Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo
2002; Springer Nature; Volume: 21; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7594736
ISSN1460-2075
AutoresStephen J. Zoog, Jennifer Schiller, Justin Wetter, Nor Chejanovsky, Paul D. Friesen,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle1 October 2002free access Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo Stephen J. Zoog Stephen J. Zoog Present address: Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT, 06877 USA Search for more papers by this author Jennifer J. Schiller Jennifer J. Schiller Present address: Department of Pediatrics, Medical School of Wisconsin, Milwaukee, WI, 53226 USA Search for more papers by this author Justin A. Wetter Justin A. Wetter Institute for Molecular Virology, and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI, 53706 USA Search for more papers by this author Nor Chejanovsky Nor Chejanovsky Entomology Department, Institute of Plant Protection, Agricultural Research Organization, Bet Dagan, Israel, 50250 Search for more papers by this author Paul D. Friesen Corresponding Author Paul D. Friesen Institute for Molecular Virology, R.M.Bock Laboratories, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, 53706-1596 USA Search for more papers by this author Stephen J. Zoog Stephen J. Zoog Present address: Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT, 06877 USA Search for more papers by this author Jennifer J. Schiller Jennifer J. Schiller Present address: Department of Pediatrics, Medical School of Wisconsin, Milwaukee, WI, 53226 USA Search for more papers by this author Justin A. Wetter Justin A. Wetter Institute for Molecular Virology, and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI, 53706 USA Search for more papers by this author Nor Chejanovsky Nor Chejanovsky Entomology Department, Institute of Plant Protection, Agricultural Research Organization, Bet Dagan, Israel, 50250 Search for more papers by this author Paul D. Friesen Corresponding Author Paul D. Friesen Institute for Molecular Virology, R.M.Bock Laboratories, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, 53706-1596 USA Search for more papers by this author Author Information Stephen J. Zoog2, Jennifer J. Schiller3, Justin A. Wetter1, Nor Chejanovsky4 and Paul D. Friesen 5 1Institute for Molecular Virology, and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI, 53706 USA 2Present address: Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT, 06877 USA 3Present address: Department of Pediatrics, Medical School of Wisconsin, Milwaukee, WI, 53226 USA 4Entomology Department, Institute of Plant Protection, Agricultural Research Organization, Bet Dagan, Israel, 50250 5Institute for Molecular Virology, R.M.Bock Laboratories, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, 53706-1596 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5130-5140https://doi.org/10.1038/sj.emboj.7594736 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors. Introduction Apoptosis is a widely conserved genetic program that deletes unwanted, abnormal or diseased cells in multicellular organisms (Jacobson et al., 1997; Vaux and Korsmeyer, 1999). Disruption of normal apoptotic regulation causes adverse effects that are correlated with tumorigenesis, immunodeficiency and degenerative disorders (Thompson, 1995; Yuan and Yankner, 2000). The caspase family of cysteine-dependent aspartate-specific proteases are essential components in the execution of apoptosis (Salvesen and Dixit, 1997; Cryns and Yuan, 1998; Thornberry and Lazebnik, 1998; Earnshaw et al., 1999; Chang and Yang, 2000). By selective proteolysis of cellular substrates, the caspases catalyze the biochemical events that lead to cellular disassembly, including DNA fragmentation, chromatin condensation and plasma membrane blebbing. Because of their pivotal role in the commitment to cell death, these proteases are important targets in therapeutic strategies for treating apoptosis-associated diseases (Jacobson, 1998; Nicholson, 2000; Yuan and