XIAP regulates apoptosis
To date, members of three protein families have been found capable of inhibiting caspase activity. The p35 protein from baculoviruses, CrmA protein from the cow pox virus and the family of proteins known as the inhibitor of apoptosis proteins (IAPs) and X-linked inhibitor of apoptosis (XIAP). The IAPs regulate apoptosis by direct caspase inhibition (Deveraux and Reed, 1999; Deveraux et al., 1998; Riedl et al., 2001; Shiozaki et al., 2003). Interestingly, IAPs seem to be multifunctional and are not only involved in regulating apoptosis, but are also involved in receptor-mediated signalling, the cell cycle and ubiquitylation (Birkey Reffey et al., 2001; Hofer-Warbinek et al., 2000; Huang et al., 2000; Levkau et al., 2001; Lu et al., 2007; MacFarlane et al., 2002; Sanna et al., 2002; Sanna et al., 1998; Varfolomeev et al., 2007).
The discovery of IAPs
IAPs were first discovered in the genomes of baculoviruses. They were found to maintain viability of virus infected cells and thus enhance virus replication (Crook et al., 1993; Miller, 1999). Subsequently it was reported that certain IAPs can function in mammalian cells to inhibit apoptosis (Duckett et al., 1996; Harper et al., 2003; Hawkins et al., 1996).
Baculoviral IAP repeat motifs and structure
All IAPs, contain one to three Baculoviral IAP repeat (BIR) motifs. The presence of at least one BIR domain is essential for the anti-apoptotic activity of members of the IAP family. Not all BIR containing proteins are IAPs (Clem and Miller, 1994; Crook et al., 1993; Vucic et al., 1998b). The BIR domain is comprised of approximately 70 amino acids. This includes three conserved cysteines and one conserved histidine residue within the following sequence CX2CX16HX6-8C (Hinds et al., 1999).
Structural analysis of two of the mammalian IAPs revealed that BIR domains typically compromise a series of four alpha helices and three-stranded beta sheet with a single zinc ion. This is coordinated by the conserved cysteine and histidine residues (Chai et al., 2001; Huang et al., 2001; Riedl et al., 2001; Sun et al., 1999). Several, but not all, IAPs also contain a carboxy-terminal RING zinc finger, which has been recognised to have E3 ubiquitin ligase activity (Yang et al., 2000).
IAPs in Drosophila
IAPs have been identified in several multi-cellular organisms from Drosophila to mammals. Convincing evidence that certain members of the IAPs are important regulators of apoptosis results from analysis of the Drosophila melanogaster IAP, DIAP-1. A reduction of DIAP-1 in cells resulted in cell death (Rodriguez et al., 2002; Zimmermann et al., 2002). DIAP-1 was found to bind directly to and inhibit the upstream DRONC and the downstream DCP-1 and DRICE caspases (Hawkins et al., 1999; Meier et al., 2000). As DIAP-1 is a potent inhibitor of cell death, its antagonism is essential in order to allow the progression of programmed cell death. Many IAP antagonists have been identified in Drosophila melanogaster including HID, Grim and Reaper. These antagonists have been shown to interact with the C-terminus of the BIR domain DIAP-1 through their N-terminus (Vucic et al., 1997; Vucic et al., 1998a, b).
Eight members of the IAP family
To date eight members of the IAP family have been identified in humans. These include c-IAP1, c-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2, Livin and Apollon (Liston et al., 2003; Liston et al., 1996). Due to the fact that IAPs are the most powerful intrinsic inhibitors of cell death, capable of inhibiting both mitochondrial and death receptor-mediated apoptotic signalling pathways, by direct binding to initiator and effector caspases, they have become of great interest therapeutically.
The X-Linked Inhibitor of Apoptosis Protein (XIAP)
XIAP is the most potent and versatile regulator of cell death. The molecular mechanism by which XIAP regulates apoptosis is beginning to be unravelled. XIAP has been found to inhibit both the initiator caspase-9 and the effector caspases-3 and-7 (Deveraux et al., 1999). XIAP can intercept and regulate both pathways of apoptosis those mediated by death receptors and those mediated by mitochondria. Interestingly in XIAP knockout mice it was reported that levels of c-IAP1 and c-IAP2 were increased. This suggests that a compensatory mechanism exists that leads to the up-regulation of other IAPs when XIAP expression is lost (Harlin et al., 2001).
