Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35.

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.

Studies with the baculovirus Autographa californica and its insect host Spodoptera frugiperda identified baculovirus-encoded proteins, p35 and IAPs (inhibitor of apoptosis protein), that suppress baculovirus-induced apoptosis in S. frugiperda cells (10 -12). These proteins are expressed by the baculovirus to counter the host's antiviral defense (i.e. apoptosis) to ensure virus latency and multiplication. Mammalian antiapoptotic proteins homologous to baculovirus IAPs have recently been identified (13)(14)(15). In contrast, no mammalian counterpart of p35 has yet been identified. Nevertheless, p35 is an effective suppressor of apoptosis in mammalian cells (16 -18). Its antiapoptotic activity is attributed to its ability to interact with and potently inhibit members of the caspase family (19,20). This suggests that the apoptotic program in S. frugiperda is similar to the mammalian program and is mediated by active caspase(s). This is further supported by the recent observation that baculovirus infection of S. frugiperda cells activates a caspase that can cleave p35 (21).
To identify and clone the S. frugiperda caspase that is responsible for execution of apoptosis in this organism, we employed a degenerate PCR approach designed to identify caspases in different species. Here we report the complete amino acid sequence of an S. frugiperda caspase named Sf caspase-1, as deduced from its cDNA. We demonstrate that this protease is capable of cleaving p35 and is potently inhibited by p35. This protease is also able to induce apoptosis in Sf9 cells and cleave the Sf9 nuclear immunophilin FKBP46 (22). Sf caspase-1 has a short prodomain and is related to human CPP32 and Mch3, implying that it is a downstream executioner of apoptosis.

MATERIALS AND METHODS
Cloning of Sf Caspase-1 Proenzyme-An aliquot (10 l) of Sf9 Uni-ZAP XR cDNA library (22) containing ϳ10 8 plaque-forming units was denatured at 99°C for 5 min and then subjected to two PCR amplification steps in the presence of degenerate primers encoding the pentapeptide GSWFI/GSWYI and QACRG as described (6,23). The secondary PCR products were then used as probes to obtain full-length Sf caspase-1 clones.
Expression of Sf Procaspase-1 and p35-The open reading frame of Sf procaspase-1 was subcloned into the bacterial expression vector pET21b in-frame with an N-terminal T7-tag and a C-terminal His-tag and transformed into BL21(DE3) bacteria. The mature protease was purified on a Ni 2ϩ -affinity resin and microsequenced by automated Edman degradation (Applied Biosystems 477A equipped with a data analyzer). ProCPP32 and proMch3 were expressed and purified in a similar fashion. p35 was expressed in the same system without T7-tag. Baculovirus encoding T7-Sf procaspase-1-His6 under the late polyhedrin promoter was generated as described previously (22). Because baculovirus replication occurs early during infection, late Sf caspase-1 synthesis has no effect on baculovirus propagation.
In Vitro Transcription/Translation and Cleavage Assays-p35 and Sf caspase-1 cDNAs were in vitro transcribed and translated in the presence of [ 35 S]methionine as described recently (7)(8)(9). Two l of translation mixture was incubated with pure enzymes or cell extracts in a 10-l volume for various times at 37°C. The products were analyzed by Tricine-SDS-PAGE.
Preparation of Sf9 Apoptotic Extracts-p35 null mutant baculovirus (vp35⌬) was propagated in TN-368 insect cells which are resistant to baculovirus-induced apoptosis. Sf9 cells were infected with wild type or vp35⌬ and harvested 24 h after infection. The cells were suspended in ICE buffer (7) and lysed by a 2-3 cycle of freeze-thaw followed by homogenization. The cell lysates were centrifuged at 16,000 ϫ g for 15 min, and the supernatants were collected and then used for the enzymatic assays.
Expression, Purification, and Microsequencing of Sf Caspase-1-To determine the enzymatic activity and primary structure of Sf caspase-1 and the exact autocatalytic processing sites in its proenzyme, it was expressed in bacteria, purified, and microsequenced. This is because bacteria do not contain any caspase activity, and mutant Cys 3 Ala active site caspases are not autoprocessed in bacteria. Expression of Sf procaspase-1, containing N-terminal T7-tag and C-terminal His6-tag, produced soluble mature enzyme. As shown in Fig. 2A, purified mature Sf caspase-1 migrates in SDS-gels as three bands of apparent molecular masses of 19, 18, and 13 kDa. The N terminus of the 13-kDa band starts with Gly-196, indicating that processing occurred after Asp-195 of Sf procaspase-1. The calculated molecular mass of this peptide excluding the Cterminal His6-tag is ϳ12 kDa. The N termini of the 19-kDa and 18-kDa bands start with Ala-29, indicating that processing occurred after Asp-28 of Sf procaspase-1. Processing at these residues removes a 4-kDa prodomain. Site-directed mutagenesis of Asp-184 and Asp-195 revealed that the difference in size between the two polypeptides is due to processing at Asp-184 in the case of the 18-kDa polypeptide and Asp-195 in the case of the 19-kDa band (data not shown).
Similar results were also obtained after incubation of 35 Slabeled Sf procaspase-1 with mature recombinant Sf caspase-1 (Fig. 2B). Sf caspase-1 was able to process its proenzyme in a time-dependent fashion to generate the p19, p18, and p12 species. Based on these data, Sf procaspase-1 can autoprocess after Asp-28, Asp-184, and Asp-195 to generate the two subunits (p19/p18, large subunit, and p12, small subunit) of mature Sf caspase-1 enzyme.
p35 Is a Substrate and a Potent Inhibitor of Sf Caspase-1-Sf9 insect cells respond to baculovirus infection by activating a novel caspase to initiate apoptosis (21). This process is counteracted by expression of the baculovirus-encoded protein p35 which is a substrate for, and a potent inhibitor of, members of the caspase family (19,20).
To determine whether p35 is a substrate for Sf caspase-1, purified recombinant Sf caspase-1 was incubated with 35 Slabeled p35 for various times (Fig. 3A). This generated the expected 10-and 25-kDa fragments, indicative of cleavage at Asp-87 (Fig. 3C). Identical results were also obtained with apoptotic extract from p35 null mutant virus-infected Sf9 cells (Fig. 3B, lane 2) and recombinant human CPP32 (lane 4) and