Yankner, 2000). Caspases are activated from dormant proenzymes by an ordered series of proteolytic cleavages. Initiator caspases are activated by mechanisms that include signal-induced interactions of specific adaptor proteins with the long N-terminal prodomain, which promotes caspase proteolytic processing (Earnshaw et al., 1999; Chang and Yang, 2000). The initiators subsequently activate the short prodomain-containing effector caspases by proteolysis. In the vertebrate mitochondrion/cytochrome c pathway, initiator caspase-9 is processed upon recruitment of its CARD-containing prodomain by adaptor Apaf-1 bound to cytochrome c released from mitochondria during apoptotic signaling (Li et al., 1997; Zou et al., 1997; Srinivasula et al., 1998). From this apoptosome complex, caspase-9 proteolytically activates effector proteases, including caspase-3 (Adrain and Martin, 2001). In invertebrates, a comparable apoptosome-mediated activation of initiator caspases is likely since initiator caspase candidates DREDD and DRONC of Drosophila melanogaster interact with the Apaf-1 homolog designated Ark (White, 2000). The existence of diverse pro- and anti-apoptotic factors that affect initiator caspase activation argues that this process is a key regulatory step in initiation of apoptosis (Chang and Yang, 2000; Adrain and Martin, 2001). Viruses have evolved novel mechanisms to prevent apoptosis of their host cell and thereby promote virus multiplication (O'Brien, 1998; Roulston et al., 1999). To date, two distinct types of apoptotic suppressor, represented by P35 and the inhibitors of apoptosis (IAPs), have been identified in the invertebrate baculoviruses (Clem, 2001). Baculovirus IAPs were the first members of the IAP protein family to be discovered (Deveraux and Reed, 1999; Miller, 1999). The best-studied viral IAP, Op-IAP, prevents apoptosis in insects and mammals by a mechanism that includes interaction with itself and pro-apoptotic proteins like Reaper, HID and GRIM of Drosophila (Birnbaum et al., 1994; Duckett et al., 1996; Hawkins et al., 1996; Vucic et al., 1997, 1998; Hozak et al., 2000). In SF21 cells from Spodoptera frugiperda (Order Lepidoptera), a model system for insect apoptosis, Op-IAP blocks proteolytic activation of the principal effector caspase, Sf-caspase-1 (Seshagiri and Miller, 1997; LaCount et al., 2000). In contrast, caspase inhibitor P35 blocks apoptosis by targeting active Sf-caspase-1 (Bertin et al., 1996; Ahmad et al., 1997; Manji et al., 1997; LaCount et al., 2000). P35 is a pancaspase inhibitor in which cleavage of its solvent-exposed reactive-site loop (RSL) leads to formation of a stoichiometric complex with the caspase target (Bump et al., 1995; Zhou et al., 1998; Fisher et al., 1999; Xu et al., 2001). Despite its broad-spectrum anti-caspase activity, P35 fails to block proteolytic activation of pro-Sf-caspase-1, suggesting the existence of a novel P35-insensitive initiator caspase (LaCount et al., 2000; Manji and Friesen, 2001). This uncharacterized caspase is designated Sf-caspase-X. Here, we describe a third type of baculovirus apoptotic suppressor, P49, that is distinguished by its capacity to inhibit the Spodoptera initiator caspase unaffected by P35. The p49 gene from baculovirus SlNPV was identified by its capacity to block apoptosis and restore replication of a p35-deletion virus (Du et al., 1999). P49 is 49% identical to P35 but is unrelated to any known cellular protein. It is characterized by its larger size (446 residues), the presence of a 120 residue insertion absent in P35 (299 residues) and a significantly different sequence (TVTD94↓G) at its predicted cleavage site (Figure 1A). These dissimilarities suggest that if P49 functions as a caspase inhibitor, it may exhibit a unique caspase specificity or target a distinct step in the death pathway. To test these possibilities, we explored the anti-caspase potential of P49 and defined the in vivo apoptotic step affected. Figure 1.P49 blocks apoptosis induced by diverse signals. (A) P49 and P35 structure. Amino