XIAP BIR Domain
XIAP possesses three BIR domains followed by a RING domain. The structure of XIAP BIR 2 and -3 domains was determined by nuclear magnetic resonance (NMR) (Sun et al., 1999; Sun et al., 2000). The caspase-3 and -7 inhibiting activity of XIAP was localized to BIR2 domain. This inhibition was also found to require residues found in the linker region that precedes BIR2. Riedl et al reported that XIAP made limited contact through its BIR domain to the surface of the enzyme and most contacts to caspase-3 originated from the N-terminal extension, similar results were obtained for the inhibition of caspase-7 (Chai et al., 2001; Riedl et al., 2001).
XIAP and Caspases
Interestingly it was found that binding of XIAP is anti-parallel to binding of the natural caspase substrates. In addition, four amino acid residues of XIAP, Gly144, Val 146, Val 147 and Asp148 respectively form either hydrogen bonds or Van der Waals interactions with the caspase -7 substrate binding sites S1, S2, S3 and S4. These crucial residues are not found in the BIR2 domain itself, but in the short linker region that precedes BIR2. The Asp 148 residue, after which cleavage occurs, is crucial for the docking interaction as it is proximal to the catalytic Cys residues in caspases. The structural analysis revealed that the binding of the XIAP fragment to caspase-7 results in the complete occupation of the catalytic groove, thus inhibiting access of other substrates (Sun et al., 1999).
XIAP binding to caspases
Binding of XIAP to caspase-3 differs slightly to that of caspase-7. This inhibition displays classical reversible tight-binding kinetics, which is explained by the noncovalent blocking of the active site by a novel structure, the helical hook. Both hook and sinker regions are needed for inhibition as single mutations within either prohibit inhibition but synthetic peptides spanning the hook-line-sinker substructures alone do not inhibit caspase-3 (Sun et al., 1999).
XIAP BIR3 Domain
The mechanism by which XIAP inhibits caspase-9 seems to be unlike that of the BIR2 interactions with caspase-3 and -7. Interestingly XIAP binds only to processed caspase-9, in the absence of proteolytic processing; XIAP is unable to interact with pro-caspase 9 (Srinivasula et al., 2001). Upon triggering by apoptotic stimuli, pro-caspase 9 undergoes auto-catalytic processing at Asp 315 after recruitment to Apaf-1 within the apoptosome. This processing was found to release the p-12 subunit of caspase-9 which results in the physical binding of the N-terminus of the caspase-9 p12 subunit to the BIR3 domain of XIAP. Interestingly deletion or mutagenesis of the four amino terminal residues of the p12 subunit prevented binding of caspase-9 to XIAP (Sun et al., 2000).
The BIR1 domain of XIAP had up to recently no ascribed function. There was no experimental evidence to suggest an ability to inhibit caspases. A possible role that has been hypothesised for the BIR1 domain is that it functions to stabilize XIAP.
Transcription of IAPs by NF kappa Beta
Nuclear factor-κB (NF-κB) has been shown to mediate the transcription of the cIAP-1, cIAP-2, and XIAP genes by tumour necrosis factor-α (TNF-α) (Chu et al., 1997; Stehlik et al., 1998). The ability of XIAP to activate NF-κB is important and could be used by cancer cells to promote survival. It has been shown that ability of XIAP to inhibit apoptosis is dependent on TAK1-dependent mediated survival signalling (Lewis et al., 2004; Sanna et al., 1998). TAK1 is a MAP kinase kinase kinase (MAP 3K), that is responsible for the activation of MAP kinases and NF-κB transcription factors by direct activation of MAP kinase (MKK) and the inhibitor of IκB kinase (IKK) (Wang et al., 2001; Yamaguchi et al., 1999; Yamaguchi et al., 1995).
XIAP and cell cycle arrest
It was also previously shown that XIAP was required for cellular survival in endothelial cells following growth factor withdrawal, by activating the NF-×§B survival pathway and also had the ability to mediate cell cycle arrest through regulation of cyclins and cyclin-dependent kinase inhibitors (CDKIs) (Levkau et al., 2001). Although it has been established that XIAP interacts with TAK1, until recently it was not known how this interaction occurred. A study preformed by Lu et al in 2007 finally assigned a function to the BIR1 domain of XIAP. They found that BIR1 interacts precisely with TAB1 (N-terminal domain of TAK1). By determining the crystal structure of BIR1, TAB1 and the interaction between BIR1/TAB1 it allowed them to conclude induced TAK1 activation is through TAB1 (Lu et al., 2007).
