ILP-1 is a potent inhibitor of apoptosis which exerts its effects, at least in part, through the direct binding and inhibition of specific caspases. For example, ILP-1 can bind active caspase 3 with nanomolar affinity and is a powerful inhibitor of its enzymatic activity (10
). Interestingly, ILP-1 is able to bind only the active, heterotetrameric form of caspase 3 and does not associate with its inactive precursor (12
). An important consequence of the high affinity of ILP-1 for caspase 3 is that ILP-1 is capable of suppressing a wide variety of apoptotic signals, including signaling through death receptors as well as DNA-damaging agents, such as irradiation and chemotoxic drugs. While these stimuli have been shown to initiate distinct apoptotic pathways involving the sequential activation of different caspases, they are thought to converge at the downstream effector caspases, particularly caspase 3, and this is thought to account for the wide-ranging protective effects of ILP-1 (10
In this report, we describe the cloning and characterization of a novel IAP gene, ILP-2
. Examination of the genomic structure of the ILP-2
locus revealed the absence of introns as well as an autosomal location. Most gene families are thought to have arisen during evolution by duplication of an ancestral gene, often followed by sequence divergence and ultimately to distinct biological functions. In this case, the general intron-exon structure of the gene family should be conserved. The lack of introns in ILP-2
strongly suggests that it was generated by a retroprocessing event (45
), that is, by the integration into the genome of a fully spliced ILP-1 cDNA generated by reverse transcription. In some cases, retrotransposons have been found to encode functional proteins (28
). One example, which has striking similarity to the situation of ILP-1 and ILP-2, is that of the X-linked gene XAP-5
and its autosomal retrotransposon-derived counterpart, X5L
). Like ILP-2, X5L
has an intronless open reading frame which is highly conserved, encodes a functional protein, and is preferentially expressed in testis. While the functions of XAP-5 and X5L are unknown, it has been proposed that the testis-specific expression of X5L serves to compensate for the absence of XAP-5 in the Y-containing gamete during spermatogenesis (36
). In this regard, ILP-2
is highly analogous to X5L
, since it is autosomal (Mir and Duckett, unpublished) and has a conserved open reading frame and in primary tissues is expressed exclusively in the testis (Fig. ). However, the retroposition event which resulted in ILP-2 most likely occurred relatively recently in primate evolution, while the X5L retroposition is thought to have taken place before the radiation of eutherian mammals (36
). The human ILP-1
DNA sequences are significantly closer to each other than they are to murine Xiap
. This, combined with Southern blot analysis data (Fig. C), suggests that a murine counterpart of ILP-2
might not exist, and therefore a direct test of the biological significance of ILP-2 by gene disruption cannot be performed. However, since ILP-1 can be proteolytically processed to a form which closely resembles ILP-2, it will be of particular interest to examine the tissue expression and functional properties of processed ILP-1.
Analysis of the ILP-2
open reading frame reveals a close similarity to ILP-1. The most significant difference between ILP-2 and ILP-1 is that ILP-2 lacks the two amino-terminal BIR domains which are present in ILP-1. Given the high primary sequence homology between the two proteins, it is more likely that this major structural difference, rather than the small degree of sequence variation, is the main determinant of the differences in biological activity between the two proteins. In support of this hypothesis, two recent reports have described the existence of a proteolytically processed form of ILP-1 (9
) which shares many structural and functional properties with ILP-2.
Despite its similarity to ILP-1, ILP-2 was found to function in a far more restricted manner. Overexpression of ILP-2 failed to inhibit apoptosis induced by signals through death domain-containing receptors but potently suppressed apoptosis induced by Bax or by ectopic expression of Apaf-1 and caspase 9. In addition, cytosolic extracts preincubated with either recombinant ILP-1 or ILP-2 before activation with ATP were found to suppress caspase activity. However, ILP-2 was unable to inhibit caspase activity when added after the extracts had been activated with ATP, while ILP-1 retained its inhibitory function under these conditions.
In many respects, the properties of ILP-2 appear to be very similar to a recently described cleaved form of ILP-1 which lacks the first two amino-terminal BIR domains (9
). Both proteins were capable of suppressing apoptosis induced by Bax or by Apaf-1 and caspase 9, and neither ILP-2 nor the equivalent version of ILP-1 was able to inhibit Fas-mediated apoptosis. However, in transfected cells, processed caspase 9, but not full-length caspase 9, was found to coprecipitate with ILP-2 or its ILP-1 equivalent, and in contrast to a previous report (11
), caspase 9 did not coprecipitate with full-length ILP-1. A recent study by Datta et al. (8
) reported that ILP-1/XIAP could coprecipitate from cells with processed caspase 9, but not full-length caspase 9, and this would appear to be consistent with our data. One possibility is that different forms of ILP-1 and caspase 9 may exist in distinct cellular compartments. For example, processed caspase 9 of the form observed in Fig. is thought to be generated in the Apaf-1-containing apoptosome (1
), and so it is formally possible that, in a cell, ILP-1 and ILP-2 might specifically be targeted to this enzyme complex.
Of particular interest was the observation that preincubation of extracts with GST–ILP-2 resulted in fractions containing a large proportion of full-length caspase 9 (Fig. B), while in cotransfection studies, processed caspase 9, but not full-length caspase 9, coprecipitated with ILP-2 (Fig. A). While we currently have no definitive explanation for these apparently contradictory findings, one possibility that would reconcile the data would be if Apaf-1, following caspase 9 recruitment, processing, and release, could recruit further full-length caspase 9 molecules. If ILP-2 were targeted to the Apaf-1-containing apoptosome, its binding to processed caspase 9 might affect the release of processed caspase 9 from Apaf-1 and the subsequent recruitment of other caspase 9 molecules. Thus, the observed result would be an abundance of full-length caspase 9, even though ILP-2 was functioning specifically on processed caspase 9, and this is consistent with our findings. While this is an attractive possibility, other models can be proposed, and further studies will be required to determine the precise mode of ILP-2 action.
These studies reinforce the notion that IAP proteins can function to suppress apoptosis at multiple points in the cell death pathway. The high degree of specificity of ILP-2 may be very useful in better understanding the apoptotic machinery, and this may also be important as other Apaf-1-independent pathways are identified. Finally, ILP-2 or the equivalent ILP-1 fragment may represent a novel therapeutic target, since disruption of these proteins from the holoenzyme would be predicted to specifically sensitize tumor cells to chemotherapy-induced apoptosis.