acid similarity between P49 and P35 is colinear with the exception of a 120 residue insert (crosshatched) within P49. The caspase cleavage site within the predicted RSL (open) is indicated. (B) Virus- and UV radiation-induced apoptosis. SF21 cells were mock-transfected or transfected with plasmids encoding P49 or P35 and subsequently infected with apoptosis-inducing virus vΔp35 or irradiated with UV-B (125 mJ/cm2). Photographs (magnification, 100×) were taken 48 or 24 h after infection (i–iii) or UV irradiation (iv–vi), respectively. Arrows depict occluded virus particles in non-apoptotic cells. A representative experiment is shown. (C) Cell survival. SF21 cells transfected with plasmids encoding wild-type P49, D94A-mutated P49, P35 or Op-IAP were infected or UV-irradiated as described in (B). Survival was quantified by computer-aided microscopy and is reported as the average number of surviving, non-apoptotic cells ± standard deviation. Download figure Download PowerPoint We report here that P49 is a caspase substrate inhibitor with a P35-like mechanism. However, unlike P35, P49 functions at an upstream step to inactivate the caspase that proteolytically activates effector caspases of Spodoptera cells. Thus, P49 exhibits a distinct in vivo target specificity for a novel P35-insensitive initiator caspase. These data indicate that despite comparable structures and mechanisms, caspase inhibitors P49 and P35 have a unique specificity for initiator or effector caspases in the context of the apoptotic cell. Our finding that P49 also has the capacity to block apoptosis in cultured cells of D.melanogaster and is a potent inhibitor of human initiator caspase-9 suggests that P49 functions at a highly conserved step in the caspase cascade and should therefore prove useful in delineating cell death pathways in many organisms. Results Baculovirus P49 blocks apoptosis induced by diverse stimuli The high sequence similarity with P35 and the presence of a caspase-like cleavage site (Figure 1A) suggested that P49 functions as a caspase inhibitor. Thus, to test the capacity of P49 to block caspase-mediated apoptosis, we expressed p49 ectopically in cultured SF21 cells that were subsequently induced to undergo apoptosis. Upon plasmid transfection, P49 blocked apoptosis triggered by infection with baculovirus mutant vΔp35, which lacks apoptotic suppressors (Figure 1B). P49 prevented premature cell death and promoted virus replication as indicated by the accumulation of intranuclear virus particles. The level of p49-mediated protection was comparable to that conferred by p35 and baculovirus inhibitor of apoptosis Op-iap (Figure 1C). Similarly, P49 was a potent suppressor of UV radiation-induced apoptosis (Figure 1B). Upon transfection, P49 was as effective as P35 in protecting SF21 cells from a dose of UV radiation that caused 95% lethality (Figure 1C). Op-IAP was comparably protective. To assess the contribution of the P49 predicted cleavage site (TVTD94↓G) to anti-apoptotic activity, Asp94 was substituted with alanine. Although readily synthesized in transfected cells (see below), D94A-mutated p49 failed to block apoptosis induced by infection or UV irradiation (Figure 1C). The loss of p49 function was confirmed by marker rescue assays (Table I) in which the anti-apoptotic activity of a plasmid-borne gene was measured by the extent to which it restored multiplication of a p35-deficient baculovirus (Bertin et al., 1996; Zoog et al., 1999). In contrast to wild-type P49, D94A-mutated P49 failed to rescue virus replication. As expected, wild-type P35 restored replication, but D87A cleavage site-mutated P35 did not (Table I). We concluded that P49 blocks apoptosis induced by distinct death signals through a mechanism requiring an aspartate residue at the predicted caspase cleavage site. Table 1. Marker rescue of vΔp35 mutant replication Transfected gene Virus yielda (× 103 p.f.u./ml) Anti-apoptotic activity (%) wt p49 125 ± 18 93.3 D94A-p49 0.0 0.0 wt p35 134 ± 18 100 D87A-p35 0.4 ± 0.1 0.3 aVirus