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, 97 (4), 1427-32

Evolutionary Conservation of Apoptosis Mechanisms: Lepidopteran and Baculoviral Inhibitor of Apoptosis Proteins Are Inhibitors of Mammalian caspase-9

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Evolutionary Conservation of Apoptosis Mechanisms: Lepidopteran and Baculoviral Inhibitor of Apoptosis Proteins Are Inhibitors of Mammalian caspase-9

Q Huang et al. Proc Natl Acad Sci U S A.

Abstract

We cloned a new inhibitor of apoptosis protein (IAP) homolog, SfIAP, from Spodoptera frugiperda Sf-21 cells, a host of insect baculoviruses. SfIAP contains two baculovirus IAP repeat domains followed by a RING domain. SfIAP has striking amino acid sequence similarity with baculoviral IAPs, CpIAP and OpIAP, suggesting that baculoviral IAPs may be host-derived genes. SfIAP and baculoviral CpIAP inhibit Bax but not Fas-induced apoptosis in human cells. Their apoptosis-suppressing activity in mammalian cells requires both baculovirus IAP repeat and RING domains. Further biochemical data suggest that SfIAP and CpIAP are specific inhibitors of mammalian caspase-9, the pinnacle caspase in the mitochondria/cytochrome c pathway for apoptosis, but are not inhibitors of downstream caspase-3 and caspase-7. Thus the mechanisms by which insect and baculoviral IAPs suppress apoptosis may involve inhibition of an insect caspase-9 homologue. Peptides representing the IAP-binding domain of the Drosophila cell death protein Grim abrogated human caspase suppression by SfIAP and CpIAP, implying evolutionary conservation of the functions of IAPs and their inhibitors.

Figures

Figure 1
Figure 1
Predicted domain topology and amino acid sequence of SfIAP. (A) The BIR and RING domains of SfIAP are depicted. (B) The predicted amino acid sequence of SfIAP is presented. Sequence alignments of the BIR1 (C), BIR2 (D), and RING (E) domains of SfIAP with the corresponding domains of other IAP-family members are shown. Black indicates identity and gray indicates homology.
Figure 2
Figure 2
SfIAP and CpIAP protect cells against Bax- but not Fas-induced apoptosis. Expression plasmids encoding Bax (A) or Fas (B) were cotransfected into 293 cells with the indicated myc-tagged IAP expression plasmids. Percentage apoptosis was measured 24–36 hr later by 4′-6-diamidino-2-phenylindole staining (mean ± SD; n = 3). (C) Lysates were prepared from 293 cells 1 day posttransfection, normalized for total protein content (50 μg), and analyzed by SDS/PAGE–immunoblotting by using anti-myc antibody with enhanced chemiluminescence-based detection to demonstrate that the desired protein was expressed in each case.
Figure 3
Figure 3
SfIAP and CpIAP suppress Cyt c- but not caspase-8-induced activation of effector caspases. Recombinant SfIAP or CpIAP (2 μM) was added to cytosolic extracts (10 mg/ml) from HEK293 cells concurrently with the addition of 1 μM Cyt c/10 mM dATP (A) or 100 ng/ml active caspase-8 (B). After incubation at 30°C for 10 min, aliquots were withdrawn and assayed for caspase activity, continuously monitoring release of AFC from Ac-DEVD-AFC substrate (100 μM) beginning from the time of substrate addition. SD from 10 replicates was smaller than the points on the graphs. (C and D) Aliquots of cell extracts from the experiments performed above were analyzed by SDS/PAGE–immunoblotting by using anticaspase-3 antiserum. Arrowheads indicate the pro- and processed forms of caspase-3. In cell extracts activated by Cyt c or active caspase-8, ≈32-kDa procaspase-3 was processed to yield ≈17- to 20-kDa forms of the large subunit, indicative of active caspase-3 (the ≈12-kDa subunit of caspase-3 is undetectable with this anticaspase-3 antibody). Recombinant SfIAP and CpIAP suppressed the processing of procaspase-3 in Cyt c-treated (C) but not in caspase-8-treated (D) extracts, whereas XIAP interrupted processing at an intermediate step (≈24-kDa band), as demonstrated previously (10, 11). (E) Grim peptide or control peptide (2–10 μM) was added with or without recombinant SfIAP (2 μM) protein to HEK293 cell lysates concurrently with the addition of Cyt c/dATP. Lysates were incubated at 30°C for 10 min, and aliquots then were withdrawn and assayed for DEVD-cleaving caspase activity as above, measuring rates from the linear portion of enzyme progress curves.Data are presented as a percentage relative to control reactions in which Cyt c/dATP were alone. Numbers below diagram indicate molar ratio of peptides relative to SfIAP protein.
Figure 4
Figure 4
SfIAP and CpIAP directly suppress caspase-9 but not caspase-3 or caspase-7. Caspase-9 expression plasmids were cotransfected into 293 cells with plasmids encoding various full-length IAPs (A) or fragments of SfIAP (B) comprising only the BIR domains (residues 1–323) or RING domain (residues 324–377) or fragments of CpIAP containing only BIR (residues 1–220) or RING (residues 221–275) domains (C). After 24–36 hr, lysates were prepared and assayed for DEVD-cleaving caspase activity, as above. Data are presented as a percentage relative to caspase activity of cells transfected with caspase-9 alone (mean ± SD; n = 3). (D) Recombinant active caspase-9 was added at 0.2 μM because of its lower specific activity and incubated at 37°C with Ac-LEHD-AFC substrate (100 μM) in the presence or absence of various concentrations (0.2–1.6 μM) of SfIAP (○) or (0.2–2.4 μM) of CpIAP (●). AFC release was measured continuously, and data are expressed as a percentage relative to control reactions lacking IAPs, using rates determined from the linear portion of enzyme progress curves. Various control GST-fusion proteins had no inhibiting effect (not shown). (E) [35S]Caspase-9 in reticulocyte lysate was incubated with GST-fusion proteins immobilized on glutathione-Sepharose: GST-SfIAP, GST-CpIAP, GST-XIAP-BIR1, and GST-XIAP. (F) Recombinant active caspase-3 (2.6 nM) was incubated at 37°C with Ac-DEVD-AFC substrate (100 μM) in the presence or absence of 0.05 μM GST-XIAP, 0.5 μM GST-SfIAP (200-fold molar excess relative to caspases), or 0.5 μM GST-CpIAP (≈200-fold molar excess). AFC release was measured as above. (G) Caspase-7 (7 nM) was incubated at 37°C with Ac-DEVD-AFC substrate (100 μM) in the presence or absence of 0.14 μM GST-XIAP, 0.7 μM GST-SfIAP (100-fold molar excess relative to caspases), or 0.6 μM GST-CpIAP (≈100-fold molar excess). AFC release was measured as above.

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