Mch3 (lane 5).
Sf caspase-1 was potently inhibited by purified recombinant p35 in a dose-dependent manner (IC 50 ϳ0.5 nM) (Fig. 4). The endogenous protease activity in Sf9 apoptotic extract was also potently inhibited by p35 (IC 50 ϳ0.5 nM) and exhibited a similar dose dependence. Interestingly, unlike recombinant Sf caspase-1, the protease activity in the Sf9 apoptotic extract was not completely inhibited by high concentrations of p35. This suggests that this extract contains an additional protease activity distinct from Sf caspase-1 which is not sensitive to p35. This activity could be another novel caspase that is activated by viral infection. The poxvirus CrmA, which is a potent inhibitor of ICE, had very little effect on the activity of recombinant Sf caspase-1 or Sf9 apoptotic extract under the same conditions (data not shown). These results clearly suggest that Sf caspase-1 could be an important target of baculovirus p35 during viral infection of S. frugiperda insect cells.
The Nuclear Sf FKBP46 Is a Target of Sf Caspase-1 in Baculovirus-induced Apoptosis-Recently, we identified and cloned an Sf9 nuclear immunophilin named FKBP46 (22). FKBP46 contains two N-terminal acidic domains with uninterrupted stretches of polyglutamic/aspartic acid residues. Because of the high content of Asp residues in these domains, we decided to test whether FKBP46 is a target of Sf caspase-1 in apoptosis. Sf9 cells were infected with wild type (WT) baculovirus (encodes p35) or p35 null mutant (vp35⌬) baculovirus and harvested 24 h after infection. Western blot analysis revealed that FKBP46 is cleaved to an ϳ25-kDa fragment in Sf9 cells infected with vp35⌬ but not with wild type virus (Fig. 5, lanes 3   FIG. 1. Predicted amino acid  and 4, respectively). Since cells infected with vp35⌬ virus but not wild type virus undergo rapid apoptosis as a result of activation of Sf caspase-1, it is most likely that Sf caspase-1 is the enzyme responsible for cleaving FKBP46. This was supported by our observations that incubation of recombinant Sf caspase-1 with purified recombinant FKBP46 or Sf9 nuclei yielded the same ϳ25-kDa cleavage product (lanes 2 and 5). Also, overexpression of Sf procaspase-1 in Sf9 cells resulted in its processing as determined by immunostaining with T7 antibody (Fig. 6A) and generation of maximal Sf caspase-1 activity at 46 h postinfection (Fig. 6B). The lower Sf caspase-1 activity observed at 16 -24 h postinfection (Fig. 6B) might be due to p35 inhibition. In addition, the lower T7-immunostaining observed at 46 h postinfection is due to removal of the T7-tagged prodo-main by the high Sf caspase-1 activity. Maximal cleavage of FKBP46 (Fig. 6C) and induction of apoptosis with characteristic internucleosomal DNA cleavage (Fig. 6D) were observed at 46 h postinfection. About 60% of cells infected with Sf caspase-1 baculovirus showed typical morphological changes of apoptosislike blebbing and nuclear condensation at 46 h postinfection.
In conclusion, we have identified and characterized two novel components of the apoptotic machinery of the insect S. frugiperda, the host of the baculovirus A. californica. The death effector component is a caspase named Sf caspase-1, related to the mammalian apoptotic effectors Mch3, CPP32, and Mch2␣. Mature Sf caspase-1 can cleave the baculovirus antiapoptotic protein p35, is potently inhibited by p35, and exhibits similar p35-inhibitory profile as the endogenous Sf9 protease present in Sf9 apoptotic extracts. Thus, Sf caspase-1 is most likely an important target of baculovirus p35. The second component is a death-associated substrate known as FKBP46, which is an Sf9 nuclear DNA binding immunophilin recently discovered in our laboratory. We demonstrated that FKBP46 is cleaved specifically during vp35⌬ baculovirus-induced apoptosis of Sf9 cells and by the death effector component Sf caspase-1. Because the basic apoptosis program has been highly conserved during evolution, the identification of these two components should facilitate the efforts to elucidate the molecular mechanism and the physiological significance of apoptosis in diverse organisms ranging from insects to mammals.