yields were determined by plaque assay using apoptosis-sensitive SF21 cells. Percentage anti-apoptotic activity is reported as the ratio of non-apoptotic, lacZ-expressing plaques produced by transfection of the gene indicated to that of wild-type p35. Values shown are the average ± standard deviation of triplicate transfections. P49 is a substrate inhibitor of caspases To determine whether P49 is a direct inhibitor of caspases, we generated C-terminal His6-tagged P49. When tested in dose-dependent assays that used the tetrapeptide DEVD-AMC as substrate, the principal effector caspase of SF21 cells, Sf-caspase-1, was inhibited by purified P49-His6, but not D94A-mutated P49-His6 (Figure 2A). Nonetheless, P49-His6 was ∼100 times less effective than P35-His6, which is a stoichiometric inhibitor of Sf-caspase-1 (Figure 2B). In contrast, P49-His6 was a potent inhibitor of human caspase-3 (Figure 2C). In these assays, P49-His6 was nearly as effective as P35-His6, which is also a stoichiometric inhibitor of caspase-3 (Bump et al., 1995; Bertin et al., 1996; Zhou et al., 1998; Fisher et al., 1999). D94A-mutated P49-His6 had little effect on human caspase-3 over the range of protein concentrations tested (Figure 2C). Figure 2.P49 is a substrate inhibitor. (A and B) Inhibition of Sf-caspase-1. Purified recombinant Sf-caspase-1 (0.1 pmol) was incubated with the indicated amounts of purified P49-His6 (filled square), D94A-mutated P49-His6 (open square), P35-His6 (filled triangle) or D87A-mutated P35-His6 (open triangle). After 30 min, residual caspase activity was measured by using DEVD-AMC as substrate. Plotted values are the average ± standard deviation of triplicate assays and are expressed as a percentage of uninhibited caspase activity. (A) and (B) differ in the range of P49 used. (C) Inhibition of human caspase-3. Purified recombinant caspase-3 (0.1 pmol) was incubated with the indicated amounts of purified P49-His6 (filled square), D94A- mutated P49-His6 (open square) or P35-His6 (filled triangle) and assayed as described above. (D) P49 cleavage. In vitro synthesized, 35S-radiolabeled, wild-type (wt) or D94A-mutated P49 was mixed with buffer (lanes 1 and 2), human caspase-3 (lanes 3 and 4) or Sf-caspase-1 (lanes 5 and 6) and analyzed by SDS–PAGE. The 40 kDa (*) and 9 kDa (*′) cleavage fragments are indicated (bottom). (E) P49–caspase-His6 complexes. Radiolabeled untagged wild-type or D94A-mutated P49 was mixed with buffer or His6-tagged human caspase-3 (300 pmol). Complexes recovered by Ni2+ affinity chromatography in the presence (lanes 3 and 4) or absence (lanes 5 and 6) of caspase-3 were analyzed by SDS–PAGE. Uncleaved P49 (input) was included (lanes 1 and 2). (F) P49-His6–caspase complexes. Purified wild-type (wt) or D94A-mutated P49-His6 was mixed with buffer or untagged caspase-3. Recovered Ni2+-bound complexes were subjected to immunoblot analysis by using α-P49 (top) or caspase-3 large subunit α-P17 (bottom) antisera. Download figure Download PowerPoint Wild-type, but not D94A-mutated P49, was cleaved by human caspase-3 and Sf-caspase-1 to produce 40 and 9 kDa fragments (Figure 2D). Amino acid sequence analysis revealed that the N-terminus of the larger 40 kDa fragment was GGGAD (data not shown), indicating that cleavage occurred between P49 residues Asp94 and Gly95 within the predicted cleavage site TVTD94↓ GGGAD. To determine whether the P49 cleavage fragments stably associate with the targeted caspase in a manner analogous to that of P35, we used His6-tagged caspase-3 in Ni2+ affinity pull-down assays. Upon recovery of His6-tagged caspase-3 from cleavage reactions containing untagged P49, both 40 and 9 kDa fragments of wild-type P49 (Figure 2E, lane 3), but not D94A-mutated P49 (lane 4) were detected. Neither wild-type nor mutated P49 was recovered in the absence of His6-tagged caspase. In reciprocal assays, where the association of untagged caspase-3 with P49-His6 was tested, caspase-3 interacted stably with cleaved P49-His6 (Figure 2F, lane 2). In contrast, D94A-mutated P49 was