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, 285 (8), 5569-80

Vaccinia Virus Protein F1L Is a caspase-9 Inhibitor

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Vaccinia Virus Protein F1L Is a caspase-9 Inhibitor

Dayong Zhai et al. J Biol Chem.

Abstract

Apoptosis plays important roles in host defense, including the elimination of virus-infected cells. The executioners of apoptosis are caspase family proteases. We report that vaccinia virus-encoded F1L protein, previously recognized as anti-apoptotic viral Bcl-2 family protein, is a caspase-9 inhibitor. F1L binds to and specifically inhibits caspase-9, the apical protease in the mitochondrial cell death pathway while failing to inhibit other caspases. In cells, F1L inhibits apoptosis and proteolytic processing of caspases induced by overexpression of caspase-9 but not caspase-8. An N-terminal region of F1L preceding the Bcl-2-like fold accounts for caspase-9 inhibition and significantly contributes to the anti-apoptotic activity of F1L. Viral F1L thus provides the first example of caspase inhibition by a Bcl-2 family member; it functions both as a suppressor of proapoptotic Bcl-2 family proteins and as an inhibitor of caspase-9, thereby neutralizing two sequential steps in the mitochondrial cell death pathway.

Figures

FIGURE 1.
FIGURE 1.
F1LΔTM inhibits cytochrome c-activated caspases in cell extracts. A and B, F1LΔTM (without GST tag), CrmA, N1L, p35, XIAP, and Bcl-XLΔTM (1 μm) proteins were preincubated with HeLa cell lysate (S100) for 10 min at 37 °C followed by the addition of cytochrome c (Cyt. c) and dATP to activate apoptosome (A) or 50 nm active caspase-8 (B) to activate procaspase-3/-7 in HEPES buffer for 20 min at 37 °C. Caspase-3/-7 activity was measured by hydrolysis of Ac-DEVD-AFC. C, F1LΔTM (2 μm without GST tag) was preincubated with HeLa cell lysate for 10 min at 37 °C followed by the addition of cytochrome c (500 nm) and dATP (200 μm) to activate apoptosome for 20 min at 37 °C. Alternatively, F1L (without GST tag) was added after the activation of apoptosome (indicated by an asterisk). Caspase-3 activity was measured by hydrolysis of Ac-DEVD-AFC (mean ± S.D.; n = 3). RFU, relative fluorescence units.
FIGURE 2.
FIGURE 2.
F1LΔTM inhibits apoptosome-mediated caspase-9 activation and apoptosome assembly. A, procaspase-9 was preincubated with F1LΔTM (lacking GST tag), N1L, XIAP, or Bcl-XLΔTM (1 μm) proteins for 10 min at 37 °C followed by the addition of cytochrome c (Cyt. c), dATP, and recombinant full-length Apaf-1 (1 μm) for 10 min at 37 °C. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). B, procaspase-9 incubated with (+) or without (−) F1L (lacking GST tag) was then added to reactions containing Apaf-1 together with cytochrome c and dATP for 10 min. The samples were size-fractionated by S-200 gel-filtration chromatography, and the resulting fractions were analyzed by SDS-PAGE/immunoblotting (WB) using caspase-9, Apaf-1, or F1L antibodies. The fifth panel represents a chromatography of F1LΔTM alone, which migrated as an apparent dimer. Molecular mass standards are indicated in kDa. C, recombinant F1LΔTM, N1L, XIAP, Bid, Bcl-XLΔTM, or SMAC (10 μm) proteins were preincubated with Apaf-1ΔWD-40 recombinant protein (1 μm) for 10 min, and then procaspase-9 (100 nm) was introduced for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). D, various concentrations of F1L (μm) proteins (without GST tag) were preincubated with active caspase-9 (200 nm) preactivated by Apaf-1ΔWD-40 recombinant protein (1 μm) for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). RFU, relative fluorescence units.
FIGURE 3.
FIGURE 3.
