As part of a search for potential anti-apoptotic genes we performed a standard BLAST search using the AcMNPV p35 gene sequence and identified AMV010, an ORF found in AmEPV with low but significant homology to p35 (). AMV010 encodes a predicted protein of 32.7 KDa which lacks identifiable sequence motifs. To be consistent with the nomenclature of other known p35 homologs, we have renamed this gene AMVp33. At the amino acid level, the identity of the predicted P33 protein to AcMNPV P35 was 25%. To determine if the predicted structure of P33 correlated to its possible function, we performed computer-assisted modeling of P33 based on the known structure of the caspase inhibitor P35. This comparison predicted a similar overall structure to that of both P35 and P49 (). While computer-based protein structure predictions are of limited value, the most significant aspect of the computer-generated model was the potential for P33 to contain a reactive site loop similar to that found in P35 and P49, with a potential caspase cleavage site, 87YNFD90, in the same position as the cleavage site 84DQMD87 found in AcMNPV P35. Since P35 is a potent caspase inhibitor, we decided to analyze the ability of P33 to inhibit apoptosis, and in particular caspases.
Figure 1 P33 alignment and predicted structure. (A) Alignment of the predicted amino acid sequences of AmEPV P33, AcMNPV P35, and SlNPV P49. Yellow bars represent identical amino acids found in all 3 sequences, blue bars represent identical amino acids found in (more ...)
P33 inhibits apoptosis induced by diverse stimuli
To test the ability of p33 to block caspase-dependent apoptosis, we transiently expressed N-terminally HA-tagged P33 in LD652Y, S2 and SF-21 cells and induced the cells to undergo apoptosis with UV radiation. Immunoblot analysis was performed to confirm expression of P33 in each cell type (). P33 expression inhibited UV-induced apoptosis in all three cell lines, to a degree comparable to that of P35 (). P33 also blocked apoptosis induced by vHSGFP/P35del, a mutant of AcMNPV that lacks p35 (). P33 prevented apoptosis and allowed for virus replication as indicated by the formation of polyhedra ().
Figure 2 P33 blocks apoptosis induced by UV irradiation in insect cell lines. (A) Anti-HA immunoblot of lysates from LD652Y, S2, and SF-21 cells collected 24 h after transfection with plasmids expressing HA-tagged P33 or point mutant P33(D90A). Lanes labeled “Mock” (more ...)
Figure 3 P33 blocks AcMNPV-induced apoptosis. (A) SF-21 cells were mock transfected or transfected with plasmids expressing P33, P35 or negative control chloramphenicol acetyl transferase (CAT) and 24 h later the cells were infected with vHSGFP/P35del at an MOI (more ...)
To assess the importance of the P33 predicted cleavage site (YNFD90) for its anti-apoptotic function, Asp90 was substituted with Ala. Although expressed at similar levels as wild type P33 (), the point mutant P33(D90A) failed to block apoptosis induced by either UV irradiation or viral infection (Fig. , ). In examining the sequence of P33, two other Asp residues (Asp83 and Asp101) were found in the predicted reactive site loop near Asp90. These Asp residues were mutated to Ala and the resulting point mutants were tested for their ability to block apoptosis. P33(D83A) and P33(D101A) were able to prevent apoptosis induced by UV irradiation or viral infection as efficiently as wild type P33 (data not shown), further illustrating the importance of residue Asp90.
P33 expression suppresses caspase activity in cells
To determine whether inhibition of apoptosis by P33 correlated with reduced caspase activity, whole cell lysates from either mock transfected, P35- or P33-expressing cells were harvested 12 h after UV or viral infection and caspase activity was determined using the fluorogenic caspase substrates Ac-IETD-AFC (to measure initiator caspase activity) or Ac-DEVD-AFC (to measure effector caspase activity). P33-expressing cells showed significantly reduced effector caspase activity as compared to mock transfected cells, comparable to that seen with P35 (). However, initiator caspase (IETD) activity was unaffected by P33 expression, again similar to what was seen in P35-expressing cells. These results suggest that P33, like P35, functions by inhibiting effector caspase activity. The point mutant P33(D90A) was unable to inhibit either type of caspase activity, which correlated with its inability to block apoptosis ().