neither cleaved nor associated with caspase-3 (lane 3). Collectively, these data indicate that caspase inhibition in vitro includes the formation of a stable P49–caspase complex, which requires cleavage at Asp94. P49 functions upstream of P35 but downstream of Op-IAP Although P49 and P35 function similarly in vitro, the structural deviations of these two caspase inhibitors suggested that they act differently in vivo, possibly by targeting distinct caspases. This notion was supported by the 100-fold difference in P49 and P35 inhibition of Sf-caspase-1 (Figure 2B). To investigate potential differences, we first defined the apoptotic step affected by P49. To this end, we constructed a recombinant baculovirus (vP49) that encodes P49 as its singular apoptotic inhibitor. By inoculating SF21 cultures with this virus, P49 was synthesized in every cell that received an apoptotic stimulus. Importantly, P49 activity could therefore be monitored in the context of physiologically relevant levels of death effectors. As expected, apoptosis was fully blocked in SF21 cells infected with vP49. Directed by the immediate early ie-1 promoter, P49 was synthesized early during infection (Figure 3A). Our α-P49 antiserum recognized full-length P49 and its 40 kDa fragment (*) but not the 9 kDa N-terminal fragment as demonstrated by comparing purified P49-His6 partially cleaved by caspase (Figure 3A). P49 cleavage was first detected between 6 and 12 h and thus coincided with virus-induced caspase activation (LaCount and Friesen, 1997; LaCount et al., 2000). To confirm the identity of the 40 kDa cleavage fragment that was present at low levels, we compared cells transfected with wild-type or mutated p49. The 40 kDa fragment was detected only in the presence of wild-type P49, not cleavage-impaired D49A-mutated P49 (Figure 3B, lanes 5 and 6). Furthermore, generation of the cleavage fragment required apoptotic signaling, since it was present in virus-infected cells (lane 5) but not mock-infected cells (lane 2). Another P49-related protein (Figure 3B, filled diamond) detected early in vP49 infection and upon transfection with wild-type and D49A-mutated P49 is a likely internal initiation product. Figure 3.In vivo P49 cleavage. (A) Time course. SF21 cell lysates prepared at the indicated hours after infection with vP49 were subjected to immunoblot analysis using α-P49, which recognized full-length P49 and the 40 kDa cleavage fragment (top), but not the N-terminal 9 kDa fragment. Due to the absence of His tags, in vivo P49 and its 40 kDa cleavage fragment (*) are smaller than P49-His6, which was partially cleaved by caspase (right lane). Another P49-related protein (filled diamond) is indicated. (B) Asp94-dependent P49 cleavage. SF21 cells were mock-transfected (−) or transfected with plasmid encoding wild-type (wt) or D94A-mutated P49. After mock infection (lanes 1–3) or infection with p35− vΔp35 to induce apoptosis (lanes 4–6), cell lysates (5 × 105 cell equiv) were prepared and subjected to α-P49 immunoblot analysis. Download figure Download PowerPoint To define the apoptotic step affected by P49, we determined where P49 functions relative to P35 by testing the effect of P49 on in vivo cleavage of P35, a sensitive indicator of effector caspase activity (Bertin et al., 1996; Manji et al., 1997). Upon infection with a p35-carrying virus (p35+), activated caspase(s) readily cleaved P35 to generate the 25 kDa cleavage (*) fragment (Figure 4A, lane 2). However, upon coinfection with vP49, P49 blocked the cleavage of P35 (lane 3). Conversely, P49 was cleaved normally even in the presence of abundant P35 (Figure 4B, lane 3). Taking into account the reduced P49 synthesis due to p35+ virus coinfection, P49 cleavage was comparable to that in cells infected with vP49 alone (lane 2). We concluded that P49 directly or indirectly inhibits the caspase(s) responsible for P35 cleavage and thus functions upstream of P35. Figure 4.P49 functions upstream of P35 but downstream of Op-IAP. (A) P49 inhibits P35 cleavage. Sf21 cells were