Keywords

1 Introduction

Caspases in animals are cysteine proteases that cleave their substrates after the P1 position in aspartic acid residues [1, 2]. The amino acid preference at the P2–P4 positions differs among caspase family members, including the peptide sequence of YVAD for caspase-1, DEVD for caspase-3, VKMD and VEID for caspase-6, and IETD for caspase-8. In plants, over the past decades, several groups have identified proteinases that exhibit caspase-like activities (defined as cleavage of the caspase-specific substrates) and are involved in PCD [3,4,5,6,7,8,9,10]. For example, vacuolar processing enzyme (VPE) exhibits a caspase-1-like activity (YVADase activity) and is related to plant developmental and stress-induced PCD [11, 12]. The subtilisin-like serine proteases, saspase and phytaspase, have caspase-6-like activities (VKMDase and VEIDase activities) and a caspase-8-like activity (IETDase activity), and they have been shown to be associated with pathogen-induced PCD in Avena sativa [13, 14]. The 26S proteasome subunit PBA1 has a caspase-3-like activity (DEVDase activity) and mediates PCD that is induced by bacterial pathogens and that occurs during xylem development [15, 16]. Additionally, the gene silencing of a metacaspase, which is distantly related to caspases and contains the caspase-conserved domains, reduces a caspase-6-like activity (VEIDase activity) and suppresses PCD in the embryo-suspensor of Norway spruce [17]. However, metacaspases lack aspartic acid specificity [18, 19], suggesting that they trigger the activation of caspase-like proteinase, either directly or indirectly, through their proteolysis.

The similarities in the substrate specificities of VPE and caspase-1 are consistent with several structural similarities, including those of the substrate pockets and active sites, between the two enzymes [20,21,22,23]. The predicted three-dimensional structures of the substrate pocket of human caspase-1 and the corresponding residues of Arabidopsis γVPE reveal an interesting enzymatic feature [11]. Two guanido groups in Arg341 and Arg179 of caspase-1 have strong affinities to the carboxylate group of the Asp residue in the substrate peptide YVAD [2]. However, γVPE has only one guanido group in Arg74, which makes the substrate pocket of γVPE less positively charged than that of caspase-1 [11]. This is consistent with γVPE having a broad substrate specificity toward Asp and Asn, while caspase-1 has a narrow substrate specificity toward Asp [12, 20, 21, 24, 25].

Since we reported that VPE has a caspase-1-like activity [26,27,28], this activity has been widely measured using specific synthetic substrates. However, some of these were measured at a neutral pH, which is used for animal caspase-1. While caspase-1 is a cytosolic enzyme, VPE is a vacuolar enzyme. VPE, with an acidic optimum pH, exhibits little activity at a neutral pH [29]. Because VPE is a proteinase that exhibits caspase-1-like activity, this activity should be measured at an acidic pH. The activities that were measured at neutral pH do not reflect the full activities in the plant cells. In addition to the method using specific synthetic substrates, this chapter provides details of another method to measure caspase-1-like/VPE activity by monitoring active forms of VPE using a biotinylated-inhibitor blot analysis . The proprotein precursor of VPE is self-catalytically converted into an intermediate form and then into the mature form at an acidic pH [29, 30]. Both intermediate and mature forms (active forms) can bind to the caspase-1 inhibitors, whereas the precursor form (inactive form) does not bind [27]. Thus, the levels of the active forms of VPE that bind to the biotinylated caspase-1 inhibitor reflect the level of caspase-1-like/VPE activity in the plant cells.

2 Materials

Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ/cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature unless indicated otherwise. Diligently follow all waste disposal regulations.

2.1 Caspase-1-Like and VPE Activity Assay Using Synthetic Peptide Substrates

  1. 1.

    Homogenizing buffer for plant leaves: 50 mM Na-acetate, pH 5.5, 50 mM NaCl, and 1 mM EDTA at 4 °C.

  2. 2.

    Bio-Rad Protein Assay Kit (Bio-Rad).

  3. 3.

    Enzyme assay buffer: 20 mM Na-acetate, pH 5.5, 0.1 mM EDTA, and 0.1 M dithiothreitol.

  4. 4.

    Stock solution for caspase-1 and VPE substrates: 20 mM fluorogenic peptide substrates (Peptide Institute) in dimethyl sulfoxide (DMSO). Acetyl-Tyr-Val-Ala-Asp-(4-methylcoumaryl-7-amide) (Ac-YVAD-MCA) for caspase-1, and acetyl-Glu-Ser-Glu-Asn-(4-methylcoumaryl-7-amide) (Ac-ESEN-MCA) and benzyloxycarbonyl-Ala-Ala-Asn-(4-methylcoumaryl-7-amide) (Z-AAN-MCA) for VPE.

