While the use of model organisms such as D. melanogaster has led to tremendous advancements in knowledge, model organisms may not be entirely representative of the large phylogenetic groups in which they reside. This study offers an example of the power of using comparative genome analysis of model and non-model organisms in combination with knowledge gained from studying model organisms. Our results illustrate that expansions and losses of caspase genes have occurred in the Diptera. Even within the genus Drosophila, many of the initiator and effector caspases have undergone gene expansions and losses when compared to D. melanogaster. Furthermore, gene expansion in the Diptera has been accompanied by the evolution of caspase genes which lack critical residues required for enzyme activity, and at least one of these caspase-like genes is able to positively regulate the activity of a paralogous caspase. The discovery of caspase-like decoy molecules in insects will complement the extensive knowledge gained from studying caspase regulation in D. melanogaster, and lead to a more in-depth and comprehensive understanding of caspase regulation in insects as a whole.
These results also add significantly to our understanding of caspase evolution in Dipteran insects, which include numerous important vectors of human disease. Several interesting points emerge from our analysis. The Dipteran initiator caspases Dronc and Dredd are present in each of the species we examined as single-copy orthologs. However, all of the other caspase clades have undergone frequent gene duplication/loss events. Extensive gene duplications/losses have been reported in the twelve Drosophila
species for many other gene families, with over 40% of all gene families analyzed differing in size (Hahn et al., 2007
). Gene losses have also been reported for caspase-18, 17 and 15 in vertebrates (Eckhart et al., 2008
), which have been lost in humans but retained in other vertebrate species. Previous analyses comparing caspase homologs in mosquitoes versus D. melanogaster
have led to speculation that there is some specific requirement for additional caspases specifically in mosquitoes, perhaps due to their requirement for blood feeding. It is now clear that expansions in the number of caspase homologs have also occurred in other Drosophila
species as well.
Enzymatically inactive caspases with the ability to regulate other caspases, known as caspase-like decoy molecules, have been reported in mammals and in nematodes. In humans, the caspase-1 gene has undergone a series of duplications resulting in the caspase-like decoy molecules COP1, INCA, and ICEBERG. These genes express truncated forms of caspase-1 that interfere with caspase-1 activation (reviewed in Lamkanfi et al., 2007
). Similarly, csp-2
from C. elegans
encode truncated caspase-like decoy molecules that bind to the zymogen form of the caspase CED-3 and inhibit CED-3 activation (Geng et al., 2008
; Geng et al., 2009
). In addition to these truncated molecules, there are examples of caspase-like decoy molecules that encode full-length caspase proteins, but which lack amino acids that are critical for enzymatic activity. In mammals, duplication of caspase-8 resulted in c-FLIP. While the splice variant c-FLIPS
encodes a truncated molecule which inhibits caspase-8, the variant c-FLIPL
is similar to full length caspase-8, but lacks several key amino acids, including the active site Cys. Interestingly, c-FLIPL
can regulate caspase-8 in both a negative and a positive manner (Micheau et al., 2002
; Budd et al., 2006
). Negative regulation by c-FLIPL
is due to inhibition of caspase-8 recruitment to its activation complex, but positive regulation is due to heterodimerization between c-FLIPL
and caspase-8, resulting in auto-activation of caspase-8. Another interesting example is human caspase-12, which has substitutions in key amino acids leading to a loss of enzymatic activity in the majority of the human population. Enzymatically active caspase-12 is associated with a higher risk of sepsis in humans (Saleh et al., 2004
), suggesting that mutations that inactivate caspase-12 activity are beneficial. In mice, caspase-12 has been shown to bind to caspase-1 and inhibit its activity, and this inhibitory function is retained in active site mutants of caspase-12 (Saleh et al., 2006
In this study, we have identified several insect caspase genes that encode full-length proteins with substitutions in amino acids that are known to be critical for enzymatic activity. One of these, A. aegypti
CASPS18, is a paralog of the effector caspase Decay in D. melanogaster
and CASPS19 in A. aegypti
. Our results show that CASPS18 enhances the activity of its functional paralog CASPS19. This is the first report of an effector-type caspase-like decoy molecule in any organism, as the other caspase-like decoy molecules known are all paralogs of initiator caspases. CASPS18 and CASPS19 are expressed in similar developmental stages and tissues in A. aegypti
(Bryant et al., 2008
), consistent with a role for CASPS18 in regulating CASPS19 activity. Interestingly, high levels of CASPS19 activity did not cause apoptosis in C6/36 cells. Although this may seem surprising given that CASPS19 is able to cleave DEVD-afc with some degree of efficiency, this does not necessarily mean that the sequence DEVD is its optimal substrate cleavage site. Presumably, the in vivo substrates for CASPS19 differ from those of effector caspases involved in apoptosis. Consistent with this result, high levels of active Decay were recently shown to be present in dying midgut during D. melanogaster
morphogenesis, but Decay was not required for midgut cell death (Denton et al., 2009
). These results suggest that Decay orthologs may function in a non-apoptotic process, such as immunity. Although speculative, a possible role in immunity is also supported by the observation that CASPS18 is up-regulated in response to Dengue virus infection in A. aegypti
(Xi et al., 2008
The direct enhancement observed using recombinant proteins suggests that the mechanism of CASPS19 enhancement by CASPS18 is direct, rather than, for example, CASPS18 binding to a cellular inhibitor of CASPS19. This direct mechanism may involve heterodimerization between CASPS18 and CASPS19 subunits, resulting either in higher enzyme activity or in more efficient auto-activation of CASPS19, similar to what is seen in caspase-8 activation by c-FLIPL. It should be noted that we cannot completely rule out the possibility that CASPS18 has catalytic activity, which somehow becomes activated when co-expressed with CASPS19, perhaps by heterodimerization. The presence of a serine rather than a cysteine in the active site could theoretically still allow nucleophile attack on the carbonyl carbon of the substrate scissile bond, and CASPS18 does still contain the necessary proton-accepting histidine residue in the active site. Although speculative, it is interesting to note that, compared to CASPS19 and Decay, CASPS18 has a four-amino acid insertion immediately downstream of the active site, perhaps to allow alternative contacts with substrate.
The discovery of these putative caspase-like decoy molecules in several Dipteran insects provides new avenues for research on the mechanisms of caspase regulation in insects. In particular, the potential positive or negative roles of caspases in transmission of disease agents by insect vectors can now be more completely investigated.