NEB, UB
    Caspase-10Mch4, FLICE-2554312IEAD/XUnknownCI, CN, NEB, UB
    17
    Caspase-13ERICEUnknownUnknown
    Caspase-14MICE291810UnknownUnknownBTL, ORP
     a Modified from Earnshaw et al. (1999).
     b The appearance of multiple entries indicates partially processed and fully processed large (Lg) and small (Sm) subunits that result from sequential cleavage at the C-terminal end of the large subunit followed by removal of the linker peptide from the small subunit and the prodomain (Pr) from the large subunit. A blank in this column indicates that the molecular weight of the processed forms has not been reported.
     c The left and right columns indicate the preferred low-molecular-weight substrate specificity reported by two groups: Talanian et al. (1997) and Thornberry et al. (1997), respectively. It is important to note, however, that additional factors also affect caspase cleavage of full-length polypeptides. Not all sites conforming to the indicated sequences are cleaved, perhaps due to limited accessibility. Conversely, polypeptides are sometimes cleaved at sites that would not be predicted based on analysis of the tetrapeptide preferences indicated in this table (Samejima et al., 1999).
     d Abbreviations: BP, BD PharMingen; BTL, BD Transduction Laboratories; CI, Chemicon International; CN, Calbiochem-Novabiochem; NEB, New England Biolabs; ORP, Oncogene Research Products; UB, Upstate Biotechnology.

Alternate Protocol 1: Cell Lysis with Guanidine Hydrochloride for Immunoblotting

 Additional Materials (also see Basic Protocol 2)
  • Guanidine hydrochloride lysis buffer (see recipe)
  • 100 mM PMSF (appendix 2A)
  • 2-Mercaptoethanol (2-ME)
  • 1.54 M (285 mg/ml) iodoacetamide in guanidine hydrochloride lysis buffer, prepared fresh
  • 4 M urea (see recipe)/50 mM Tris×Cl, pH 7.4 at 4°C (appendix 2A)
  • 0.1% (w/v) SDS
  • 1-cm dialysis tubing (MWCO 8000 to 10,000), double knotted at one end, and dialysis clips
  • Additional reagents and equipment for determining protein concentration (appendix 3B)

CAUTION: 2-ME has a strong odor and its use is confined to the hood in some laboratories .

NOTE: All steps involving iodoacetamide should be performed under subdued light because of the light sensitivity of the carbon-iodine bond.


Support Protocol 1: Removing (Stripping) Primary and Secondary Antibodies from Blots

 Materials
  • Nitrocellulose or PVDF membrane with bound antibodies (see Basic Protocol 2 or Alternate Protocol 1)
  • Blot erasure buffer (see recipe)
  • CMF-DPBS (appendix 2A)
  • Resealable plastic bags
  • 65°C water bath

Basic Protocol 3: Labeling and Detecting Active Caspases Using Biotinylated Substrate Analogs

 Materials
  • Incomplete KPM buffer (see recipe), 4°C
  • Complete KPM buffer (see recipe), 4°C
  • 100 µM N-(N-benzyloxycarbonylglutamyl-N-biotinyllysyl)aspartic acid ([2,6-dimethylbenzoyl]oxy)methylketone [zEK(bio)D-aomk; Osaka Peptide Institute] in dimethyl sulfoxide (DMSO), stored in aliquots up to 2 years at –80°C
  • 3× SDS sample buffer (see recipe)
  • 5% (w/v) nonfat dry milk in PBS-T
  • PBS-T (see recipe)
  • Peroxidase-coupled streptavidin (e.g., Amersham Pharmacia Biotech)
  • Enhanced chemiluminescence reagents (e.g., ECL; Amersham Pharmacia Biotech)
  • Cell scraper (optional)
  • 8 × 34–mm polycarbonate ultracentrifuge tubes (e.g., Beckman)
  • Beckman Optima TLX tabletop ultracentrifuge and TL100.1 rotor, or equivalent, 4°C
  • Additional reagents and equipment for inducing apoptosis (see Basic Protocol 1), cell trypsinization (optional; unit 1.1), protein determination (appendix 3B), SDS-polyacrylamide gel electrophoresis (unit 6.1), and electrophoretic transfer of polypeptides to a solid support (unit 6.2)

Alternate Protocol 2: In Vitro Activation of Caspases in Naive Lysates Followed by Affinity Labeling

 Additional Materials (also see Basic Protocol 3)
  • Incomplete KHM buffer (see recipe), 4°C
  • Complete KHM buffer (see recipe), 4°C
  • 5 µg/ml active caspase-8 (BD PharMingen) in complete KHM buffer or 5 mg/ml cytochrome c (Sigma) in complete KHM buffer
  • 10 mM dATP (Sigma) in complete KHM, pH 7.4, optional
  • 100 µM N-(N-benzyloxycarbonylglutamyl-N-biotinyllysyl)aspartic acid ([2,6-dimethylbenzoyl]oxy)methylketone [zEK(bio)D-aomk; Osaka Peptide Institute] in dimethyl sulfoxide (DMSO), stored in aliquots up to 2 years at –80°C

Support Protocol 3: Stripping Membrane in the Presence of d-Biotin for Reprobing with Antibody

 Additional Materials (also see Basic Protocol 3)
  • Membrane containing affinity-labeled caspases (see Basic Protocol 3)
  • 2 mM d-biotin (Sigma) in PBS-T
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Figures

  • Figure 18.2.1
    Measurement of caspase activity using a fluorogenic substrate. (A) Conversion of the substrate acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (acetyl-DEVD-AFC) to tetrapeptide and free 7-amino-4-trifluoromethylcoumarin (AFC). (B) Velocity versus substrate concentration curves for caspase-3. Purified recombinant human caspase-3 was incubated for 2 hr with increasing concentrations of acetyl-DEVD-AFC or acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin (acetyl-LEHD-AFC). From these data it is possible to determine that the maximal velocity is 5-fold lower for acetyl-LEHD-AFC than for acetyl-DEVD-AFC, whereas the concentration that results in half-maximal velocity (KM) is the same (~12.5 µM) for each substrate.