Figure 4 P33 expression prevents effector but not initiator caspase activity in cells. (A) SF-21, S2 or LD652Y cells were either left untreated, infected with control virus vHSGFP, or mock transfected or transfected with plasmids encoding P35, P33 or P33(D90A) (more ...)
P33 is a direct inhibitor of caspases
Next, we determined if P33 was a direct inhibitor of caspases by purifying recombinant C-terminally His6-tagged P33 from bacteria and testing its ability to inhibit purified recombinant caspases. We used the fluorogenic caspase substrates Ac-DEVD-AFC (DrICE, DCP-1, Sf-Caspase-1, caspase-3), Ac-IETDAFC (DRONC), or Ac-LEHD-AFC (caspase-9) performed a dose-response assay, with increasing amounts of P33-His6. The results showed that the effector caspases Sf-caspase-1 from Spodoptera frugiperda, human caspase-3, and DrICE and DCP-1 from Drosophila were efficiently inhibited by purified P33-His6 (), but the initiator caspases DRONC from Drosophila and human caspase-9 were not efficiently inhibited by P33- His6, with approximately 4-fold excess showing no inhibition. A 200-fold excess of P33-His was able to inhibit DRONC and caspase-9 (), indicating that P33 has a limited ability to inhibit initiator caspases. However, given its lack of inhibition at lower concentrations, P33 is probably not able to inhibit initiator caspases under physiological conditions.
Figure 5 P33 directly inhibits effector caspase activity in vitro. Recombinant purified (A) DrICE, (B) Sf-caspase-1, (C) DCP-1, (D) DRONC, (E) caspase-3, or (F) caspase-9 were incubated with increasing concentrations of recombinant P33-His6 and caspase activity (more ...)
To assess the ability of the P33(D90A) mutant to inhibit caspase activity in vitro, recombinant P33(D90A)-His6 was incubated in increasing amounts with effector caspases. P33(D90A)-His6 was unable to block the caspase activity of DrICE, DCP-1, caspase-3, or Sf-caspase-1 (), indicating that residue Asp90 is important for caspase inhibition.
Figure 6 P33 (D90A) does not inhibit caspase activity in vitro. P33 (D90A) (100 μM) was tested for its ability to inhibit recombinant effector caspases (A) DrICE, (B) DCP-1, (C) caspase-3, or (D) Sf-caspase-1 (0.5 μM each) using the substrate Ac-DEVD-AFC. (more ...)
Mechanism of caspase inhibition by P33
AcMNPV P35 is a substrate inhibitor of caspases. The P35 protein is cleaved at position Asp87
by caspases and then the P35 cleavage products become covalently linked to the caspase by a thioester bond (Xu et al., 2001
). To determine whether P33 acts by a similar mechanism, in vitro
S-labeled P33 was incubated with lysate prepared from LD652Y cells that had been UV irradiated or infected with vHSGFP/P35del. P33 was cleaved by the apoptotic lysates and this cleavage was inhibited by the pan-caspase inhibitor z-VAD-fmk, indicating that caspases were involved in the cleavage (). To further determine if P33 was a substrate for caspases, recombinant purified DrICE, DCP-1, Sf-caspase-1, caspase-3, caspase-9 or DRONC were incubated with in vitro
translated P33. P33 was cleaved by the effector caspases caspase-3, DrICE, DCP-1 and Sf-caspase-1 (). Cleavage was not observed with DRONC or caspase-9 (). To determine whether the cleavage fragments associated with the target caspase, we immunoprecipitated the His-tagged caspases using anti-His antibody. 35
S-labeled P33 cleavage fragments associated with the effector caspases caspase-3, DrICE, DCP-1, Sf-caspase-1, but not with DRONC or caspase-9 (). The point mutant P33(D90A) was not cleaved by any of the caspases tested () and did not associate with any of the caspases (), indicating that Asp90
is likely the site of caspase cleavage in P33.
Figure 7 P33 is cleaved and associates stably with effector caspases. (A) Diagram of P33 showing the putative cleavage site D90 and the predicted 22 kDa (*’) and 11 kDa (*) cleavage fragments. (B) 35S-labeled P33 was incubated with lysates from untreated (more ...)