mock-infected (mi) or infected with viruses vP49 (+p49), vOp-IAP (+iap) or vΔp35 (−p35) with (lanes 2–5) or without (lanes 6–8) wild-type p35+ virus (+P35). Lysates (3 × 105 cell equiv) prepared 24 h later were subjected to immunoblot analysis using α-P35. Top: P35 and its C-terminal cleavage product (*) are indicated. (B) Op-IAP inhibits P49 cleavage. Lysates (106 cell equiv) prepared 24 h after infection with the viruses described in (A) with (lanes 2–5) or without (lanes 6–8) vP49 (+P49) were subjected to immunoblot analysis with α-P49. SF21 cells constitutively expressing Op-iap (IAP) were included (lane 9). (Top) P49 and its C-terminal cleavage fragment (*) are indicated. The P49-related protein (filled diamond) is marked. Download figure Download PowerPoint To determine where P49 functions relative to an IAP, we also assessed the effect of Op-IAP on P49 cleavage. In cells coinfected with vP49 and an Op-iap+ virus (vOp-IAP), Op-IAP blocked P49 cleavage (Figure 4B, lane 4). To confirm this finding, we infected SF21 cells that constitutively express Op-iap with vP49. In contrast to parental (Op-iap−) cells, P49 cleavage was fully blocked in Op-iap+ cells (Figure 4B, compare lanes 9 and 10). As expected, Op-IAP also prevented P35 cleavage (Figure 4A, lane 4) confirming its upstream function relative to P35 (Manji et al., 1997). These data indicated that Op-IAP functions at or upstream of the caspase that cleaves P49, which is upstream of the caspase(s) inhibited by P35. P49, but not P35, prevents in vivo proteolytic processing of effector caspases During apoptosis of SF21 cells, pro-Sf-caspase-1 (Figure 5A) and pro-Sf-caspase-2 (Figure 5C) are proteolytically processed (Ahmad et al., 1997; LaCount, 1998; LaCount et al., 2000). This signal-induced activation is initiated by a P35-insensitive caspase (LaCount et al., 2000; Manji and Friesen, 2001). Sf-caspase-1 undergoes a second cleavage, which removes the prodomain mediated by a P35-inhibitable protease, probably Sf-caspase-1 itself (LaCount et al., 2000). To determine whether P49 affects the caspase responsible for processing these effector caspases, we compared Sf-caspase activation in the presence of P49 and P35. Upon apoptotic signaling caused by p35+ virus infection, pro-Sf-caspase-1 was processed to its large subunit fragments p25 and p19 (Figure 5B). Thus, cleavage of pro-Sf-caspase-1 occurred at TETD↓G despite an abundance of P35. In contrast, P49 blocked the processing of pro-Sf-caspase-1 during infection with vP49 (Figure 5B). Levels of pro-Sf-caspase-1 were unchanged and cleavage products, including p25, were not detected until very late. In a similar pattern, P49 prevented proteolytic processing of pro-Sf-caspase-2, whereas P35 did not (Figure 5D). The large subunit p18 of Sf-caspase-2 accumulated in the presence of P35 but not P49. These data confirmed that P49 acts at a distinct step upstream from P35 in the apoptotic pathway of these lepidopteran cells. Furthermore, P49 likely targets the P35-insensitive initiator caspase responsible for activation of pro-Sf-caspase-1 and -2. Figure 5.P49 inhibits effector caspase proteolytic processing. (A and C) Processing of Sf-caspase-1 and -2. Activation of pro-Sf-caspase-1 initiates with cleavage at TETD↓G to generate intermediate p25, which is subsequently cleaved at DEGD↓G to generate the mature p19 and p12 subunits. Pro-Sf-caspase-2 is cleaved at AETD↓G to generate p18 and p12 subunits. (B and D) Effect of P49 on Sf-caspase-1 and -2 processing. SF21 lysates (3.8 × 105 cell equiv) prepared at the hours (h) indicated after infection with wild-type p35+ virus (+P35), vP49 (+P49) or vΔp35 (−P49, −P35) were subjected to immunoblot analysis using α-Sfcasp1 (B) or α-Sfcasp2 (D) antiserum. Download figure Download PowerPoint Does the P49 cleavage site confer caspase specificity? The similarity between the P49 cleavage site TVTD↓G (Figure 6A) and the initial processing sites TETD↓G and AETD↓G of pro-Sf-caspase-1 and -2, respectively, suggested that the apical caspase selectivity of P49 