  5. 5.

    Stock solution for caspase-1 and VPE inhibitors: 20 mM peptide inhibitors in DMSO. Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO) (Peptide Institute) and benzyloxycarbonyl-Tyr-Val-Ala-Asp-fluoromethylketone (Z-YVAD-fmk) (Calbiochem) for caspase-1, and acetyl-Glu-Ser-Glu-Asn-CHO (Ac-ESEN-CHO) (Peptide Institute) for VPE.

  6. 6.

    A 1-cm-diameter cork borer.

  7. 7.

    TissueLyser and 3-mm tungsten carbide beads (Qiagen).

  8. 8.

    A 96-well opaque (white), flat-bottom microplate (Corning) (see Note 1 ).

  9. 9.

    Fluorescence spectrophotometer (microplate reader, GENios; TECAN).

2.2 Caspase-1-Like and VPE Activity Assay by Biotinylated-Inhibitor Blot Analysis

2.2.1 Preparation of Plant Extract and Reaction Solution

  1. 1.

    Homogenizing buffer for plant leaves: 50 mM Na-acetate, pH 5.5, 50 mM NaCl and 1 mM EDTA at 4 °C.

  2. 2.

    Bio-Rad Protein Assay Kit (Bio-Rad).

  3. 3.

    TissueLyser and 3-mm tungsten carbide beads (Qiagen).

  4. 4.

    5× Enzyme assay buffer: 100 mM Na-acetate, pH 5.5, 0.5 mM EDTA and 0.5 M dithiothreitol.

  5. 5.

    Stock solution for caspase-1 and VPE inhibitors: 20 mM peptide inhibitors (Peptide Institute) in DMSO. Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO) for caspase-1 and acetyl-Glu-Ser-Glu-Asn-CHO (Ac-ESEN-CHO) for VPE.

  6. 6.

    Stock solution for biotinylated-caspase-1 inhibitor: 100 mM biotin-Tyr-Val-Ala-Asp-fluoromethylketone (biotin-YVAD-fmk) (Peptide Institute) in DMSO.

2.2.2 Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)

  1. 1.

    SDS-PAGE apparatus.

  2. 2.

    Power supply.

  3. 3.

    1.5 M Tris–HCl, pH 8.8: 18.17 g tris-aminomethane (Tris) in 80 mL of water. Mix and adjust the pH to 8.8 with HCl. Bring the total volume to 100 mL by the addition of water.

  4. 4.

    0.5 M Tris–HCl, pH 6.8: 6.06 g Tris in 100 mL of water. Prepare solution as in Subheading 2.2.2, item 3.

  5. 5.

    A 40% (w/v) acrylamide–bis-acrylamide mixed solution (29:1) (Bio-Rad).

  6. 6.

    Ammonium persulfate (APS): 10% APS solution in water. Store at −20 °C.

  7. 7.

    N,N,N′,N′-Tetramethyl ethylenediamine (TEMED): Store at 4 °C.

  8. 8.

    5× SDS sample buffer: Mix 2.5 mL of 1 M Tris–HCl, pH 6.8, 1 g of SDS, 2.5 mL of β-mercaptoethanol, 5 mL of glycerol and a few mg of bromophenol blue (BPB) to turn the solution blue.

  9. 9.

    SDS–PAGE running buffer: 0.025 M Tris, 0.192 M glycine and 0.1% SDS. Prepare 10× buffer. Weigh 30.3 g Tris and 144 g glycine, and dissolve in 800 mL of water. Add the detergent (10 g SDS) last, and minimize vigorous mixing to avoid the production of foam. After completely dissolving all of the chemicals, bring the final volume up to 1 L.

  10. 10.

    Molecular weight markers: A prestained molecular weight marker is a convenient guide for cutting the gel. A prestained ladder of low range markers (15–85 kDa) is recommended. Markers that react with peroxidase are convenient.

2.2.3 Biotinylated-Inhibitor Blot Analysis

  1. 1.

    Semidry protein blotter apparatus.

  2. 2.

    Power supply.

  3. 3.

    PVDF membrane: Immobilon-P (Millipore).

  4. 4.

    Filter paper, cut to the size of the gel.

  5. 5.

    Blotting buffer: 0.048 M Tris, 0.039 M glycine, and 20% methanol.

  6. 6.