XIAP, cIAP-1 and cIAP-2 also contain a carboxy-terminal RING motif, which has been recognised to have E3 ubiquitin ligase activity. RING domain containing proteins are involved in the process of ubiquitination within a cell (Salvesen and Duckett, 2002).
Ubiquitination and RING domains
RING domains are characterised by the existence of six to seven cysteines and one or to histidines that together form a crossbone architecture that also comprises two zinc ions (Weissman, 2001). The E3 ligase activity in cooperation with the a ubiquitin activating enzymes E1, and E2; catalyse the transfer of ubiquitin to target proteins.(Lorick et al., 1999). This RING domain of the IAPs has the ability to partake in the ubiquitination of proteins. Such as caspase-3,-7 in an E3 ubiquitin protein ligase dependent manner leading to proteasome-mediated degradation (Weissman, 1997). Another feature of the RING domain in the IAPs is that they can mediate their own auto-ubiquitination and degradation (Huang et al., 2000; Yang et al., 2000). This auto-ubiquitination and degradation, is an important regulatory step as lacking this E3 activity is reported to confer resistance to apoptotic stimuli (Yang et al., 2000).
SMAC/DIABLO inhibit XIAP
Caspase inhibition by XIAP may be counteracted by Smac/DIABLO/, or the serine protease Omi/HtrA2. Both of which are released by mitochondria into the cytosol during apoptosis. In addition there is a third protein, located within the nucleus with an ability to inhibit XIAP, known as XIAP associated factor 1 (XAF-1).
The role XIAP plays in tumour development & progression
Resistance to apoptotic stimuli is a hallmark feature of various cancers. Defects in apoptotic mechanisms also play an important role in resistance to chemotherapy and radiation and have been implicated in the acceleration of tumour progression and metastasis (Thompson, 1995). One mechanism of resistance to apoptotic stimuli involves the over-expression of the IAPs. Clearly, the up-regulation of IAP family members would be advantageous for tumours (Nachmias et al., 2004).
Studies using biopsy material from cancer patients indicate a direct association between IAP expression levels and the malignancy of individual tumours. High expression of XIAP has been reported in many malignant tumour types. Such as carcinomas of the breast, ovaries, lung (Gerhard et al., 2002),pancreas, cervix (Liu et al., 2001) and prostrate as well as leukaemia (Tamm et al., 2000). High levels of XIAP have been detected in primary cells of Hodgkins disease (HD). The malignant Hodgkin and Reed Sternberg cells (HR-S) of Hodgkin Lymphoma (HL) and HL-derived B cell lines had previously been shown to be resistant to different apoptotic stimuli. Kashkar et al observed that XIAP is expressed constitutively and at high levels in HL-derived B cells as well as in HR-S cells of tumour biopsies.
In addition, they detected that cytochrome c, as well as caspase-8 or granzyme B-induced activation of caspase 3 is severely impaired in lysates of HL-derived B cell lines. Functional neutralisation by the inhibitor Smac/DIABLO restored the apoptotic response in both lysates and intact HL- derived B cells. This suggests that XIAP is a main mediator of apoptotic resistance in Hodgkin Lymphoma (Kashkar et al., 2003).
XIAP and Smac/DIABLO in tumour progression
A study carried out by Yan et al in renal cell carcinomas (RCCs) to explore the relevance and function of XIAP and Smac/DIABLO in tumour progression revealed that XIAP and Smac/DIABLO mRNA expression was found in all RCCs. Significantly, XIAP mRNA expression levels considerably increased from early to advanced tumour stages and also with tumour differentiation. In contrast mRNA and protein expression levels of Smac/DIABLO did not significantly alter between high and low tumour grades. This investigation demonstrated for the first time a stage and grade-dependent increase of anti-apoptotic XIAP expression in human RCCs. The balance between XIAP and Smac/DIABLO expression is gradually disturbed during progression of RCC, possibly contributing to the resistance to apoptosis in RCCs (Yan et al., 2004).
These studies show the role XIAP plays in tumour development and progression. IAPs appear to be crucial in keeping the tumour cells alive and triggering resistance to chemotherapeutic drugs. Thus understanding the biological role of these inhibitors will facilitate the design of more efficient and selective drugs that could overcome apoptosis resistance in certain cell types.