is conferred by its RSL cleavage residues. We hypothesized that the in vivo selectivity of P49 and P35 could be switched by exchanging cleavage site residues. Thus, we substituted the P4–P1 residues of P49 and P35 and determined the effect of these swaps by monitoring Sf-caspase activation in transfected cells. Figure 6.In vivo caspase selectivity by P49 and P35. (A) Cleavage sites. The P4–P1′ residues of P49 and P35 are compared with the initial processing sites of pro-Sf-caspase-1 and -2. (B) Anti-apoptotic activity. The capacity of plasmid-borne wild-type (wt) or mutated p49 and p35 to block virus-induced apoptosis was determined by marker rescue. The average ± standard deviation of triplicate transfections is reported as a percentage of the capacity of wild-type P35 to restore virus replication. (C and D) Processing of Sf-caspases. SF21 cells were transfected with plasmids encoding wt or mutated P49 (lanes 1–4) or wt or mutated P35 (lanes 5–8), and infected 24 h later to induce apoptosis. Lysates (3.8 × 105 cell equiv) prepared 24 h after infection were subjected to immunoblot analysis with α-Sfcasp2 (C) or α-Sfcasp1 (D). Download figure Download PowerPoint The exchange of P49 or P35 cleavage sites had no effect on the anti-apoptotic activity of either caspase inhibitor. Marker rescue assays demonstrated that DQMD94G-substituted P49 and TVTD87G-substituted P35 were as effective as the wild-type proteins in blocking virus-induced apoptosis (Figure 6B). In contrast, loss-of- function D94A- or I76P-mutated P49 and D87A- or V71P-mutated P35 failed to block apoptosis. Our previous studies indicated that substitution V71P disrupted the P35 RSL and thus caused loss of function (Fisher et al., 1999; Zoog et al., 1999). Although fully functional, the cleavage site substitutions failed to alter the effect of P49 or P35 on procaspase processing during virus infection. Both wild-type and DQMD94G-substituted P49 prevented pro-Sf-caspase-2 processing (Figure 6C, lanes 1 and 4), whereas loss-of-function D94A- or I76P-mutated P49 did not (lanes 2 and 3). The higher p18 levels (lanes 1 and 4) were attributed to those infected cells that failed to produce P49 because they were not transfected (transfection efficiencies ranged from 75 to 90%). Furthermore, neither wild-type (lane 5) nor TVTD87G-P35 (lane 8) blocked pro-Sf-caspase-2 processing. As expected, loss-of-function D87A- and V71P-mutated P35 (lanes 6 and 7) failed to block processing. Likewise, wild-type and DQMD94G-P49 prevented pro-Sf-caspase-1 processing (Figure 6D, lanes 1 and 4). TVTD87G-P35 exhibited a slightly increased capacity to prevent processing compared with wild-type P35 (Figure 6D, lanes 5 and 8). Overall, the exchange of cleavage sites had a minimal effect on processing of pro-Sf-caspase-1 and -2. We concluded that the P4–P1 residues are not sufficient to confer P49 or P35 target specificity in vivo. Thus, specificity is conferred by a determinant other than or in addition to the cleavage site residues. P49 is a substrate inhibitor of human initiator caspase-9 Our data suggested that P49 functions in vivo to block activity of an initiator caspase. Therefore, to determine whether P49 is capable of inhibiting a well characterized initiator caspase, we tested the effect of P49 on purified human caspase-9, which functions in the mitochondrion/cytochrome c death pathway of mammals (Earnshaw et al., 1999; Chang and Yang, 2000; Adrain and Martin, 2001). When tested in dose-dependent assays that used the tetrapeptide LEHD-AFC as substrate, purified P49-His6 effectively inhibited caspase-9 but D94A-mutated P49-His6 did not (Figure 7A). Interestingly, wild-type but not D87A-mutated P35-His6 inhibited caspase-9. Under identical conditions, baculovirus vector purified P49 was ∼8-fold less potent than Escherichia coli-produced P35. Wild-type but not D94A-mutated P49 was cleaved by caspase-9 into 40 and 9 kDa fragments that were electrophoretically indistinguishable from those generat
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