    Tris-buffered saline (TBS, 10×): 0.2 M Tris–HCl, pH 7.5, and 1.5 M NaCl.

  7. 7.

    TBS containing 0.05% Tween 20 (TBST).

  8. 8.

    Blocking solution: 5% skim milk in TBST (see Note 2 ).

  9. 9.

    Plastic containers or stainless-steel trays.

  10. 10.

    Streptavidin-conjugated horseradish peroxidase (Streptavidin-HRP) (GE Healthcare).

  11. 11.

    Western blot detection kit: ECL Western Blotting Detection Reagents (GE Healthcare).

  12. 12.

    A system for the development and fixation of X-ray films, or a lumino-image analyzer.

3 Methods

Carry out all procedures on ice or 4 °C unless indicated otherwise.

3.1 Preparation of Plant Extract

  1. 1.

    A plant leaf disk punched with a 1-cm-diameter cork borer was homogenized using TissueLyser and 3-mm tungsten carbide beads in 100 μL of homogenizing buffer (see Note 3 ).

  2. 2.

    Centrifuge the extract at 15,000 × g for 20 min to obtain the supernatant.

3.2 Protein Quantification Assay

  1. 1.

    Prepare five standards with bovine gamma globulin concentrations ranging from 0 to 22.4 μg/mL.

  2. 2.

    Dilute samples into at least two dilutions (such as 1:2, 1:5, 1:10, and 1:20).

  3. 3.

    Prepare bicinchoninic acid (BCA) reagent: Add BCA reagent A to reagent B in a 50 to 1 ratio.

  4. 4.

    Add 200 μL of the BCA reagent mixture to each well of the 96-well microplate.

  5. 5.

    Add 10 μL of diluted standards and diluted samples.

  6. 6.

    Cover and incubate the plate at 37 °C for 20 min.

  7. 7.

    Read the absorbance at 562 nm using a spectrophotometer plate reader at room temperature.

  8. 8.

    Using the standard curve constructed with the standards and blank, determine the protein concentrations of the plant extracts.

3.3 Caspase-1-Like and VPE Activity Assay Using Synthetic Peptide Substrates

3.3.1 Enzyme Activity

  1. 1.

    Transfer 65 μL of plant extract (Subheading 3.1) to each well of a 96-well microplate.

  2. 2.

    Add 20 μL of enzyme assay buffer to each well.

  3. 3.

    Incubate the reaction mixture in the microplate at 30 °C for 30 min.

  4. 4.

    Add 10 μL of 1 mM fluorogenic substrate to each well (final concentration of 100 μM) and mix well by quickly pipetting up and down three to five times with a multichannel pipette.

  5. 5.

    Read immediately at 30 °C in kinetic/continuous mode with intervals as short as possible (EXλ: 405 nm, EMλ: 510 nm).

  6. 6.

    The presence of 7-amino-4-methyl coumarin (AMC) was determined at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Based on the standard curve of AMC and the results of protein quantification (Subheading 3.2), caspase-1-like and VPE activities were calculated as μmol/L AMC per mg protein.

3.3.2 Inhibition of Enzyme Activity

  1. 1.

    Transfer 60 μL of plant extract to each well of a 96-well microplate.

  2. 2.

    Add 20 μL of enzyme assay buffer to each well.

  3. 3.

    Add 5 μL of diluted inhibitors to each well (final concentration of 0–200 μM).

  4. 4.

    Incubate the reaction mixture in microplate at 30 °C for 30 min.

  5. 5.

    Follow steps 4–6 above (Subheading 3.3.1).

3.4 Caspase-1-Like and VPE Activity Assay Monitoring Active Forms of VPE (Biotinylated-Inhibitor Blot Analysis)

3.4.1 Reaction with Biotinylated-Caspase-1 Inhibitor

  1. 1.

    Transfer 16 μL of plant extract (Subheading 3.1) to 1.5-mL sample tube.

  2. 2.

    Add 4 μL of 5× Enzyme assay buffer.

  3. 3.

    Add 5 μL of 100 μM biotin-YVAD-fmk (final concentration of 20 μM).

  4. 4.

    Incubate the reaction mixture at 30 °C for 1 h.

  5. 5.

    Add 5 μL of 5× SDS sample buffer.

  6. 6.

    Heat at 95 °C for 5 min. The samples can be stored at −20 °C or immediately used in the subsequent steps.

3.4.2 12.5% SDS-Page

  1. 1.