    View Image
  • Figure 18.2.2
    Assessment of caspase activation by immunoblotting. (A) Caspase activation. The prodomain, large subunit, and small subunit are indicated. Two types of epitopes, a conventional linear epitope in the large subunit and a neoepitope at the C terminus of the large subunit, are indicated. The lower box indicates the peptide sequences of procaspase-9 and procaspase-8 at the junction between the large and small subunits. When cleavage occurs on the C-terminal side of aspartate, new C-terminal epitopes (neoepitopes) are formed. (B) Results obtained when HL-60 human leukemia cells are treated with 68 µM etoposide, a topoisomerase II–directed agent that causes DNA damage, for 0, 1, 1.5, 2, 2.5, 3, and 4 or 6 hr. Adjacent lanes were loaded with 50 µg of total cellular protein harvested according to the guanidine hydrochloride cell lysis procedure (see Alternate Protocol 1). Blots were probed with monoclonal anti–poly(ADP-ribose) polymerase (PARP), a sensitive marker of caspase activation (Lazebnik et al., 1994); reagents that detect procaspases-2, -8, and -9; or anti-neoepitope sera that react with active caspase-9 (Mesner et al., 1999) or active caspase-8 species. Two major splice variants of procaspase-8 are detected in these cells. The arrow indicates the product resulting from PARP cleavage by caspases. The anti-neoepitope antisera were raised by immunizing rabbits with the neoepitope tetrapeptides indicated in (A). Note that the increased signal for active caspase-9 is at least as readily detected with the anti-caspase-9 neoepitope serum as it is with the anti-procaspase-9 serum. Also note that the signal for procaspase-2 does not change during the course of apoptosis in this cell line, illustrating the selective cleavage of some procaspases and not others during the course of apoptosis in situ(Martins et al., 1997).

    View Image
  • Figure 18.2.3
    Labeling of active caspases by acyloxymethylketones. (A) The reaction mechanism of zEK(bio)D-aomk (Martins et al., 1997). The active site cysteine initially forms a reversible thiohemiketal. In a slower reaction, the methyl group of the substituted methylketone undergoes SN2 attack by the same active site thiolate to form the thioether (alkylated enzyme) and liberate the leaving group 2,6-dimethylbenzoic acid. (B) Results obtained when N-(N-benzyloxycarbonylglutamyl-N-biotinyllysyl)aspartic acid ([2,6-dimethylbenzoyl]oxy)methylketone (zEK[bio]D-aomk) is used to detect caspase activation. Cytosol from 1 × 106 cells (40 µg) was incubated for 1 hr at 37°C with diluent (lane 1), 100 ng cytochrome c (lane 2), or 50 ng active caspase-8 (lane 3). Samples were then covalently modified with zEK(bio)D-aomk as described in Basic Protocol 3. Lanes 4 and 5: 50 ng of active caspase-8 without cell lysate. Samples in lanes 1 to 4 were visualized using peroxidase-coupled streptavidin and enhanced chemiluminescence reagents. Lane 5 was probed with anti-caspase-8 antiserum followed by peroxidase-coupled secondary antibody. Collectively, these results demonstrate that endogenous biotinylated species are visualized by this technique (lane 1), that multiple zEK(bio)D-aomk-modified bands between 15 and 50 kDa are detected after caspase activation (lanes 2 and 3), and that active caspase-8 does not label with this reagent (lanes 4 and 5). Numbers at left, molecular weights of marker proteins in kilodaltons.

    View Image

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    Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T., and Nicholson, D.W. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem 72:17907-17911.
    Wilson, K.P., Black, J.A., Thomson, J.A., Kim, E.E., Griffith, J.P., Navia, M.A., Murcko, M.A., Chambers, S.P., Aldape, R.A., Raybuck, S.A.,et al. 1994. Structure and mechanism of interleukin-1 converting enzyme. Nature 370:270-275.
    Zapata, J.M., Takahashi, R., Salvesen, G.S., and Reed, J.C. 1998. Granzyme release and caspase activation in activated human T-lymphocytes. J. Biol. Chem 273:6916-6920.
    Zhou, Q. and Salvesen, G.S. 1997. Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity. Biochem. J. 324:361-364.
 Key References
    Enari, M., Talanian, R.V., Wong, W.W., and Nagata, S. 1996. Sequential activation of ICE-like and CPP32-like proteases during fas-mediated apoptosis. Nature 380:723-726.

This paper illustrates the use of different tetrapeptide substrates to demonstrate the sequential activation of multiple caspases during apoptosis.

    Schlegel, J., Peters, I., Orrenius, S., Miller, D.K., Thornberry, N.A., Yamin, T.T., and Nicholson, D.W. 1996. CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in fas-mediated apoptosis. J. Biol. Chem. 271:1841-1844.

This paper illustrates one of the first uses of immunoblotting to demonstrate caspase activation during apoptosis.

    Takahashi et al., 1996b. See above.

This paper describes the first use of affinity labeling to detect active caspases in extracts from apoptotic cells.

 Internet Resources
    www.peptide.co.jp

The Web site of the Osaka Peptide Institute contains a variety of caspase substrates as well as acycloxymethyl ketones that can be used as affinity labels.

     
 
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