Comparison of caspase inhibition by P33 and P35
To compare the caspase inhibiting ability of P33 in relation to P35, increasing concentrations of recombinant P33 or P35 were incubated with recombinant effector caspases DrICE, DCP-1, Sf-caspase-1, caspase-3 or initiator caspases DRONC or caspase-9 and caspase activity was determined. Both P33 and P35 inhibited effector caspase activity to a similar extent (), but were unable to inhibit the initiator caspase DRONC, except at 200-fold excess (). It has been shown previously that P35 inhibits caspase-9 in vitro
, but not in vivo
(Ryan et al., 2002
). While our results verified inhibition of caspase-9 by P35, we did not observe inhibition of caspase-9 by P33 unless P33 was in high molar excess (). In addition, P35 was cleaved by caspase-9 in vitro
, but P33 was not (data not shown). These results suggest that P33 is more similar to P35 than to P49, since P49 is able to inhibit DRONC in addition to effector caspases, but that P33 differs from P35 in being unable to inhibit human caspase-9 in vitro
Figure 8 Comparison of P33 and P35 caspase inhibiting activity. Increasing concentrations of recombinant purified P33-His6 or P35-His6 were incubated with the indicated caspases (0.5 μM) and caspase activity was determined using the indicated substrates. (more ...)
P33 protects mammalian cells against UV-induced apoptosis
Expression of AcMNPV P35 inhibits apoptosis in a wide variety of organisms, ranging from nematodes to human cells. To determine whether P33 could also protect against apoptosis in a phylogenetically diverse organism, P33 was expressed in the HT-1080 human fibroblastoma cell line and the cells were UV-irradiated to induce apoptosis. P33-expressing cells were protected against apoptosis compared to cells that were mock transfected () and this protection correlated with reduced caspase activity (). P33 was also able to block apoptosis in human embryonic kidney 293 cells induced by UV irradiation ().
Figure 9 P33 blocks UV-induced apoptosis in HT-1080 and HEK293 cells. (A) HT-1080 or (B) 293 cells were mock transfected or transfected with plasmids expressing P33 and 24 h after transfection the cells were UV irradiated. Twenty four h later viability (i) or (more ...)
In this study we have identified and characterized AMVp33, the first homolog of p35 genes found outside of the baculoviruses. Computer-assisted modeling of P33 showed an overall predicted structure similar to that of P35, including a potential reactive site loop and caspase cleavage site. Expression of P33 was found to inhibit apoptosis induced by UV radiation and baculovirus infection in cells from diverse organisms, including insects and human. P33 was able to potently inhibit effector caspases from phylogenetically diverse organisms, but had only limited ability to inhibit initiator caspases. Thus P33 appears to be more similar in action to P35 than to P49. Mutation of the potential cleavage site, Asp90, resulted in complete loss of anti-apoptotic activity and caspase inhibition, and also eliminated caspase cleavage of P33, indicating that Asp90 is likely a site for caspase cleavage similar to that seen in P35 and P49. These results indicate that P33 functions like P35 by acting as a substrate inhibitor of effector caspases.
P35 family members are able to block apoptosis in diverse organisms (Hay et al., 1994
; Rabizadeh et al., 1993
; Sugimoto et al., 1994
), and are among the most widely acting anti-apoptotic proteins known. Despite this, P35 homologs have only been identified to date in baculoviruses and entomopoxviruses, both of which infect only insects. Whether or not P35 homologs exist in cellular genomes or in the genomes of viruses that infect higher organisms is still an unanswered question. The reason for this may be that such homologs have evolutionarily diverged to the point where the sequence identity is too low to be recognized by currently available algorithms. However it is interesting to note that iap
genes, which are also found in insect viruses including baculoviruses, entomopoxviruses, and iridoviruses, do have readily recognizable homologs in higher organisms, but have not been found in viruses that infect higher organisms (with the exception of African swine fever virus, which has an obligate stage in ticks). This work significantly expands the P35 family of protease inhibitors and may aid in the eventual identification of cellular P35 homologs.