    Prepare a separating gel solution: Mix 2.5 mL of 1.5 M Tris–HCl, pH 8.8, 4.16 mL of 30% acrylamide solution, 0.1 mL of 10% SDS, 1.14 mL of water, 2.0 mL of 50% glycerol and 0.1 mL of APS. If necessary, leave the mixture on ice (see Note 4 ).

  2. 2.

    Prepare a stacking gel solution. Mix 0.63 mL of 0.5 M Tris–HCL, pH 6.8, 0.83 mL of 30% acrylamide solution, 0.05 mL of 10% SDS, 3.44 mL of water, and 0.05 mL of APS. If necessary, leave the mixture on ice (see Note 4 ).

  3. 3.

    Cast a mini gel cassette and mark the position 1 cm below the bottom of the gel comb.

  4. 4.

    Add 4 μL of TEMED to the separating gel solution, immediately mix well, and pour it into a mini wide gel cassette. Without a pause, add 5 μL of TEMED to the stacking gel solution, immediately mix well, and gently overlay it on the separating gel. Insert a gel comb immediately without introducing air bubbles and let the gel polymerize for 1 h.

  5. 5.

    Load 15 μL of each sample to the wells of the 12.5% SDS–PAGE gel. Also load 5 μL of the molecular weight marker ladder to one lane on each side of the gel (two lanes of molecular weight markers per gel). Perform electrophoresis at 25 mA until the dye front reaches the bottom of the gel.

  6. 6.

    Cut a PVDF membrane and a filter paper to the size of the gel. Six filter papers are needed. Immerse the cut PVDF membrane in methanol for 20 s. Transfer the membrane and the filter papers to each container with blotting buffer.

  7. 7.

    Following electrophoresis, pry the gel plates open using a spatula. The gel remains on one of the glass plates. Remove the gel wells and measure the size of the gel. Transfer carefully to a container of blotting buffer.

3.4.3 Biotinylated-Inhibitor Blot Analysis

  1. 1.

    Place three filter papers on the anode of a semidry protein blotter. Layer the membrane, the gel, and the remaining three filter papers on top without introducing air bubbles (see Note 5 ).

  2. 2.

    Place the cathode on the blotter and run at the appropriate current for 90 min (see Note 6 ).

  3. 3.

    Wash the blotted membrane briefly in TBST.

  4. 4.

    Cover the blotted membrane with blocking solution and shake at room temperature for 1 h on a platform rotary shaker.

  5. 5.

    Wash the blotted membrane with TBST twice for 5 min each.

  6. 6.

    Incubate the blotted membrane with streptavidin-HRP at room temperature for 1 h. Streptavidin-HRP is diluted to 1:3000 with TBST.

  7. 7.

    Wash the blotted membrane with TBST three times for 10 min each.

  8. 8.

    Detect the streptavidin-HRP signals with the detection kit. Signals are directly detected by the lumino-image analyzer, or detected as sensitized spots on an X-ray film that was exposed to the PVDF membrane.

3.4.4 Competitive Inhibition Assay

  1. 1.

    Transfer 15 μL of plant extract (Subheading 3.1) to a 1.5-mL sample tube.

  2. 2.

    Add 4 μL of 5× Enzyme assay buffer.

  3. 3.

    Add 1 μL of diluted inhibitors (final concentration of 1 mM) or DMSO as a control.

  4. 4.

    Incubate the reaction mixture at 30 °C for 1 h.

  5. 5.

    Add 5 μL of 100 μM biotin-YVAD-fmk (final concentration of 20 μM).

  6. 6.

    Incubate the reaction mixture at 30 °C for 30 min.

  7. 7.

    Add 5 μL of 5× SDS sample buffer.

  8. 8.

    Heat at 95 °C for 5 min. The samples can be stored at −20 °C or immediately used for SDS–PAGE and biotinylated-inhibitor blot analysis (Subheadings 3.4.2 and 3.4.3).

4 Notes

  1. 1.

    Opaque black plates may also be used, but the fluorescence yields will be much lower.

  2. 2.

    It is best to prepare this solution fresh.

  3. 3.

    Plant tissues are stored and kept in a −80 °C freezer until use.

  4. 4.

    Polymerization starts with the addition of TEMED. Because the speed of polymerization depends on the temperature, you may cool the mixture to slow the polymerization.

  5. 5.

    Proteins denatured by SDS are negatively charged and move from the cathode to the anode. We use a semidry blotter equipped with the anode in the body and the cathode as the cover.

  6. 6.

    The gel size (cm2) is used as the value of the appropriate current (mA). For example, 50 mA is used for the gel of 50 cm2.