F1L selectively inhibits caspase-9 activity. A, various concentrations of F1LΔTM (without GST tag), XIAP BIR1–3 domain, p35, CrmA, or N1L (μm) proteins were preincubated with active caspase-9 (200 nm) preactivated by sodium citrate. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). B–F, active caspase-2 (B), caspase-3 (C), caspase-7 (D), caspase-8 (E), and caspase-9 (F) were incubated with (+) or without (−) F1LΔTM (2 mm without GST tag) protein for 10 min before the addition of fluorogenic substrate peptides. Caspase activity was measured by hydrolysis of Ac-DEVD-AFC (caspase-2, -3, and -7), Ac-IETD-AFC (caspase-8), or Ac-LEHD-AFC (caspase-9) (mean ± S.D.; n = 3). RFU, relative fluorescence units. G, apoptosome-preactivated caspase-9 (100 nm) was mixed with various concentrations of F1LΔTM (without GST tag). Caspase activity was measured immediately by hydrolysis of Ac-LEHD-AFC at 37 °C for 30 min. The rate of inhibition (kobs) and the second order rate constant (ka) were calculated.
FIGURE 4.
FIGURE 4.
F1L binds caspase-9. A, recombinant GST or GST-F1LΔTM proteins (1 μg) were incubated with 1 μg of active caspase-9 (Casp-9) for 4 h together with 10 μl of glutathione-Sepharose 4B resin. The resin was washed three times, and associated proteins were analyzed by SDS-PAGE/immunoblotting using anti-caspase-9 or GST antibodies. B, caspase-9 binding to GST-F1L demonstrated by enzyme-linked immunosorbent assays. F1LΔTM (black symbols) or GST (white symbols) was incubated with various concentrations of caspase-9. C and D, procaspase-9 (C) or sodium citrate-activated caspase-9 (D) was incubated with (+) or without (−) GST-F1LΔTM and then analyzed by 2S00 gel-sieve chromatography. Eluted fractions were analyzed by SDS-PAGE/immunoblotting (WB) using caspase-9 antibody. Molecular mass standards are indicated in kDa. Note that bacterially expressed procaspase-9 contained both uncleaved p50 and cleaved p35 caspase-9 (top), which was reduced to cleaved p35 caspase-9 following activation with Sodium citrate. The addition of F1LΔTM shifted both unprocessed (p50) and processed (p35) caspase-9.
FIGURE 5.
FIGURE 5.
Studies of the mechanism of F1L-mediated inhibition of caspase-9. A, the fluorescence polarization assay method was used to monitor binding of rhodamine-labeled SMAC peptide to F1LΔTM (no GST tag). Various concentrations of F1LΔTM, XIAP BIR1–3 domain, Bid, or Bcl-XLΔTM were incubated with 20 mm rhodamine-conjugated SMAC peptide. Fluorescence polarization (millipolars (mP)) was measured after 10 min. XIAP BIR1–3 domain (0.5 μm) or F1LΔTM (no GST tag) (1 μm) proteins were preincubated with active caspase-9 (200 nm) with or without SMAC protein (2 μm) for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). B, XIAP BIR1–3 domain (0.5 μm) or F1LΔTM (1 μm) proteins were preincubated with active caspase-9 (200 nm) with or without SMAC protein (2 μm) for 10 min. Caspase-9 (Casp-9) activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). C, various concentrations of F1LΔTM (lacking GST) or XIAP BIR1–3 domain (μm) proteins were preincubated with active caspase-9–5A (single chain) mutant (200 nm) for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). D and E, F1LΔTM (no GST tag) or XIAP (BIR1–3) proteins (2 μm) were preincubated with active full-length caspase-9 (activated by sodium citrate) or caspase-9 protein lacking the CARD for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). F, various concentrations of F1LΔTM (no GST tag) or CrmA proteins (μm) were preincubated with caspase-9/-8 protein (10 nm) for 10 min prior to the addition of substrate. Caspase-8 activity was measured by hydrolysis of Ac-IETD-AFC (mean ± S.D.; n = 3) RFU, relative fluorescence units.
FIGURE 6.
FIGURE 6.
F1L suppresses caspase-9-induced apoptosis. A–C, HEK293T cells were transfected with various amounts of plasmids (pcDNA3 alone (−)) encoding full-length FLAG-F1L or Bcl-XL (0–2.0 μg) with or without FLAG-caspase-9 (0.5 μg (A and B)) or caspase-8 (1 μg (C)) plasmid while maintaining total DNA constant at 3 μg by the addition of pcDNA3. Cell lysates were prepared 20 h after transfection, normalized for protein content (10 μg), and incubated with the caspase-3/-7 substrate Ac-DEVD-AFC. Enzyme activity was determined by the generation of fluorescent AFC product, and Vmax was calculated (mean ± S.D.; n = 3). D, HEK293T cells were transfected with various amounts of plasmids encoding GFP (−), GFP-F1L, or Bcl-XL (0–2.0 μg) with or without FLAG-caspase-9 (0.5 μg) or caspase-8 (1 μg) plasmid while maintaining total DNA constant at 3 μg by the addition of pcDNA3. At 20 h post-transfection, both floating and adherent cells were collected, fixed, and stained with 0.1 μg/ml DAPI. The percentages of apoptotic cells were determined by counting the GFP-positive cells having nuclear fragmentation and/or chromatin condensation (mean ± S.D.; n = 3). RFU, relative fluorescence units.
FIGURE 7.
FIGURE 7.
Analysis of F1L mutants. A, depiction of F1L and Bcl-XL proteins. The locations of some of the α-helices of F1L and Bcl-XL are shown as well as the C-terminal transmembrane (TM) domains. The regions required for caspase-9 and Bak/Bim/Bid binding are shown, illustrating the alanine substitutions that ablate protein interactions at C7A (caspase-9) and M67P (Bak). The numbers indicate amino acid residues that define the borders of the indicated structural elements. B, immunoprecipitation (IP) of caspase-9 or Bak with GFP-tagged wild-type and mutant F1L. HEK293T cells were co-transfected with GFP-F1L and FLAG-caspase-9 plasmids as indicated. After 24 h, cell lysates were prepared and analyzed by co-immunoprecipitation assay using anti-GFP antibody. Immunocomplexes (top) and cell lysates (bottom) were analyzed by SDS-PAGE/immunoblotting (WB) using antibodies specific for GFP, caspase-9, or Bak. Procaspase-9 (∼50 kDa) and the processed caspase-9 large subunit (Casp-9 LS) (∼35 kDa) are indicated. Selected molecular mass markers are indicated in kilodaltons. C, recombinant GST-tagged F1LΔTM or GST-F1LΔTM M67P (1 μm) was preincubated with Sodium citrate-activated caspase-9 (200 nm). Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). RFU, relative fluorescence units. D, mouse embryonic fibroblast Apaf-1−/− cells were transfected with various amounts of plasmids (pcDNA3 alone (−)) encoding GFP-F1L, F1L (C7A), or F1L (M67P) (0–2.0 g), with or without FLAG-procaspase-9 (0.5 μg) plasmid, while maintaining total DNA constant at 3 μg by the addition of pcDNA3. At 20 h post-transfection, both floating and adherent cells were collected, fixed, and stained with 0.1 μg/ml DAPI. The percentages of apoptotic cells were determined by counting the GFP-positive cells having nuclear fragmentation and/or chromatin condensation (mean ± S.D.; n = 3). E, HEK293T cells were transfected with various amounts of plasmid DNA encoding GFP (−)), GFP-F1L, GFP-F1L (C7A), or GFP-F1L (M67P) while maintaining total DNA constant at 3 μg by the addition of GFP plasmid. After 20 h, cells were treated with 0.1 μm staurosporine for 16 h. Both floating and adherent cells were collected, fixed, and stained with 0.1 μg/ml DAPI. The percentages of apoptotic cells (condensed chromatin and/or fragmented nucleus) among GFP-positive cells were determined by UV microscopy. Data represent the percent nonapoptotic GFP-positive cells (mean ± S.D.; n = 3).

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